Patent Grant
10676789
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jun. 5, 2018, is named 43487703309SL.txt and is 5 kilobytes in size.
The processing of polynucleotides and polynucleotide fragments is a critical aspect of a wide variety of technologies, including polynucleotide sequencing. Polynucleotide sequencing continues to find more widespread use in medical applications such as genetic screening and genotyping of tumors. Many polynucleotide sequencing methods rely on sample processing techniques solely utilizing random fragmentation of polynucleotides. Such random, uncontrolled fragmentation can introduce several problems in downstream processing. For example, these methods may produce fragments with large variation in length, including a large number or fraction of sequences that are too long to be sequenced accurately. This results in a loss of sequence information. Current methods of processing may also damage polynucleotides, resulting in incorrect sequence information, and/or the loss of sequence information. These, and other, problems may be significantly amplified by relatively minor operator variability. Thus, there is a significant need for improved methods that provide better control over all aspects of polynucleotide fragmentation and processing. In particular, there is need for polynucleotide processing methods that consistently provide fragments of appropriate size and composition for any downstream application, including sequencing.
I. Non-Overlapping Fragmentation
This disclosure provides methods, compositions, systems, and devices for processing polynucleotides. In one example, a method provided herein comprises: (a) providing a target polynucleotide; (b) fragmenting said target polynucleotide to generate a plurality of non-overlapping first polynucleotide fragments; (c) partitioning said first polynucleotide fragments to generate partitioned first polynucleotide fragments, wherein at least one partition of said partitioned first polynucleotide fragments comprises a first polynucleotide fragment with a unique sequence within said at least one partition; and (d) fragmenting said partitioned first polynucleotide fragments, to generate a plurality of non-overlapping second polynucleotide fragments.
In some of the methods provided in this disclosure, a third and fourth set of polynucleotide fragments are generated by performing the method described above and additionally performing a method comprising: (a) fragmenting said target polynucleotide to generate a plurality of non-overlapping third polynucleotide fragments; (b) partitioning said third polynucleotide fragments to generate partitioned third polynucleotide fragments, wherein at least one partition of said partitioned third polynucleotide fragments comprises a third polynucleotide fragment with a unique sequence within said at least one partition; and (c) fragmenting said partitioned third polynucleotide fragments to generate a plurality of non-overlapping fourth polynucleotide fragments.
The third polynucleotide fragments may overlap with the first polynucleotide fragments. The fourth polynucleotide fragments may overlap with the second polynucleotide fragments.
The target polynucleotide may be, for example, DNA, RNA, cDNA, or any other polynucleotide.
In some cases, at least one of the first, second, third, and fourth polynucleotide fragments are generated by an enzyme. The enzyme may be a restriction enzyme. The restriction enzyme used to generate the first polynucleotide fragments may be different from the restriction enzyme used to generate the third polynucleotide fragments. The restriction enzyme used to generate the second polynucleotide fragments may be different from the restriction enzyme used to generate the fourth polynucleotide fragments. The restriction enzymes may have a recognition site of at least about six nucleotides in length.
The fragments can be of a variety of lengths. For example, the first and/or third polynucleotide fragments may have a median length of least about 10,000 nucleotides. The second or fourth polynucleotide fragments may have a median length of less than about 200 nucleotides.
The fragments can be attached to barcodes. For example, the second polynucleotide fragments and/or the fourth polynucleotide fragments may be attached to barcodes, to generate barcoded second and/or fourth polynucleotide fragments. The barcodes may be polynucleotide barcodes. The attachment of the barcodes to the polynucleotide fragments may be performed using an enzyme. The enzyme may be a ligase. The barcoded fragments may be pooled. Unpooled or pooled barcoded fragments may be sequenced.
In some cases, one or more steps of the methods described in this disclosure may be performed within a device. The device may comprise at least one well. The well may be a microwell. Any of the partitioning steps described in this disclosure may be performed by dispensing into a microwell.
The microwell (or well) may comprise reagents. These reagents may be any reagent, including, for example, barcodes, enzymes, adapters, and combinations thereof. The reagents may be physically separated from a polynucleotide sample placed in the microwell. This physical separation may be accomplished by containing the reagents within a microcapsule that is placed within a microwell. The physical separation may also be accomplished by dispensing the reagents in the microwell and overlaying the reagents with a layer that is, for example, dissolvable, meltable, or permeable prior to introducing the polynucleotide sample into the microwell. This layer may be, for example, an oil, wax, membrane, or the like. The microwell may be sealed at any point, for example after addition of the microcapsule, after addition of the reagents, or after addition of either of these components plus a polynucleotide sample.
Partitioning may also be performed by a variety of other means, including through the use of fluid flow in microfluidic channels, by emulsification, using spotted arrays, by surface acoustic waves, and by piezoelectric droplet generation.
Additional methods of fragmenting nucleic acids that are compatible with the methods provided herein include mechanical disruption, sonication, chemical fragmentation, treatment with UV light, and heating, and combinations thereof. These methods may be used to fragment, for example, the partitioned first or third polynucleotide fragments described above.
Partitioning may be done at any time. For example, the first polynucleotide fragments and/or the third polynucleotide fragments may each be further partitioned into two or more partitions before further processing.
Pseudo-Random Fragmentation
This disclosure provides methods for pseudo-random fragmentation of polynucleotides. In some cases, such methods comprise: (a) providing a target polynucleotide; (b) fragmenting said target polynucleotide to generate a plurality of first polynucleotide fragments; (c) partitioning said first polynucleotide fragments to generate partitioned first polynucleotide fragments, such that at least one partition comprises a first polynucleotide fragment with a unique sequence within said at least one partition; and (d) fragmenting said partitioned first polynucleotide fragments with at least one restriction enzyme in at least one partition, to generate a plurality of second polynucleotide fragments, wherein said partitioned first polynucleotide fragment is fragmented with at least two restriction enzymes across all partitions.
In some cases, at least two restriction enzymes are disposed within the same partition. In some cases, at least two restriction enzymes are disposed across a plurality of different partitions.
The pseudo-random fragmentation methods can be performed in order to yield fragments of a certain size. In some cases, at least about 50% of the nucleotides within a target polynucleotide are within about 100 nucleotides of a restriction site of a restriction enzyme used to perform pseudo-random fragmentation. In some cases, at most about 25% of the nucleotides within a target polynucleotide are within about 50 nucleotides of a restriction site of a restriction enzyme used to perform pseudo-random fragmentation. In some cases, at most about 10% of the nucleotides within a target polynucleotide are more than about 200 nucleotides from a restriction site a restriction enzyme used to perform pseudo-random fragmentation.
A polynucleotide may be treated with two or more restriction enzymes concurrently or sequentially.
The pseudo-randomly fragmented polynucleotides may be attached to barcodes, to generate barcoded polynucleotide fragments. The barcoded polynucleotides may be pooled and sequenced.
The number of partitions holding the partitioned first polynucleotide fragments may be at least about 1,000 partitions. The volume of these partitions may be less than about 500 nanoliters.
Each enzyme may occupy an equivalent number of partitions, or each enzyme may occupy a different number of partitions.
III. Restriction Enzyme-Mediated Recycling
This disclosure provides methods for recycling certain unwanted reaction side products back into starting materials that can be used to generate a desired product. In some cases, these methods comprise: (a) providing a first polynucleotide, a second polynucleotide, a first restriction enzyme, and a second restriction enzyme, wherein said first polynucleotide comprises a target polynucleotide or a fragment thereof; and (b) attaching said first polynucleotide to said second polynucleotide, to generate a polynucleotide product, wherein said first restriction enzyme cuts a polynucleotide generated by attachment of said first polynucleotide to itself, said second restriction enzyme cuts a polynucleotide generated by attachment of said second polynucleotide to itself, and neither said first restriction enzyme nor said second restriction enzyme cuts said polynucleotide product.
The first polynucleotide may be generated in the same reaction volume as the polynucleotide product, or in a different reaction volume. The target polynucleotide may be, for example, a fragment of genomic DNA.
The second polynucleotide may be generated in the same reaction volume as the polynucleotide product, or in a different reaction volume. The second polynucleotide may be, for example, a barcode or an adapter.
The first restriction enzyme may have a recognition site of at most about four nucleotides in length. The second restriction enzyme may have a recognition site of at least about six nucleotides in length. The first restriction enzyme may have a recognition site of about four nucleotides in length. The second restriction enzyme may have a recognition site of at least about five nucleotides in length.
The first and second restriction enzymes may generate ligation compatible ends. These ends may have single-stranded overhangs (i.e., “sticky ends”) or be blunt. The sticky ends may match in sequence and orientation, to allow ligation. The attachment step may be performed by ligation.
The sequence 5′ to the ligation compatible end generated by the first restriction enzyme may be different from the sequence 5′ to the ligation compatible end generated by the second restriction enzyme. This will ensure that the desired product cannot be re-cut by either restriction enzyme.
The sequence 3′ to the ligation compatible end generated by the first restriction enzyme may be different from the sequence 3′ to the ligation compatible end generated by the second restriction enzyme. This will ensure that the desired product cannot be re-cut by either restriction enzyme. Given the criteria provided throughout this specification, one of ordinary skill in the art will recognize that many pairs of enzymes are suitable for use with this method.
The recycling may provide increased yield of the desired product, for example at least about 75% (w/w).
Also provided by this disclosure is a polynucleotide fragment generated by any of the methods provided herein, devices for performing the methods provided herein, and systems for performing the methods provided herein.
The methods provided in this disclosure (and portions thereof) may also be used with each other. For example, the non-overlapping fragmentation methods may be used alone and/or with the pseudo-random fragmentation methods and/or with the restriction enzyme-mediated recycling methods. Likewise, the pseudo-random fragmentation methods may be used alone and/or with the non-overlapping fragmentation methods and/or with the restriction enzyme-mediated recycling methods. Similarly, the restriction enzyme-mediated recycling methods may be used alone and/or with the non-overlapping fragmentation methods and/or with the pseudo-random fragmentation methods.
Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.
All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
The novel features of methods, compositions, systems, and devices of this disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of this disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the methods, compositions, systems, and devices of this disclosure are utilized, and the accompanying drawings of which:
While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.
This disclosure provides methods, compositions, systems, and devices for processing polynucleotides. Applications include processing polynucleotides for polynucleotide sequencing. Polynucleotides sequencing includes the sequencing of whole genomes, detection of specific sequences such as single nucleotide polymorphisms (SNPs) and other mutations, detection of nucleic acid (e.g., deoxyribonucleic acid) insertions, and detection of nucleic acid deletions.
Utilization of the methods, compositions, systems, and devices described herein may incorporate, unless otherwise indicated, conventional techniques of organic chemistry, polymer technology, microfluidics, molecular biology and recombinant techniques, cell biology, biochemistry, and immunology. Such conventional techniques include microwell construction, microfluidic device construction, polymer chemistry, restriction digestion, ligation, cloning, polynucleotide sequencing, and polynucleotide sequence assembly. Specific, non-limiting, illustrations of suitable techniques are described throughout this disclosure. However, equivalent procedures may also be utilized. Descriptions of certain techniques may be found in standard laboratory manuals, such as Genome Analysis: A Laboratory Manual Series (Vols. I-IV), Using Antibodies: A Laboratory Manual, Cells: A Laboratory Manual, PCR Primer: A Laboratory Manual, and Molecular Cloning: A Laboratory Manual (all from Cold Spring Harbor Laboratory Press), and “Oligonucleotide Synthesis: A Practical Approach” 1984, IRL Press London, all of which are herein incorporated in their entirety by reference for all purposes.
I. Definitions
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.
As used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, “such as”, or variants thereof, are used in either the specification and/or the claims, such terms are not limiting and are intended to be inclusive in a manner similar to the term “comprising”.
The term “about,” as used herein, generally refers to a range that is 15% greater than or less than a stated numerical value within the context of the particular usage. For example, “about 10” would include a range from 8.5 to 11.5.
The term “barcode”, as used herein, generally refers to a label that may be attached to a polynucleotide, or any variant thereof, to convey information about the polynucleotide. For example, a barcode may be a polynucleotide sequence attached to all fragments of a target polynucleotide contained within a particular partition. This barcode may then be sequenced with the fragments of the target polynucleotide. The presence of the same barcode on multiple sequences may provide information about the origin of the sequence. For example, a barcode may indicate that the sequence came from a particular partition and/or a proximal region of a genome. This may be particularly useful when several partitions are pooled before sequencing.
The term “bp,” as used herein, generally refers to an abbreviation for “base pairs”.
The term “Mer,” as used herein to refer to restriction enzymes, generally refers to the number of nucleotides in one strand of a restriction enzyme's recognition site. For example, the enzyme CviQI has a recognition site of GTAC (4 nucleotides on one strand) and is thus referred to as a “4Mer cutter.” The enzyme StuI has a recognition site of AGGCCT (6 nucleotides on one strand) and is thus referred to as a “6Mer cutter.”
The term “microwell,” as used herein, generally refers to a well with a volume of less than 1 mL. Microwells may be made in various volumes, depending on the application. For example, microwells may be made in a size appropriate to accommodate any of the partition volumes described herein.
The terms “non-overlapping” and “overlapping,” as used to refer to polynucleotide fragments, generally refer to a collection of polynucleotide fragments without overlapping sequence or with overlapping sequence, respectively. By way of illustration, consider a hypothetical partition containing three copies of a genome (
The term “partition,” as used herein, may be a verb or a noun. When used as a verb (e.g., “partitioning”), the term refers to the fractionation of a substance (e.g., a polynucleotide) between vessels that can be used to sequester one fraction from another. Such vessels are referred to using the noun “partition.” Partitioning may be performed, for example, using microfluidics, dilution, dispensing, and the like. A partition may be, for example, a well, a microwell, a droplet, a test tube, a spot, or any other means of sequestering one fraction of a sample from another. In the methods and systems described herein, polynucleotides are often partitioned into microwells.
The terms “polynucleotide” or “nucleic acid,” as used herein, are used herein to refer to biological molecules comprising a plurality of nucleotides. Exemplary polynucleotides include deoxyribonucleic acids, ribonucleic acids, and synthetic analogues thereof, including peptide nucleic acids.
The term “rare-cutter enzyme,” as used herein, generally refers to an enzyme with a recognition site that occurs only rarely in a genome. The size of restriction fragments generated by cutting a hypothetical random genome with a restriction enzyme may be approximated by 4N, where N is the number of nucleotides in the recognition site of the enzyme. For example, an enzyme with a recognition site consisting of 7 nucleotides would cut a genome once every 47 bp, producing fragments of about 16,384 bp. Generally rare-cutter enzymes have recognition sites comprising 6 or more nucleotides. For example, a rare cutter enzyme may have a recognition site comprising or consisting of 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides. Examples of rare-cutter enzymes include NotI (GCGGCCGC), XmaIII (CGGCCG), SstII (CCGCGG), SalI (GTCGAC), NruI (TCGCGA), NheI (GCTAGC), Nb.BbvCI (CCTCAGC), BbvCI (CCTCAGC), AscI (GGCGCGCC), AsiSI (GCGATCGC), FseI (GGCCGGCC), PacI (TTAATTAA), PmeI (GTTTAAAC), SbfI (CCTGCAGG), SgrAI (CRCCGGYG), SwaI (ATTTAAAT), BspQI (GCTCTTC), SapI (GCTCTTC), SfiI (GGCCNNNNNGGCC (SEQ ID NO: 1)), CspCI (CAANNNNNGTGG (SEQ ID NO: 2)), AbsI (CCTCGAGG), CciNI (GCGGCCGC), FspAI (RTGCGCAY), MauBI (CGCGCGCG), MreI (CGCCGGCG), MssI (GTTTAAAC), PalAI (GGCGCGCC), RgaI (GCGATCGC), RigI (GGCCGGCC), SdaI (CCTGCAGG), SfaAI (GCGATCGC), SgfI (GCGATCGC), SgrDI (CGTCGACG), SgsI (GGCGCGCC), SmiI (ATTTAAAT), SrfI (GCCCGGGC), Sse2321 (CGCCGGCG), Sse83871 (CCTGCAGG), LguI (GCTCTTC), PciSI (GCTCTTC), AarI (CACCTGC), AjuI (GAANNNNNNNTTGG (SEQ ID NO: 3)), AloI (GAACNNNNNNTCC (SEQ ID NO: 4)), BarI (GAAGNNNNNNTAC (SEQ ID NO: 5)), PpiI (GAACNNNNNCTC (SEQ ID NO: 6)), PsrI (GAACNNNNNNTAC (SEQ ID NO: 7)), and others.
The term “target polynucleotide,” as used herein, generally refers to a polynucleotide to be processed. For example, if a user intends to process genomic DNA into fragments that may be sequenced, the genomic DNA would be the target polynucleotide. If a user intends to process fragments of a polynucleotide, then the fragments of the polynucleotide may be the target polynucleotide.
II. Non-Overlapping Fragmentation
This disclosure provides methods, compositions, systems, and devices for the generation of non-overlapping polynucleotide fragments. These fragments may be useful for downstream analyses such as DNA sequencing. For example, with reference to
In order to facilitate DNA sequence assembly, the second fragments may be attached to a barcode, which may be attached to all of the second fragments disposed in a particular partition. The barcode may be, for example, a DNA barcode. With continued reference to
Certain methods of genome sequence assembly rely on the presence of overlapping fragments in order to generate higher order sequence data (e.g., whole genome sequences) from sequenced fragments. The methods, compositions, systems, and devices provided herein may also be used to provide overlapping fragments. For example, with continued reference to
Specifically, the third polynucleotide fragments may be partitioned such that at least one partition 108 comprises a third polynucleotide fragment with a unique sequence within that partition and, optionally, an additional third polynucleotide fragment with a different sequence 109. These partitioned fragments may then be further fragmented to produce a plurality of non-overlapping fourth polynucleotide fragments 110. The fourth polynucleotides fragments and the second polynucleotide fragments may overlap. As for the second polynucleotide fragments, the fourth polynucleotide fragments may be generated by, for example, enzymatic digestion, exposure to ultraviolet (UV) light, ultrasonication, and/or mechanical agitation. The fourth fragments may be of a size that is appropriate for DNA sequencing, i.e., a size that enables a DNA sequencer to obtain accurate sequence data for the entire fragment.
In order to facilitate DNA sequencing, the fourth fragments may be attached to a barcode, which may be attached to all of the fourth fragments disposed in a particular partition. The barcode may be, for example, a DNA barcode. After attachment of the barcode, the barcoded fragments may be pooled, into a partition comprising pooled, barcoded, sequences 111. Three barcodes are depicted as [4], [5], and [6] in 111. The pooled fragments may be sequenced. The overlap between the sequences of the second fragments and the fourth fragments may be used to assemble higher order sequences, such whole genome sequences.
The steps described above may be performed using a variety of techniques. For example, certain steps of the methods may be performed in a device comprising microwell chambers (microwells), for example a microfluidic device. These microwells may be connected to each other, or to a source of reagents, by channels. The first and third fragments may be generated outside of the device and then introduced into the device (or separate devices) for further processing. Partitioning of the first and third fragments may accomplished using fluidic techniques. Generation of the second and fourth fragments may then occur within the microwells of the device or devices. These microwells may contain reagents for barcoding of the second and fourth fragments, such as DNA barcodes, ligase, adapter sequences, and the like. Microwells may feed or be directed into a common outlet, so that barcoded fragments may be pooled or otherwise collected into one or more aliquots which may then be sequenced.
In another example, the entire process could be performed within a single device. For example, a device could be split into two sections. A first section may comprise a partition comprising rare-cutter enzyme 1 (generating first polynucleotide fragments) and a second section may comprise a partition comprising rare-cutter enzyme 2 (generating third polynucleotide fragments). An aliquot of the target polynucleotide sequence may be placed into each of these partitions. Following digestion, the enzyme may be inactivated and the samples may be partitioned, fragmented, barcoded, pooled, and sequenced as described above. For convenience, this example has been described using rare-cutter enzymes as the means of generating the first and third fragments. However, this is not intended to be limiting, here or anywhere else in this disclosure. One of ordinary skill in the art will readily recognize that other means of generating non-overlapping, or predominantly non-overlapping, fragments would be just as suitable as the use of rare-cutter enzymes.
III. Pseudo-Random Fragmentation
This disclosure also provides methods, compositions, systems, and devices for fragmenting polynucleotides in a pseudo-random manner. This may be performed by treating partitioned polynucleotides with more than one restriction enzyme. For example, polynucleotides partitioned into microwells may be treated with combinations of restriction enzymes. Within each partition containing a particular combination of enzymes, the cutting is defined and predictable. However, across all of the partitions (through the use of multiple combinations of restriction enzymes in different partitions), the polynucleotide fragments generated approximate those obtained from methods of random fragmentation. However, these polynucleotide fragments are generated in a much more controlled manner than random fragments generated by methods known in the art (e.g., shearing). The partitioned, pseudo-randomly fragmented polynucleotides may be barcoded, as described throughout this disclosure, pooled, and sequenced. The pseudo-random fragmentation methods may be used with the non-overlapping fragmentation methods described herein, or with any other method described herein such as the high yield adapter/barcode attachment method. Pseudo-random fragmentation may occur by exposing a polynucleotide to multiple enzymes simultaneously, sequentially, or simultaneously and sequentially.
Thus, this disclosure provides methods and systems for processing polynucleotides comprising generating pseudo-random fragments of said polynucleotides. These pseudo random fragments are generated by treating a polynucleotide with more than one restriction enzyme. For example, a polynucleotide may be treated with about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 45, 50, or more restriction enzymes. A polynucleotide may be treated with at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 45, 50, or more restriction enzymes. A polynucleotide may be treated with at least 2 but fewer than 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 45, or 50 restriction enzymes. A polynucleotide may be treated with about 2-4, 4-6, 6-8, 8-10, 10-12, 12-14, 14-16, 16-18, 18-20, 20-25, 25-30, 35-40, 40-45, or 45-50 restriction enzymes.
The restriction enzymes may be chosen in order to maximize the number or fraction of fragments that will provide accurate sequencing data, based on the size of the fragments generated by the pseudo-random fragmentation. For present day sequencing technology, accuracy degrades beyond a read length of about 100 nucleotides. Therefore, fragments of about 200 or fewer nucleotides generally provide the most accurate sequence data since they can be sequenced from either end. Fragments below about 50 nucleotides are generally less desirable because, although the produce accurate sequencing data, they underutilize the read length capacity of current sequencing instruments which are capable of 150 to 200 base reads. Fragments of about 200 to about 400 nucleotides may be sequenced with systematic errors introduced as the read length increases beyond the initial 100 bases from each end. Sequence information from fragments greater than about 400 nucleotides is typically completely lost for those bases greater than 200 bases from either end. One of skill in the art will recognize that sequencing technology is constantly advancing and that the ability to obtain accurate sequence information from longer fragments is also constantly improving. Thus, the pseudo-random fragmentation methods presented herein may be used to produce optimal fragment lengths for any sequencing method.
In some cases, fragments may be defined by the distance of their component nucleotides from a restriction site (measured in nucleotides). For example, each nucleotide within a polynucleotide fragment generated by the pseudo-random fragmentation method may be less than about 10, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 550, 600, 1000, 5000, 10000, or 100000 nucleotides from the restriction site of an enzyme to which the polynucleotide is exposed. Each nucleotide within a polynucleotide fragment may be about 10, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 550, 600, 1000, 5000, 10000, or 100000 nucleotides from the restriction site of an enzyme to which the polynucleotide is exposed. Each nucleotide within a polynucleotide fragment may be at least about 10, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 550, 600, 1000, 5000, 10000, or 100000 nucleotides from the restriction site of an enzyme to which the polynucleotide is exposed.
In some cases, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, of the nucleotides comprising a target polynucleotide sequence are within about 10, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 550, 600, 1000, 5000, 10000, or 100000 nucleotides from the restriction site of an enzyme to which the polynucleotide is exposed. All combinations of these percentages and polynucleotide lengths are contemplated.
In some cases, at less than about 1%, 5%, 10%, 25%, 30%, 35%, 40%, 45%, or 50% of the nucleotides comprising a target polynucleotide sequence are within about 1, 5, 10, 50, 200, 250, 300, 350, 400, 550, 600, 1000, 5000, 10000, or 100000 nucleotides from the restriction site of an enzyme to which the polynucleotide is exposed. All combinations of these percentages and polynucleotide lengths are contemplated.
The pseudo-random fragmentation methods may be used to obtain fragments of about 10 to 50 nucleotides, 46 to 210 nucleotides, 50 to 250 nucleotides, 250 to 400 nucleotides, 400 to 550 nucleotides, 550 to 700 nucleotides, 700 to 1000 nucleotides, 1000 to 1300 nucleotides, 1300 to 1600 nucleotides, 1600 to 1900 nucleotides, 1900 to 2200 nucleotides, or 2200 to 3000 nucleotides. The pseudo-random fragmentation methods may be used to obtain fragments with a mean or median of about 40 nucleotides, 60 nucleotides, 80 nucleotides, 100 nucleotides, 120 nucleotides, 130 nucleotides, 140 nucleotides, 160 nucleotides, 180 nucleotides, 200 nucleotides, 250 nucleotides, 300 nucleotides, 400 nucleotides, 500 nucleotides, 600 nucleotides, 700 nucleotides, 800 nucleotides, 900 nucleotides, 1000 nucleotides, 1200 nucleotides, 1400 nucleotides, 1600 nucleotides, 1800 nucleotides, 2000 nucleotides, 2500 nucleotides, 3000 nucleotides, or more. The pseudo-random fragmentation methods may be used to obtain fragments with a mean or median of at least about 40 nucleotides, 60 nucleotides, 80 nucleotides, 100 nucleotides, 120 nucleotides, 130 nucleotides, 140 nucleotides, 160 nucleotides, 180 nucleotides, 200 nucleotides, 250 nucleotides, 300 nucleotides, 400 nucleotides, 500 nucleotides, 600 nucleotides, 700 nucleotides, 800 nucleotides, 900 nucleotides, 1000 nucleotides, 1200 nucleotides, 1400 nucleotides, 1600 nucleotides, 1800 nucleotides, 2000 nucleotides, 2500 nucleotides, 3000 nucleotides, or more. The pseudo-random fragmentation methods may be used to obtain fragments with a mean or median of less than about 40 nucleotides, 60 nucleotides, 80 nucleotides, 100 nucleotides, 120 nucleotides, 130 nucleotides, 140 nucleotides, 160 nucleotides, 180 nucleotides, 200 nucleotides, 250 nucleotides, 300 nucleotides, 400 nucleotides, 500 nucleotides, 600 nucleotides, 700 nucleotides, 800 nucleotides, 900 nucleotides, 1000 nucleotides, 1200 nucleotides, 1400 nucleotides, 1600 nucleotides, 1800 nucleotides, 2000 nucleotides, 2500 nucleotides, or 3000 nucleotides.
In some examples, the pseudo-random fragmentation methods provided herein are used to generate fragments wherein a particular percentage (or fraction) of the fragments generated fall within any of the size ranges described herein. For example, about 0%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, 30%, 32%, 34%, 36%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, or 100% of the fragments generated may fall within any of the size ranges described herein.
In some examples multiple 4Mer cutters may be used to provide a distribution of about 18% of fragments of about 50 nucleotides or less, about 38% of fragments of about 200 nucleotides or less, about 25% of fragments between about 200 and about 400 nucleotides, and about 37% of fragments greater than about 400 nucleotides (e.g., see
Additionally, the pseudo-random fragmentation method may be designed to minimize the percentage of fragments greater than a certain number of nucleotides in length, in order to minimize the loss of sequence information. For example, the method may be designed to yield less than about 0.1%, 0.5%, 1%, 2%, 5%, 10%, 20%, or 50% fragments greater than 100 nucleotides. The method may be designed to yield less than about 0.1%, 0.5%, 1%, 2%, 5%, 10%, 20%, or 50% fragments greater than 150 nucleotides. The method may be designed to yield less than about 0.1%, 0.5%, 1%, 2%, 5%, 10%, 20%, or 50% fragments greater than 200 nucleotides. The method may be designed to yield less than about 0.1%, 0.5%, 1%, 2%, 5%, 10%, 20%, or 50% fragments greater than 300 nucleotides, and so on. As the ability of sequencing technologies to accurately read long DNA fragments increases, the pseudo-random fragmentation methods of the invention may be used to generate sequences suitable for any chosen read length.
Enzymes for use with the pseudo-random fragmentation method described herein may be chosen, for example, based on the length of their recognition site and their compatibility with certain buffer conditions (to allow for combination with other enzymes). Enzymes may also be chosen so that their cutting activity is methylation insensitive, or sensitive to methylation. For example, restriction enzymes with shorter recognition sites generally cut polynucleotides more frequently. Thus, cutting a target polynucleotide with a 6Mer cutter will generally produce more large fragments than cutting the same polynucleotide with a 4Mer cutter (e.g., compare
This disclosure also provides methods of selecting a plurality of enzymes for pseudo-random fragmentation of a polynucleotide sequence. For example, a target polynucleotide may be exposed separately to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 restriction enzymes. The size distribution of the target polynucleotide fragments is then determined, for example, by electrophoresis. The combination of enzymes providing the greatest number of fragments that are capable of being sequenced can then be chosen. The method can also be carried out in silico.
The enzymes may be disposed within the same partition, or within a plurality of partitions. For example, any of the plurality of enzyme number described herein may be disposed within a single partition, or across partitions. For example, a polynucleotide may be treated with about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 45, 50, or more restriction enzymes in the same partition, or across partitions. A polynucleotide may be treated with at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 45, 50, or more restriction enzymes in the same partition, or across partitions. A polynucleotide may be treated with at least 2 but fewer than 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 45, or 50 restriction enzymes in the same partition, or across partitions. A polynucleotide may be treated with about 2-4, 4-6, 6-8, 8-10, 10-12, 12-14, 14-16, 16-18, 18-20, 20-25, 25-30, 35-40, 40-45, or 45-50 restriction enzymes in the same partition, or across partitions.
The distribution of the restriction enzymes among the partitions will vary depending on the restriction enzymes, the target polynucleotide, and the desired fragment size. In some cases, each restriction enzyme may be distributed across an equivalent number of partitions, so that the number of partitions occupied by each restriction enzyme is equivalent. For example, if 10 restriction enzymes are used in a device containing 1,000 partitions, each enzyme may be present in 100 partitions. In other cases, each restriction enzyme may be distributed across a non-equivalent number of partitions, so that the number of partitions occupied by each restriction enzyme is not equivalent. For example, if 10 restriction enzymes are used in a device containing 1,000 partitions, enzymes 1-8 may be present in 100 partitions each, enzyme 9 may be present in 50 partitions, and enzyme 10 may be present in 150 partitions. Placement of restriction enzymes in an unequal number of partitions may be beneficial, for example, when an enzyme generates a desired product at a low yield. Placing this low-yield enzyme in more partitions will therefore expose more of the target polynucleotide to the enzyme, increasing the amount of the desired product (e.g., fragment of a certain size or composition) that can be formed from the enzyme. Such an approach may be useful for accessing portions of a target polynucleotide (e.g., a genome) that are not cut by enzymes producing polynucleotide fragments at a higher yield. The restriction site and efficiency of an enzyme, composition of the target polynucleotide, and efficiency and side-products generated by the enzyme may all be among the factors considered when determining how many partitions should receive a particular enzyme.
In some cases, different numbers of restriction enzymes may be used in a single partition and across all partitions. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 45, or 50 restriction enzymes or more may be used in each partition, while 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 45, or 50 restriction enzymes or more may be used across all partitions. All combinations of these numbers are included within the invention. Non-limiting specific examples include the use of 1 restriction enzyme per partition and 2, 3, 4, 5, 6, 7, 8, 9, or 10 restriction enzymes across all partitions; 2 restriction enzymes per partition and 3, 4, 5, 6, 7, 8, 9, or 10 restriction enzymes across all partitions; 3 restriction enzymes per partition and 4, 5, 6, 7, 8, 9, or 10 restriction enzymes across all partitions; 4 restriction enzymes per partition and 5, 6, 7, 8, 9, or 10 restriction enzymes across all partitions; 5 restriction enzymes per partition and 6, 7, 8, 9, or 10 restriction enzymes across all partitions; 6 restriction enzymes per partition and 7, 8, 9, or 10 restriction enzymes across all partitions; 7 restriction enzymes per partition and 8, 9, or 10 restriction enzymes across all partitions; 8 restriction enzymes per partition and 9 or 10 restriction enzymes across all partitions; and 9 restriction enzymes per partition and 10 or more restriction enzymes across all partitions.
In some cases, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 45, or 50 restriction enzymes or more may be used in each partition, while at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 45, or 50 restriction enzymes or more may be used across all partitions. All combinations of these numbers are included within the invention. Non-limiting specific examples include the use of at least 1 restriction enzyme per partition and at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 restriction enzymes across all partitions; at least 2 restriction enzymes per partition and at least 3, 4, 5, 6, 7, 8, 9, or 10 restriction enzymes across all partitions; at least 3 restriction enzymes per partition and at least 4, 5, 6, 7, 8, 9, or 10 restriction enzymes across all partitions; at least 4 restriction enzymes per partition and at least 5, 6, 7, 8, 9, or 10 restriction enzymes across all partitions; at least 5 restriction enzymes per partition and at least 6, 7, 8, 9, or 10 restriction enzymes across all partitions; at least 6 restriction enzymes per partition and at least 7, 8, 9, or 10 restriction enzymes across all partitions; at least 7 restriction enzymes per partition and at least 8, 9, or 10 restriction enzymes across all partitions; at least 8 restriction enzymes per partition and at least 9 or 10 restriction enzymes across all partitions; and at least 9 restriction enzymes per partition and at least 10 or more restriction enzymes across all partitions.
In some cases, at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 45, or 50 restriction enzymes or more may be used in each partition, while at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 45, or 50 restriction enzymes or more may be used across all partitions. All combinations of these numbers are included within the invention. Non-limiting specific examples include the use of at most 1 restriction enzyme per partition and at most 2, 3, 4, 5, 6, 7, 8, 9, or 10 restriction enzymes across all partitions; at most 2 restriction enzymes per partition and at most 3, 4, 5, 6, 7, 8, 9, or 10 restriction enzymes across all partitions; at most 3 restriction enzymes per partition and at most 4, 5, 6, 7, 8, 9, or 10 restriction enzymes across all partitions; at most 4 restriction enzymes per partition and at most 5, 6, 7, 8, 9, or 10 restriction enzymes across all partitions; at most 5 restriction enzymes per partition and at most 6, 7, 8, 9, or 10 restriction enzymes across all partitions; at most 6 restriction enzymes per partition and at most 7, 8, 9, or 10 restriction enzymes across all partitions; at most 7 restriction enzymes per partition and at most 8, 9, or 10 restriction enzymes across all partitions; at most 8 restriction enzymes per partition and at most 9 or 10 restriction enzymes across all partitions; and at most 9 restriction enzymes per partition and at most 10 or more restriction enzymes across all partitions.
In some cases, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 45, or 50 restriction enzymes or more may be used in each partition, while at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 45, or 50 restriction enzymes or more may be used across all partitions. All combinations of these numbers are included within the invention. Non-limiting specific examples include the use of at least 1 restriction enzyme per partition and at most 2, 3, 4, 5, 6, 7, 8, 9, or 10 restriction enzymes across all partitions; at least 2 restriction enzymes per partition and at most 3, 4, 5, 6, 7, 8, 9, or 10 restriction enzymes across all partitions; at least 3 restriction enzymes per partition and at most 4, 5, 6, 7, 8, 9, or 10 restriction enzymes across all partitions; at least 4 restriction enzymes per partition and at most 5, 6, 7, 8, 9, or 10 restriction enzymes across all partitions; at least 5 restriction enzymes per partition and at most 6, 7, 8, 9, or 10 restriction enzymes across all partitions; at least 6 restriction enzymes per partition and at most 7, 8, 9, or 10 restriction enzymes across all partitions; at least 7 restriction enzymes per partition and at most 8, 9, or 10 restriction enzymes across all partitions; at least 8 restriction enzymes per partition and at most 9 or 10 restriction enzymes across all partitions; and at least 9 restriction enzymes per partition and at most 10 or more restriction enzymes across all partitions.
In some cases, at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 45, or 50 restriction enzymes or more may be used in each partition, while at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 45, 45, or 50 restriction enzymes or more may be used across all partitions. All combinations of these numbers are included within the invention. Non-limiting specific examples include the use of at most 1 restriction enzyme per partition and at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 restriction enzymes across all partitions; at most 2 restriction enzymes per partition and at least 3, 4, 5, 6, 7, 8, 9, or 10 restriction enzymes across all partitions; at most 3 restriction enzymes per partition and at least 4, 5, 6, 7, 8, 9, or 10 restriction enzymes across all partitions; at most 4 restriction enzymes per partition and at least 5, 6, 7, 8, 9, or 10 restriction enzymes across all partitions; at most 5 restriction enzymes per partition and at least 6, 7, 8, 9, or 10 restriction enzymes across all partitions; at most 6 restriction enzymes per partition and at least 7, 8, 9, or 10 restriction enzymes across all partitions; at most 7 restriction enzymes per partition and at least 8, 9, or 10 restriction enzymes across all partitions; at most 8 restriction enzymes per partition and at least 9 or 10 restriction enzymes across all partitions; and at most 9 restriction enzymes per partition and at least 10 or more restriction enzymes across all partitions.
IV. Restriction Enzyme-Mediated Recycling
As described throughout this disclosure, certain methods of the invention involve the addition of barcodes, adapters, or other sequences to fragmented target polynucleotides. Barcodes may be polynucleotide barcodes, which may be ligated to the fragmented target polynucleotides or added via an amplification reaction. As described throughout this disclosure, fragmentation of target polynucleotides may be performed using one or more restriction enzymes contained within a partition (e.g., a microwell) where the fragmentation is performed. The partition may also contain a polynucleotide barcode and a ligase, which enables the attachment of the barcode to the fragmented polynucleotide. In some cases, an adapter may be used to make a fragmented target polynucleotide compatible for ligation with a barcode. The presence of adapters, fragmented target polynucleotide, barcodes, restriction enzymes, and ligases in the same partition may lead to the generation of undesirable side products that decrease the yield of a desired end product. For example, self-ligation may occur between adapters, target polynucleotide fragments, and/or barcodes. These self-ligations reduce the amount of starting material and decrease the yield of the desired product, for example, a polynucleotide fragment properly ligated to a barcode and/or and adapter.
This disclosure provides methods, compositions, systems, and devices for addressing this problem and increasing the yield of a desired product. The problem is addressed by pairing a first restriction enzyme and a second restriction enzyme. The two restriction enzymes create compatible termini upon cutting, but each enzyme has a different recognition sequence.
Ligation of two pieces of DNA generated after cutting with the first restriction enzyme will regenerate the recognition site for the first restriction enzyme, allowing the first restriction enzyme to re-cut the ligated DNA. Likewise, ligation of two pieces of DNA generated after cutting with the second restriction enzyme will regenerate the recognition site for the second restriction enzyme, allowing the second restriction enzyme to re-cut the ligated DNA. However, ligation of one piece of DNA generated after cutting with the first restriction enzyme and one piece of DNA generated after cutting with the second restriction enzyme will result in ligated DNA that is unrecognizable (and therefore uncuttable) by both the first and second enzymes. The result is that any multimers of fragmented target polynucleotides are re-cut and any multimers of adapter (or other molecules, e.g., barcodes) are also re-cut. However, when a fragmented target polynucleotide is properly ligated to an adapter (or barcode), the restriction sites for both enzymes are not present and the correctly ligated molecule may not be re-cut by either enzyme.
An example of this method is illustrated in
This method may be used to increase the yield of any of the barcoding methods described herein. The regeneration of the starting materials (e.g., fragmented target polynucleotide, adapters, and barcodes) allows these starting materials another opportunity to form the desired products (i.e., fragmented target polynucleotides ligated to barcodes, optionally with adapters). This greatly increases the yield of the reaction and therefore decreases the amount of starting material required to produce the necessary amount of the desired products while limiting the amount of undesirable side products and lost sequence information.
The methods described above may be used to achieve about 75%, 85%, 95%, 96%, 97%, 98%, 99%, or 99.5% yield (w/w). The methods may be used to achieve at least about 75%, 85%, 95%, 96%, 97%, 98%, 99%, or 99.5% yield (w/w).
The methods described above may use, for example, a pair of restriction enzyme selected from the group consisting of MspI-NarI, BfaI-NarI, BfaI-NdeI, HinPlI-ClaI, MseI-NdeI, CviQI-NdeI, Taqoa-AcII, RsaI-PmeI, AluI-EcoRV, BstUI-PmeI, DpnI-StuI, HaeIII-PmeI, and HpyCH4V-SfoI. This list of enzymes is provided for purposes of illustration only, and is not meant to be limiting.
The methods described above may generally use any two enzymes that create ligatable termini upon cutting but that have different recognition sequences. However, the method is not limited to ligation. For example, multimers formed after amplification of side products formed by association of compatible ends could also be re-cut using the methods described above.
More than one pair of enzymes may also be used. The number of pairs of enzymes chosen will vary depending on the number of undesirable side products formed in a reaction. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more pairs of enzymes may be used. Treatment of a polynucleotide with the enzymes may be sequential, simultaneous, or both.
V. Preparation of Target Polynucleotides
Target polynucleotides processed according to the methods provided in this disclosure may be DNA, RNA, peptide nucleic acids, and any hybrid thereof, where the polynucleotide contains any combination of deoxyribo- and ribo-nucleotides. Polynucleotides may be single stranded or double stranded, as specified, or contain portions of both double stranded or single stranded sequence. Polynucleotides may contain any combination of nucleotides, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, isoguanine and any nucleotide derivative thereof. As used herein, the term “nucleotide” may include nucleotides and nucleosides, as well as nucleoside and nucleotide analogs, and modified nucleotides, including both synthetic and naturally occurring species. Target polynucleotides may be cDNA, mitochondrial DNA (mtDNA), messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), nuclear RNA (nRNA), small interfering RNA (siRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), small Cajal body-specific RNA (scaRNA), microRNA (miRNA), double stranded (dsRNA), ribozyme, riboswitch or viral RNA. Target polynucleotides may be contained on a plasmid, cosmid, or chromosome, and may be part of a genome. In some cases, a target polynucleotide may comprise one or more genes and/or one or more pseudogenes. A pseudogene generally refers to a dysfunctional relative of a gene that has lost its protein coding ability and/or is otherwise no longer expressed in the cell.
Target polynucleotides may be obtained from a sample using any methods known in the art. A target polynucleotide processed as described herein may be obtained from whole cells, cell preparations and cell-free compositions from any organism, tissue, cell, or environment. In some instances, target polynucleotides may be obtained from bodily fluids which may include blood, urine, serum, lymph, saliva, mucosal secretions, perspiration, or semen. In some instances, polynucleotides may be obtained from environmental samples including air, agricultural products, water, and soil. In other instances polynucleotides may be the products of experimental manipulation including, recombinant cloning, polynucleotide amplification (as generally described in PCT/US99/01705), polymerase chain reaction (PCR) amplification, purification methods (such as purification of genomic DNA or RNA), and synthesis reactions.
Genomic DNA may be obtained from naturally occurring or genetically modified organisms or from artificially or synthetically created genomes. Target polynucleotides comprising genomic DNA may be obtained from any source and using any methods known in the art. For example, genomic DNA may be isolated with or without amplification. Amplification may include PCR amplification, multiple displacement amplification (MDA), rolling circle amplification and other amplification methods. Genomic DNA may also be obtained by cloning or recombinant methods, such as those involving plasmids and artificial chromosomes or other conventional methods (see Sambrook and Russell, Molecular Cloning: A Laboratory Manual, cited supra.) Polynucleotides may be isolated using other methods known in the art, for example as disclosed in Genome Analysis: A Laboratory Manual Series (Vols. I-IV) or Molecular Cloning: A Laboratory Manual. If the isolated polynucleotide is an mRNA, it may be reverse transcribed into cDNA using conventional techniques, as described in Sambrook and Russell, Molecular Cloning: A Laboratory Manual, cited supra.
Target polynucleotides may also be isolated from “target organisms” or “target cells”. The terms “target organism” and “target cell” refer to an organism or cell, respectively, from which target polynucleotides may be obtained. Target cells may be obtained from a variety of organisms including human, mammal, non-human mammal, ape, monkey, chimpanzee, plant, reptilian, amphibian, avian, fungal, viral or bacterial organisms. Target cells may also be obtained from a variety of clinical sources such as biopsies, aspirates, blood, urine, formalin fixed embedded tissues, and the like. Target cells may comprise a specific cell type, such as a somatic cell, germline cell, wild-type cell, cancer or tumor cells, or diseased or infected cell. A target cell may refer to a cell derived from a particular tissue or a particular locus in a target organism. A target cell may comprise whole intact cells, or cell preparations.
Target polynucleotides may also be obtained or provided in specified quantities. Amplification may be used to increase the quantity of a target polynucleotide. Target polynucleotides may quantified by mass. For example, target polynucleotides may be provided in a mass ranging from about 1-10, 10-50, 50-100, 100-200, 200-1000, 1000-10000 ng. Target polynucleotides may be provided in a mass of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 50, 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, or 10000 ng. Target polynucleotides may be provided in a mass of less than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 50, 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, or 10000 ng.
Target polynucleotides may also be quantified as “genome equivalents.” A genome equivalent is an amount of polynucleotide equivalent to one haploid genome of an organism from which the target polynucleotide is derived. For example, a single diploid cell contains two genome equivalents of DNA. Target polynucleotides may be provided in an amount ranging from about 1-10, 10-50, 50-100, 100-1000, 1000-10000, 10000-100000, or 100000-1000000 genome equivalents. Target polynucleotides may be provided in an amount of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 50, 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 20000, 30000, 40000, 50000, 60000 70000, 80000, 90000, 100000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, or 1000000 genome equivalents. Target polynucleotides may be provided in an amount less than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 50, 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 20000, 30000, 40000, 50000, 60000 70000, 80000, 90000, 100000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, or 1000000 genome equivalents.
Target polynucleotide may also be quantified by the amount of sequence coverage provided. The amount of sequence coverage refers to the average number of reads representing a given nucleotide in a reconstructed sequence. Generally, the greater the number of times a region is sequenced, the more accurate the sequence information obtained. Target polynucleotides may be provided in an amount that provides a range of sequence coverage from about 0.1×-10×, 10-×-50×, 50×-100×, 100×-200×, or 200×-500×. Target polynucleotide may be provided in an amount that provides at least about 0.1×, 0.2×, 0.3×, 0.4×, 0.5×, 0.6×, 0.7×, 0.8×, 0.9×, 1.0×, 5×, 10×, 25×, 50×, 100×, 125×, 150×, 175×, or 200× sequence coverage. Target polynucleotide may be provided in an amount that provides less than about 0.2×, 0.3×, 0.4×, 0.5×, 0.6×, 0.7×, 0.8×, 0.9×, 1.0×, 5×, 10×, 25×, 50×, 100×, 125×, 150×, 175×, or 200× sequence coverage.
VI. Fragmentation of Target Polynucleotides
Fragmentation of polynucleotides is used as a step in a variety of processing methods described herein. The size of the polynucleotide fragments, typically described in terms of length (quantified by the linear number of nucleotides per fragment), may vary depending on the source of the target polynucleotide, the method used for fragmentation, and the desired application. Moreover, while certain methods of the invention are illustrated using a certain number of fragmentation steps, the number of fragmentation steps provided is not meant to be limiting, and any number of fragmentation steps may be used. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more fragmentation steps may be used.
Fragments generated using the methods described herein may be about 1-10, 10-20, 20-50, 50-100, 50-200, 100-200, 200-300, 300-400, 400-500, 500-1000, 1000-5000, 5000-10000, 10000-100000, 100000-250000, or 250000-500000 nucleotides in length. Fragments generated using the methods described herein may be at least about 10, 20, 100, 200, 300, 400, 500, 1000, 5000, 10000, 100000, 250000, 500000, or more nucleotides in length. Fragments generated using the methods described herein may be less than about 10, 20, 100, 200, 300, 400, 500, 1000, 5000, 10000, 100000, 250000, 500000, nucleotides in length.
Fragments generated using the methods described herein may have a mean or median length of about 1-10, 10-20, 20-50, 50-100, 50-200, 100-200, 200-300, 300-400, 400-500, 500-1000, 1000-5000, 5000-10000, 10000-100000, 100000-250000, or 250000-500000 nucleotides. Fragments generated using the methods described herein may have a mean or median length of at least about 10, 20, 100, 200, 300, 400, 500, 1000, 5000, 10000, 100000, 250000, 500000, or more nucleotides. Fragments generated using the methods described herein may have a mean or median length of less than about 10, 20, 100, 200, 300, 400, 500, 1000, 5000, 10000, 100000, 250000, 500000, nucleotides.
Numerous fragmentation methods are described herein and known in the art. For example, fragmentation may be performed through physical, mechanical or enzymatic methods. Physical fragmentation may include exposing a target polynucleotide to heat or to UV light. Mechanical disruption may be used to mechanically shear a target polynucleotide into fragments of the desired range. Mechanical shearing may be accomplished through a number of methods known in the art, including repetitive pipetting of the target polynucleotide, sonication and nebulization. Target polynucleotides may also be fragmented using enzymatic methods. In some cases, enzymatic digestion may be performed using enzymes such as using restriction enzymes.
While the methods of fragmentation described in the preceding paragraph, and in some paragraphs of the disclosure, are described with reference to “target” polynucleotides, this is not meant to be limiting, above or anywhere else in this disclosure. Any means of fragmentation described herein, or known in the art, can be applied to any polynucleotide used with the invention. In some cases, this polynucleotide may be a target polynucleotide, such as a genome. In other cases, this polynucleotide may be a fragment of a target polynucleotide which one wishes to further fragment. In still other cases, still further fragments may be still further fragmented. Any suitable polynucleotide may be fragmented according the methods described herein.
A fragment of a polynucleotide generally comprises a portion of the sequence of the targeted polynucleotide from which the fragment was generated. In some cases, a fragment may comprise a copy of a gene and/or pseudogene, including one included in the original target polynucleotide. In some cases, a plurality of fragments generated from fragmenting a target polynucleotide may comprise fragments that each comprise a copy of a gene and/or pseudogene.
Restriction enzymes may be used to perform specific or non-specific fragmentation of target polynucleotides. The methods of the present disclosure may use one or more types of restriction enzymes, generally described as Type I enzymes, Type II enzymes, and/or Type III enzymes. Type II and Type III enzymes are generally commercially available and well known in the art. Type II and Type III enzymes recognize specific sequences of nucleotide base pairs within a double stranded polynucleotide sequence (a “recognition sequence” or “recognition site”). Upon binding and recognition of these sequences, Type II and Type III enzymes cleave the polynucleotide sequence. In some cases, cleavage will result in a polynucleotide fragment with a portion of overhanging single stranded DNA, called a “sticky end.” In other cases, cleavage will not result in a fragment with an overhang, creating a “blunt end.” The methods of the present disclosure may comprise use of restriction enzymes that generate either sticky ends or blunt ends.
Restriction enzymes may recognize a variety of recognition sites in the target polynucleotide. Some restriction enzymes (“exact cutters”) recognize only a single recognition site (e.g., GAATTC). Other restriction enzymes are more promiscuous, and recognize more than one recognition site, or a variety of recognition sites. Some enzymes cut at a single position within the recognition site, while others may cut at multiple positions. Some enzymes cut at the same position within the recognition site, while others cut at variable positions.
The present disclosure provides method of selecting one or more restriction enzymes to produce fragments of a desired length. Polynucleotide fragmentation may be simulated in silico, and the fragmentation may be optimized to obtain the greatest number or fraction of polynucleotide fragments within a particular size range, while minimizing the number or fraction of fragments within undesirable size ranges. Optimization algorithms may be applied to select a combination of two or more enzymes to produce the desired fragment sizes with the desired distribution of fragments quantities.
A polynucleotide may be exposed to two or more restriction enzymes simultaneously or sequentially. This may be accomplished by, for example, adding more than one restriction enzyme to a partition, or by adding one restriction enzyme to a partition, performing the digestion, deactivating the restriction enzyme (e.g., by heat treatment) and then adding a second restriction enzyme. Any suitable restriction enzyme may be used alone, or in combination, in the methods presented herein.
Fragmenting of a target polynucleotide may occur prior to partitioning of the target polynucleotide or fragments generated from fragmenting. For example, genomic DNA (gDNA) may be fragmented, using, for example, a restriction enzyme, prior to the partitioning of its generated fragments. In another example, a target polynucleotide may be entered into a partition along with reagents necessary for fragmentation (e.g., including a restriction enzyme), such that fragmentation of the target polynucleotide occurs within the partition. For example, gDNA may be fragmented in a partition comprising a restriction enzyme, and the restriction enzyme is used to fragment the gDNA.
In some cases, a plurality of fragments may be generated prior to partitioning, using any method for fragmentation described herein. Some or all of the fragments of the plurality, for example, may each comprise a copy of a gene and/or a pseudogene. The fragments can be separated and partitioned such that each copy of the gene or pseudogene is located in a different partition. Each partition, for example, can comprise a different barcode sequence such that each copy of the gene and/or pseudogene can be associated with a different barcode sequence, using barcoding methods described elsewhere herein. Via the different barcode sequences, each gene and/or pseudogene can be counted and/or differentiated during sequencing of the barcoded fragments. Any sequencing method may be used, including those described herein.
For example, using restriction enzymes, genomic DNA (gDNA) can be fragmented to generate a plurality of non-overlapping fragments of the gDNA. At least some of the fragments of the plurality may each comprise a copy of a gene and/or a pseudogene. The fragments may be separated and partitioned such that each copy of the gene or pseudogene is located in a different partition. Each partition, for example, can comprise a different barcode sequence such that each copy of the gene and/or pseudogene may be barcoded with a different barcode sequence. Via the different barcode sequences, the genes and/or pseudogenes may be counted and or differentiated after sequencing of the barcoded fragments. Any sequencing method may be used, including those described herein.
IV. Partitioning of Polynucleotides
As described throughout the disclosure, certain methods, systems, and compositions of the disclosure may utilize partitioning of polynucleotides into separate partitions (e.g., microwells, droplets of an emulsion). These partitions may be used to contain polynucleotides for further processing, such as, for example, cutting, ligating, and/or barcoding.
Any number of devices, systems or containers may be used to hold, support or contain partitions of polynucleotides and their fragments. In some cases, partitions are formed from droplets, emulsions, or spots on a substrate. Weizmann et al. (Nature Methods, 2006, Vol. 3 No. 7 pages 545-550). Suitable methods for forming emulsions, which can be used as partitions or to generate microcapsules, include the methods described in Weitz et al. (U.S. Pub. No. 2012/0211084). Partitions may also be formed through the use of wells, microwells, multi-well plates, and microwell arrays. Partitioning may be performed using piezoelectric droplet generation (e.g., Bransky et al., Lab on a Chip, 2009, 9, 516-520). Partitioning may be performed using surface acoustic waves (e.g., Demirci and Montesano, Lab on a Chip, 2007, 7, 1139-1145).
Each partition may also contain, or be contained within any other suitable partition. For example, a well, microwell, hole, a surface of a bead, or a tube may comprise a droplet (e.g., a droplet in an emulsion), a continuous phase in an emulsion, a spot, a capsule, or any other suitable partition. A droplet may comprise a capsule, bead, or another droplet. A capsule may comprise a droplet, bead, or another capsule. These descriptions are merely illustrative, and all suitable combinations and pluralities are also envisioned. For example, any suitable partition may comprise a plurality of the same or different partitions. In one example, a well or microwell comprises a plurality of droplets and a plurality of capsules. In another example, a capsule comprises a plurality of capsules and a plurality of droplets. All combinations of partitions are envisioned. Table 1 shows non-limiting examples of partitions that may be combined with each other.
Any partition described herein may comprise multiple partitions. For example, a partition may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 50, 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, or 50000 partitions. A partition may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 50, 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, or 50000 partitions. In some cases, a partition may comprise less than 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 50, 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, or 50000 partitions. In some cases, each partition may comprise 2-50, 2-20, 2-10, or 2-5 partitions.
The number of partitions employed may vary depending on the application. For example, the number of partitions may be about 5, 10, 50, 100, 250, 500, 750, 1000, 1500, 2000, 2500, 5000, 7500, or 10,000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100,000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, 1,000,000, 2,000,000, 3,000,000, 4,000,000, 5,000,000, 10000000, 20000000, or more. The number of partitions may be at least about 5, 10, 50, 100, 250, 500, 750, 1000, 1500, 2000, 2500, 5000, 7500, 10,000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100,000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, 1,000,000, 2,000,000, 3,000,000, 4,000,000, 5,000,000, 10000000, 20000000, or more. The number of partitions may be less than about 5, 10, 50, 100, 250, 500, 750, 1000, 1500, 2000, 2500, 5000, 7500, 10,000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100,000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, 1,000,000, 2,000,000, 3,000,000, 4,000,000, 5,000,000, 10000000, 20000000. The number of partitions may be about 5-10000000, 5-5000000, 5-1,000,000, 10-10,000, 10-5,000, 10-1,000, 1,000-6,000, 1,000-5,000, 1,000-4,000, 1,000-3,000, or 1,000-2,000.
Such partitions may be pre-loaded with reagents to perform a particular reaction. For example, a capsule containing one or more reagents may be placed within a microwell. After adding a polynucleotide sample to the well, the capsule may be made to release its contents. The contents of the capsule may include, for example, restriction enzymes, ligases, barcodes, and adapters for processing the polynucleotide sample placed in the microwell.
In some cases, such partitions may be droplets of an emulsion. For example, a droplet of an emulsion may be an aqueous droplet in an oil phase. The droplet may comprise, for example, one or more reagents (e.g., restriction enzymes, ligases, polymerases, reagents necessary for nucleic acid amplification (e.g., primers, DNA polymerases, dNTPs, buffers)), a polynucleotide sample, and a barcode sequence. In some cases, the barcode sequence, polynucleotide sample, or any reagent may be associated with a solid surface within a droplet. In some cases, the solid surface is a bead. In some cases, the bead is a gel bead (see e.g., Agresti et al., U.S. Patent Publication No. 2010/0136544). In some cases the droplet is hardened into a gel bead (e.g., via polymerization). In some cases, isolation methods such as magnetic separation or sedimentation of particles may be used. Such methods may include, for example, a step of attaching a polynucleotide to be amplified, a primer corresponding to said polynucleotide to be amplified, and/or a polynucleotide product of amplification to a bead. In some cases, attachment of a polynucleotide to be amplified, primer corresponding to said polynucleotide to be amplified, and/or a polynucleotide to be amplified, primer corresponding to said polynucleotide to be amplified, and/or a polynucleotide product to a bead may be via a photolabile linker, such as, for example, PC Amino C6. In cases where a photolabile linker is used, light may be used to release a linked polynucleotide from the bead. The bead may be, for example, a magnetic bead or a latex bead. The bead may then enable separation by, for example, magnetic sorting or sedimentation. Sedimentation of latex particles may be performed, for example, by centrifugation in a liquid that is more dense than latex, such as glycerol. In some cases, density gradient centrifugation may be used.
A species may be contained within a droplet in an emulsion containing, for example, a first phase (e.g., oil or water) forming the droplet and a second (continuous) phase (e.g., water or oil). An emulsion may be a single emulsion, for example, a water-in-oil or an oil-in-water emulsion. An emulsion may be a double emulsion, for example a water-in-oil-in-water or an oil-in-water-in-oil emulsion. Higher-order emulsions are also possible. The emulsion may be held in any suitable container, including any suitable partition described in this disclosure.
In some cases, droplets in an emulsion comprise other partitions. A droplet in an emulsion may comprise any suitable partition including, for example, another droplet (e.g., a droplet in an emulsion), a capsule, a bead, and the like. Each partition may be present as a single partition or a plurality of partitions, and each partition may comprise the same species or different species.
In one example, a droplet in an emulsion comprises a capsule comprising reagents for sample processing. As described elsewhere in this disclosure, a capsule may contain one or more capsules, or other partitions. A sample comprising an analyte to be processed is contained within the droplet. A stimulus is applied to cause release of the contents of the capsule into the droplet, resulting in contact between the reagents and the analyte to be processed. The droplet is incubated under appropriate conditions for the processing of the analyte. Processed analyte may then be recovered. While this example describes an embodiment where a reagent is in a capsule and an analyte is in the droplet, the opposite configuration—i.e., reagent in the droplet and analyte in the capsule—is also possible.
The droplets in an emulsion may be of uniform size or heterogeneous size. In some cases, the diameter of a droplet in an emulsion may be about 0.001 μm, 0.01 μm, 0.05 μm, 0.1 μm, 0.5 μm, 1 μm, 5 μm, 10 μm, 50 μm, 100 μm, 150 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, or 1 mm. A droplet may have a diameter of at least about 0.001 μm, 0.01 μm, 0.05 μm, 0.1 μm, 0.5 μm, 1 μm, 5 μm, 10 μm, 50 μm, 100 μm, 150 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, or 1 mm. In some cases, a droplet may have a diameter of less than about 0.001 μm, 0.01 μm, 0.05 μm, 0.1 μm, 0.5 μm, 1 μm, 5 μm, 10 μm, 50 μm, 100 μm, 150 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, or 1 mm. In some cases, a droplet may have a diameter of about 0.001 μm to 1 mm, 0.01 μm to 900 μm, 0.1 μm to 600 μm, 100 μm to 200 μm, 100 μm to 300 μm, 100 μm to 400 μm, 100 μm to 500 μm, 100 μm to 600 μm, 150 μm to 200 μm, 150 μm to 300 μm, or 150 μm to 400 μm.
Droplets in an emulsion also may have a particular density. In some cases, the droplets are less dense than an aqueous fluid (e.g., water); in some cases, the droplets are denser than an aqueous fluid. In some cases, the droplets are less dense than a non-aqueous fluid (e.g., oil); in some cases, the droplets are denser than a non-aqueous fluid. Droplets may have a density of about 0.05 g/cm3, 0.1 g/cm3, 0.2 g/cm3, 0.3 g/cm3, 0.4 g/cm3, 0.5 g/cm3, 0.6 g/cm3, 0.7 g/cm3, 0.8 g/cm3, 0.81 g/cm3, 0.82 g/cm3, 0.83 g/cm3, 0.84 g/cm3, 0.85 g/cm3, 0.86 g/cm3, 0.87 g/cm3, 0.88 g/cm3, 0.89 g/cm3, 0.90 g/cm3, 0.91 g/cm3, 0.92 g/cm3, 0.93 g/cm3, 0.94 g/cm3, 0.95 g/cm3, 0.96 g/cm3, 0.97 g/cm3, 0.98 g/cm3, 0.99 g/cm3, 1.00 g/cm3, 1.05 g/cm3, 1.1 g/cm3, 1.2 g/cm3, 1.3 g/cm3, 1.4 g/cm3, 1.5 g/cm3, 1.6 g/cm3, 1.7 g/cm3, 1.8 g/cm3, 1.9 g/cm3, 2.0 g/cm3, 2.1 g/cm3, 2.2 g/cm3, 2.3 g/cm3, 2.4 g/cm3, or 2.5 g/cm3. Droplets may have a density of at least about 0.05 g/cm3, 0.1 g/cm3, 0.2 g/cm3, 0.3 g/cm3, 0.4 g/cm3, 0.5 g/cm3, 0.6 g/cm3, 0.7 g/cm3, 0.8 g/cm3, 0.81 g/cm3, 0.82 g/cm3, 0.83 g/cm3, 0.84 g/cm3, 0.85 g/cm3, 0.86 g/cm3, 0.87 g/cm3, 0.88 g/cm3, 0.89 g/cm3, 0.90 g/cm3, 0.91 g/cm3, 0.92 g/cm3, 0.93 g/cm3, 0.94 g/cm3, 0.95 g/cm3, 0.96 g/cm3, 0.97 g/cm3, 0.98 g/cm3, 0.99 g/cm3, 1.00 g/cm3, 1.05 g/cm3, 1.1 g/cm3, 1.2 g/cm3, 1.3 g/cm3, 1.4 g/cm3, 1.5 g/cm3, 1.6 g/cm3, 1.7 g/cm3, 1.8 g/cm3, 1.9 g/cm3, 2.0 g/cm3, 2.1 g/cm3, 2.2 g/cm3, 2.3 g/cm3, 2.4 g/cm3, or 2.5 g/cm3. In other cases, droplet densities may be at most about 0.7 g/cm3, 0.8 g/cm3, 0.81 g/cm3, 0.82 g/cm3, 0.83 g/cm3, 0.84 g/cm3, 0.85 g/cm3, 0.86 g/cm3, 0.87 g/cm3, 0.88 g/cm3, 0.89 g/cm3, 0.90 g/cm3, 0.91 g/cm3, 0.92 g/cm3, 0.93 g/cm3, 0.94 g/cm3, 0.95 g/cm3, 0.96 g/cm3, 0.97 g/cm3, 0.98 g/cm3, 0.99 g/cm3, 1.00 g/cm3, 1.05 g/cm3, 1.1 g/cm3, 1.2 g/cm3, 1.3 g/cm3, 1.4 g/cm3, 1.5 g/cm3, 1.6 g/cm3, 1.7 g/cm3, 1.8 g/cm3, 1.9 g/cm3, 2.0 g/cm3, 2.1 g/cm3, 2.2 g/cm3, 2.3 g/cm3, 2.4 g/cm3, or 2.5 g/cm3. Such densities can reflect the density of the capsule in any particular fluid (e.g., aqueous, water, oil, etc.)
Polynucleotides may be partitioned using a variety of methods. For example, polynucleotides may be diluted and dispensed across a plurality of partitions. A terminal dilution of a medium comprising polynucleotides may be performed such that the number of partitions or wells exceeds the number of polynucleotides. The ratio of the number of polynucleotides to the number of partitions may range from about 0.1-10, 0.5-10, 1-10, 2-10, 10-100, 100-1000, or more. The ratio of the number of polynucleotides to the number of partitions may be about 0.1, 0.5, 1, 2, 4, 8, 10, 20, 50, 100, or 1000. The ratio of the number of polynucleotides to the number of partitions may be at least about 0.1, 0.5, 1, 2, 4, 8, 10, 20, 50, 100, or 1000. The ratio of the number of polynucleotides to the number of partitions may be less than about 0.1, 0.5, 1, 2, 4, 8, 10, 20, 50, 100, or 1000.
The number of partitions employed may vary depending on the application. For example, the number of partitions may be about 5, 10, 50, 100, 250, 500, 750, 1000, 1500, 2000, 2500, 5000, 7500, or 10,000, or more. The number of partitions may be at least about 5, 10, 50, 100, 250, 500, 750, 1000, 1500, 2000, 2500, 5000, 7500, or 10,000, or more. The number of partitions may be less than about 5, 10, 50, 100, 250, 500, 750, 1000, 1500, 2000, 2500, 5000, 7500, or 10,000.
The volume of the partitions may vary depending on the application. For example, the volume of the partitions may be about 1000 μl, 900 μl, 800 μl, 700 μl, 600 μl, 500 μl, 400 μl, 300 μl, 200 μl, 100 μl, 50 μl, 25 μl, 10 μl, 5 μl, 1 μl, 900 nL, 800 nL, 700 nL, 600 nL, 500 nL, 400 nL, 300 nL, 200 nL, 100 nL, 50 nL, 25 nL, 10 nL, or 5 nL. The volume of the partitions may be at least about 1000 μl, 900 μl, 800 μl, 700 μl, 600 μl, 500 μl, 400 μl, 300 μl, 200 μl, 100 μl, 50 μl, 25 μl, 10 μl, 5 μl, 1 μl, 900 nL, 800 nL, 700 nL, 600 nL, 500 nL, 400 nL, 300 nL, 200 nL, 100 nL, 50 nL, 25 nL, 10 nL, or 5 nL. The volume of the partitions may be less than about 1000 μl, 900 μl, 800 μl, 700 μl, 600 μl, 500 μl, 400 μl, 300 μl, 200 μl, 100 μl, 50 μl, 25 μl, 10 μl, 5 μl, 1 μl, 900 nL, 800 nL, 700 nL, 600 nL, 500 nL, 400 nL, 300 nL, 200 nL, 100 nL, 50 nL, 25 nL, 10 nL, or 5 nL.
Species may also be partitioned at a particular density. For example, species may be partitioned so that each partition contains about 1, 5, 10, 50, 100, 1000, 10000, 100000, or 1000000 species per partition. Species may be partitioned so that each partition contains at least about 1, 5, 10, 50, 100, 1000, 10000, 100000, 1000000 or more species per partition. Species may be partitioned so that each partition contains less than about 1, 5, 10, 50, 100, 1000, 10000, 100000, or 1000000 species per partition. Species may be partitioned such that each partition contains about 1-5, 5-10, 10-50, 50-100, 100-1000, 1000-10000, 10000-100000, or 100000-1000000 species per partition.
Species may be partitioned such that at least one partition comprises a species that is unique within that partition. This may be true for about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the partitions. This may be true for at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the partitions. This may be true for less than about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the partitions.
Particular polynucleotides may also be targeted to specific partitions. For example, in some cases, a capture reagent such as an oligonucleotide probe may be immobilized in a partition to capture specific polynucleotides through hybridization.
Polynucleotides may also be partitioned at a particular density. For example, polynucleotides may be partitioned such that each partition contains about 1-5, 5-10, 10-50, 50-100, 100-1000, 1000-10000, 10000-100000, or 100000-1000000 polynucleotides per partition. Polynucleotides may be partitioned so that each partition contains about 1, 5, 10, 50, 100, 1000, 10000, 100000, 1000000 or more polynucleotides per partition. Polynucleotides may be partitioned so that each partition contains less than about 1, 5, 10, 50, 100, 1000, 10000, 100000, or 1000000 polynucleotides per partition. Polynucleotides may be partitioned so that each partition contains at least about 1, 5, 10, 50, 100, 1000, 10000, 100000, or 1000000 polynucleotides per partition.
Polynucleotides may be partitioned such that at least one partition comprises a polynucleotide sequence with a unique sequence compared to all other polynucleotide sequences contained within the same partition. This may be true for about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the partitions. This may be true for less than about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the partitions. This may be true for more than about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the partitions.
V. Barcoding
Downstream applications, for example DNA sequencing, may rely on the barcodes to identify the origin of a sequence and, for example, to assemble a larger sequence from sequenced fragments. Therefore, it may be desirable to add barcodes to the polynucleotide fragments generated by the methods described herein. Barcodes may be of a variety of different formats, including polynucleotide barcodes. Depending upon the specific application, barcodes may be attached to polynucleotide fragments in a reversible or irreversible manner. Barcodes may also allow for identification and/or quantification of individual polynucleotide fragments during sequencing.
Barcodes may be loaded into partitions so that one or more barcodes are introduced into a particular partition. Each partition may contain a different set of barcodes. This may be accomplished by directly dispensing the barcodes into the partitions, enveloping the barcodes (e.g., in a droplet of an emulsion), or by placing the barcodes within a container that is placed in a partition (e.g., a microcapsule).
The number of partitions employed may vary depending on the application. For example, the number of partitions may be about 5, 10, 50, 100, 250, 500, 750, 1000, 1500, 2000, 2500, 5000, 7500, or 10,000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100,000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, 1,000,000, 2,000,000, 3,000,000, 4,000,000, 5,000,000, 10000000, 20000000, or more. The number of partitions may be at least about 5, 10, 50, 100, 250, 500, 750, 1000, 1500, 2000, 2500, 5000, 7500, 10,000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100,000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, 1,000,000, 2,000,000, 3,000,000, 4,000,000, 5,000,000, 10000000, 20000000, or more. The number of partitions may be less than about 5, 10, 50, 100, 250, 500, 750, 1000, 1500, 2000, 2500, 5000, 7500, 10,000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100,000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, 1,000,000, 2,000,000, 3,000,000, 4,000,000, 5,000,000, 10000000, 20000000. The number of partitions may be about 5-10000000, 5-5000000, 5-1,000,000, 10-10,000, 10-5,000, 10-1,000, 1,000-6,000, 1,000-5,000, 1,000-4,000, 1,000-3,000, or 1,000-2,000.
The number of different barcodes or different sets of barcodes that are partitioned may vary depending upon, for example, the particular barcodes to be partitioned and/or the application. Different sets of barcodes may be, for example, sets of identical barcodes where the identical barcodes differ between each set. Or different sets of barcodes may be, for example, sets of different barcodes, where each set differs in its included barcodes. For example, about 1, 5, 10, 50, 100, 1000, 10000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1000000, 2000000, 3000000, 4000000, 5000000, 6000000, 7000000, 8000000, 9000000, 10000000, 20000000, 50000000, 100000000, or more different barcodes or different sets of barcodes may be partitioned. In some examples, at least about 1, 5, 10, 50, 100, 1000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1000000, 2000000, 3000000, 4000000, 5000000, 6000000, 7000000, 8000000, 9000000, 10000000, 20000000, 50000000, 100000000, or more different barcodes or different sets of barcodes may be partitioned. In some examples, less than about 1, 5, 10, 50, 100, 1000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1000000, 2000000, 3000000, 4000000, 5000000, 6000000, 7000000, 8000000, 9000000, 10000000, 20000000, 50000000, or 100000000 different barcodes or different sets of barcodes may be partitioned. In some examples, about 1-5, 5-10, 10-50, 50-100, 100-1000, 1000-10000, 10000-100000, 100000-1000000, 10000-1000000, 10000-10000000, or 10000-100000000 barcodes may be partitioned.
Barcodes may be partitioned at a particular density. For example, barcodes may be partitioned so that each partition contains about 1, 5, 10, 50, 100, 1000, 10000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1000000, 2000000, 3000000, 4000000, 5000000, 6000000, 7000000, 8000000, 9000000, 10000000, 20000000, 50000000, or 100000000 barcodes per partition. Barcodes may be partitioned so that each partition contains at least about 1, 5, 10, 50, 100, 1000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1000000, 2000000, 3000000, 4000000, 5000000, 6000000, 7000000, 8000000, 9000000, 10000000, 20000000, 50000000, 100000000, or more barcodes per partition. Barcodes may be partitioned so that each partition contains less than about 1, 5, 10, 50, 100, 1000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1000000, 2000000, 3000000, 4000000, 5000000, 6000000, 7000000, 8000000, 9000000, 10000000, 20000000, 50000000, or 100000000 barcodes per partition. Barcodes may be partitioned such that each partition contains about 1-5, 5-10, 10-50, 50-100, 100-1000, 1000-10000, 10000-100000, 100000-1000000, 10000-1000000, 10000-10000000, or 10000-100000000 barcodes per partition.
Barcodes may be partitioned such that identical barcodes are partitioned at a particular density. For example, identical barcodes may be partitioned so that each partition contains about 1, 5, 10, 50, 100, 1000, 10000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1000000, 2000000, 3000000, 4000000, 5000000, 6000000, 7000000, 8000000, 9000000, 10000000, 20000000, 50000000, or 100000000 identical barcodes per partition. Barcodes may be partitioned so that each partition contains at least about 1, 5, 10, 50, 100, 1000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1000000, 2000000, 3000000, 4000000, 5000000, 6000000, 7000000, 8000000, 9000000, 10000000, 20000000, 50000000, 100000000, or more identical barcodes per partition. Barcodes may be partitioned so that each partition contains less than about 1, 5, 10, 50, 100, 1000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1000000, 2000000, 3000000, 4000000, 5000000, 6000000, 7000000, 8000000, 9000000, 10000000, 20000000, 50000000, or 100000000 identical barcodes per partition. Barcodes may be partitioned such that each partition contains about 1-5, 5-10, 10-50, 50-100, 100-1000, 1000-10000, 10000-100000, 100000-1000000, 10000-1000000, 10000-10000000, or 10000-100000000 identical barcodes per partition.
Barcodes may be partitioned such that different barcodes are partitioned at a particular density. For example, different barcodes may be partitioned so that each partition contains about 1, 5, 10, 50, 100, 1000, 10000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1000000, 2000000, 3000000, 4000000, 5000000, 6000000, 7000000, 8000000, 9000000, 10000000, 20000000, 50000000, or 100000000 different barcodes per partition. Barcodes may be partitioned so that each partition contains at least about 1, 5, 10, 50, 100, 1000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1000000, 2000000, 3000000, 4000000, 5000000, 6000000, 7000000, 8000000, 9000000, 10000000, 20000000, 50000000, 100000000, or more different barcodes per partition. Barcodes may be partitioned so that each partition contains less than about 1, 5, 10, 50, 100, 1000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1000000, 2000000, 3000000, 4000000, 5000000, 6000000, 7000000, 8000000, 9000000, 10000000, 20000000, 50000000, or 100000000 different barcodes per partition. Barcodes may be partitioned such that each partition contains about 1-5, 5-10, 10-50, 50-100, 100-1000, 1000-10000, 10000-100000, 100000-1000000, 10000-1000000, 10000-10000000, or 10000-100000000 different barcodes per partition.
The number of partitions employed to partition barcodes may vary, for example, depending on the application and/or the number of different barcodes to be partitioned. For example, the number of partitions employed to partition barcodes may be about 5, 10, 50, 100, 250, 500, 750, 1000, 1500, 2000, 2500, 5000, 7500, or 10,000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100,000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, 1,000,000, 2,000,000, 3,000,000, 4,000,000, 5,000,000, 10000000, 20000000 or more. The number of partitions employed to partition barcodes may be at least about 5, 10, 50, 100, 250, 500, 750, 1000, 1500, 2000, 2500, 5000, 7500, 10,000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, 1000000, 2000000, 3000000, 4000000, 5000000, 10000000, 20000000 or more. The number of partitions employed to partition barcodes may be less than about 5, 10, 50, 100, 250, 500, 750, 1000, 1500, 2000, 2500, 5000, 7500, 10,000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, 1000000, 2000000, 3000000, 4000000, 5000000, 10000000, or 20000000. The number of partitions employed to partition barcodes may be about 5-10000000, 5-5000000, 5-1,000,000, 10-10,000, 10-5,000, 10-1,000, 1,000-6,000, 1,000-5,000, 1,000-4,000, 1,000-3,000, or 1,000-2,000. As described above, different barcodes or different sets of barcodes (e.g., each set comprising a plurality of identical barcodes or different barcodes) may be partitioned such that each partition comprises a different barcode or different barcode set. In some cases, each partition may comprise a different set of identical barcodes. Where different sets of identical barcodes are partitioned, the number of identical barcodes per partition may vary. For example, about 100,000 or more different sets of identical barcodes may be partitioned across about 100,000 or more different partitions, such that each partition comprises a different set of identical barcodes. In each partition, the number of identical barcodes per set of barcodes may be about 1,000,000 identical barcodes. In some cases, the number of different sets of barcodes may be equal to or substantially equal to the number of partitions. Any suitable number of different barcodes or different barcode sets (including numbers of different barcodes or different barcode sets to be partitioned described elsewhere herein), number of barcodes per partition (including numbers of barcodes per partition described elsewhere herein), and number of partitions (including numbers of partitions described elsewhere herein) may be combined to generate a diverse library of partitioned barcodes with high numbers of barcodes per partition. Thus, as will be appreciated, any of the above-described different numbers of barcodes may be provided with any of the above-described barcode densities per partition, and in any of the above-described numbers of partitions.
For example, a population of microcapsules may be prepared such that a first microcapsule in the population comprises multiple copies of identical barcodes (e.g., polynucleotide bar codes, etc.) and a second microcapsule in the population comprises multiple copies of a barcode that differs from the barcode within the first microcapsule. In some cases, the population of microcapsules may comprise multiple microcapsules (e.g., greater than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 500, 1000, 5000, 10000, 100000, 1000000, 10000000, 100000000, or 1000000000 microcapsules), each containing multiple copies of a barcode that differs from that contained in the other microcapsules. In some cases, the population may comprise greater than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 500, 1000, 5000, 10000, 100000, 1000000, 10000000, 100000000, or 1000000000 microcapsules with identical sets of barcodes. In some cases, the population may comprise greater than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 500, 1000, 5000, 10000, 100000, 1000000, 10000000, 100000000, or 1000000000 microcapsules, wherein the microcapsules each comprise a different combination of barcodes. For example, in some cases the different combinations overlap, such that a first microcapsule may comprise, e.g., barcodes A, B, and C, while a second microcapsule may comprise barcodes A, B, and D. In another example, the different combinations do not overlap, such that a first microcapsule may comprise, e.g., barcodes A, B, and C, while a second microcapsule may comprise barcodes D, E, and F. The use of microcapsules is, of course, optional. All of the combinations described above, and throughout this disclosure, may also be generated by dispending barcodes (and other reagents) directly into partitions (e.g., microwells).
The barcodes may be loaded into the partitions at an expected or predicted ratio of barcodes per species to be barcoded (e.g., polynucleotide fragment, strand of polynucleotide, cell, etc.). In some cases, the barcodes are loaded into partitions such that more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 500, 1000, 5000, 10000, or 200000 barcodes are loaded per species. In some cases, the barcodes are loaded in the partitions so that less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 500, 1000, 5000, 10000, or 200000 barcodes are loaded per species. In some cases, the average number of barcodes loaded per species is less than, or greater than, about 0.0001, 0.001, 0.01, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 500, 1000, 5000, 10000, or 200000 barcodes per species.
When more than one barcode is present per polynucleotide fragment, such barcodes may be copies of the same barcode, or multiple different barcodes. For example, the attachment process may be designed to attach multiple identical barcodes to a single polynucleotide fragment, or multiple different barcodes to the polynucleotide fragment.
The methods provided herein may comprise loading a partition (e.g., a microwell, droplet of an emulsion) with the reagents necessary for the attachment of barcodes to polynucleotide fragments. In the case of ligation reactions, reagents including restriction enzymes, ligase enzymes, buffers, adapters, barcodes and the like may be loaded into a partition. In the case barcoding by amplification, reagents including primers, DNA polymerases, DNTPs, buffers, barcodes and the like may be loaded into a partition. As described throughout this disclosure, these reagents may be loaded directly into the partition, or via a container such as a microcapsule. If the reagents are not disposed within a container, they may be loaded into a partition (e.g., a microwell) which may then be sealed with a wax or oil until the reagents are used.
Barcodes may be ligated to a polynucleotide fragment using sticky or blunt ends. Barcoded polynucleotide fragments may also be generated by amplifying a polynucleotide fragment with primers comprising barcodes.
Barcodes may be assembled combinatorially, from smaller components designed to assemble in a modular format. For example, three modules, 1A, 1B, and 1C may be combinatorially assembled to produce barcode 1ABC. Such combinatorial assembly may significantly reduce the cost of synthesizing a plurality of barcodes. For example, a combinatorial system consisting of 3 A modules, 3 B modules, and 3 C modules may generate 3*3*3=27 possible barcode sequences from only 9 modules.
VI. Microcapsules and Microwell Capsule Arrays
Microcapsules and microwell capsule array (MCA) devices may be used to perform the polynucleotide processing methods described herein. MCA devices are devices with a plurality of microwells. Microcapsules are introduced into these microwells, before, after, or concurrently with the introduction of a sample.
Microwells may comprise free reagents and/or reagents encapsulated in microcapsules. Any of the reagents described in this disclosure may be encapsulated in a microcapsule, including any chemicals, particles, and elements suitable for sample processing reactions involving a polynucleotide. For example, a microcapsule used in a sample preparation reaction for DNA sequencing may comprise one or more of the following reagents: enzymes, restriction enzymes (e.g., multiple cutters), ligase, polymerase, fluorophores, oligonucleotide barcodes, adapters, buffers, dNTPs, ddNTPs and the like.
Additional exemplary reagents include: buffers, acidic solution, basic solution, temperature-sensitive enzymes, pH-sensitive enzymes, light-sensitive enzymes, metals, metal ions, magnesium chloride, sodium chloride, manganese, aqueous buffer, mild buffer, ionic buffer, inhibitor, enzyme, protein, polynucleotide, antibodies, saccharides, lipid, oil, salt, ion, detergents, ionic detergents, non-ionic detergents, oligonucleotides, nucleotides, deoxyribonucleotide triphosphates (dNTPs), dideoxyribonucleotide triphosphates (ddNTPs), DNA, RNA, peptide polynucleotides, complementary DNA (cDNA), double stranded DNA (dsDNA), single stranded DNA (ssDNA), plasmid DNA, cosmid DNA, chromosomal DNA, genomic DNA, viral DNA, bacterial DNA, mtDNA (mitochondrial DNA), mRNA, rRNA, tRNA, nRNA, siRNA, snRNA, snoRNA, scaRNA, microRNA, dsRNA, ribozyme, riboswitch and viral RNA, polymerase, ligase, restriction enzymes, proteases, nucleases, protease inhibitors, nuclease inhibitors, chelating agents, reducing agents, oxidizing agents, fluorophores, probes, chromophores, dyes, organics, emulsifiers, surfactants, stabilizers, polymers, water, small molecules, pharmaceuticals, radioactive molecules, preservatives, antibiotics, aptamers, and pharmaceutical drug compounds.
In some cases, a microcapsule comprises a set of reagents that have a similar attribute (e.g., a set of enzymes, a set of minerals, a set of oligonucleotides, a mixture of different bar-codes, a mixture of identical bar-codes). In other cases, a microcapsule comprises a heterogeneous mixture of reagents. In some cases, the heterogeneous mixture of reagents comprises all components necessary to perform a reaction. In some cases, such mixture comprises all components necessary to perform a reaction, except for 1, 2, 3, 4, 5, or more components necessary to perform a reaction. In some cases, such additional components are contained within a different microcapsule or within a solution within a partition (e.g., microwell) of the device.
In some cases, only microcapsules comprising reagents are introduced. In other cases, both free reagents and reagents encapsulated in microcapsules are loaded into the device, either sequentially or concurrently. In some cases, reagents are introduced to the device either before or after a particular step. In some cases, reagents and/or microcapsules comprising reagents are introduced sequentially such that different reactions or operations occur at different steps. The reagents (or microcapsules) may be also be loaded at steps interspersed with a reaction or operation step. For example, microcapsules comprising reagents for fragmenting polynucleotides (e.g., restriction enzymes) may be loaded into the device, followed by loading of microcapsules comprising reagents for ligating bar-codes and subsequent ligation of the bar-codes to the fragmented molecules.
Microcapsules may be pre-formed and filled with reagents by injection. For example, the picoinjection methods described in Abate et al. (Proc. Natl. Acad. Sci. U.S.A., 2010, 107(45), 19163-19166) and Weitz et al. (U.S. Pub. No. 2012/0132288) may be used to introduce reagents into the interior of microcapsules described herein. These methods can also be used to introduce a plurality of any of the reagents described herein into microcapsules.
Microcapsules may be formed by any emulsion technique known in the art. For example, the multiple emulsion technique of Weitz et al. (U.S. Pub. No. 2012/0211084) may be used to form microcapsules (or partitions) for use with the methods disclosed herein.
Numerous chemical triggers may be used to trigger the disruption of partitions (e.g., Plunkett et al., Biomacromolecules, 2005, 6:632-637). Examples of these chemical changes may include, but are not limited to pH-mediated changes to the integrity of a component of a partition, disintegration of a component of a partition via chemical cleavage of crosslink bonds, and triggered depolymerization of a component of a partition. Bulk changes may also be used to trigger disruption of partitions.
A change in pH of a solution, such as a decrease in pH, may trigger disruption of a partition via a number of different mechanisms. The addition of acid may cause degradation or disassembly a portion of a partition through a variety of mechanisms. Addition of protons may disassemble cross-linking of polymers in a component of a partition, disrupt ionic or hydrogen bonds in a component of a partition, or create nanopores in a component of a partition to allow the inner contents to leak through to the exterior. A change in pH may also destabilize an emulsion, leading to release of the contents of the droplets.
In some examples, a partition is produced from materials that comprise acid-degradable chemical cross-linkers, such a ketals. A decrease in pH, particular to a pH lower than 5, may induce the ketal to convert to a ketone and two alcohols and facilitate disruption of the partition. In other examples, the partitions may be produced from materials comprising one or more polyelectrolytes that are pH sensitive. A decrease in pH may disrupt the ionic- or hydrogen-bonding interactions of such partitions, or create nanopores therein. In some cases, partitions made from materials comprising polyelectrolytes comprise a charged, gel-based core that expands and contracts upon a change of pH.
Disruption of cross-linked materials comprising a partition can be accomplished through a number of mechanisms. In some examples, a partition can be contacted with various chemicals that induce oxidation, reduction or other chemical changes. In some cases, a reducing agent, such as beta-mercaptoethanol, can be used, such that disulfide bonds of a partition are disrupted. In addition, enzymes may be added to cleave peptide bonds in materials forming a partition, thereby resulting in a loss of integrity of the partition.
Depolymerization can also be used to disrupt partitions. A chemical trigger may be added to facilitate the removal of a protecting head group. For example, the trigger may cause removal of a head group of a carbonate ester or carbamate within a polymer, which in turn causes depolymerization and release of species from the inside of a partition.
In yet another example, a chemical trigger may comprise an osmotic trigger, whereby a change in ion or solute concentration in a solution induces swelling of a material used to make a partition. Swelling may cause a buildup of internal pressure such that a partition ruptures to release its contents. Swelling may also cause an increase in the pore size of the material, allowing species contained within the partition to diffuse out, and vice versa.
A partition may also be made to release its contents via bulk or physical changes, such as pressure induced rupture, melting, or changes in porosity.
VII. Polynucleotide Sequencing
Generally, the methods and compositions provided herein are useful for preparation of polynucleotide fragments for downstream applications such as sequencing. Sequencing may be performed by any available technique. For example, sequencing may be performed by the classic Sanger sequencing method. Sequencing methods may also include: high-throughput sequencing, pyrosequencing, sequencing-by-synthesis, single-molecule sequencing, nanopore sequencing, sequencing-by-ligation, sequencing-by-hybridization, RNA-Seq (Illumina), Digital Gene Expression (Helicos), next generation sequencing, single molecule sequencing by synthesis (SMSS) (Helicos), massively-parallel sequencing, clonal single molecule Array (Solexa), shotgun sequencing, Maxim-Gilbert sequencing, primer walking, and any other sequencing methods known in the art.
In some cases varying numbers of fragments are sequenced. For example, in some cases about 30%-90% of the fragments are sequenced. In some cases, about 35%-85%, 40%-80%, 45%-75%, 50%-70%, 55%-65%, or 50%-60% of the fragments are sequenced. In some cases, at least about 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the fragments are sequenced. In some cases less than about 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the fragments are sequenced.
In some cases sequences from fragments are assembled to provide sequence information for a contiguous region of the original target polynucleotide that is longer than the individual sequence reads. Individual sequence reads may be about 10-50, 50-100, 100-200, 200-300, 300-400, or more nucleotides in length.
The identities of the barcode tags may serve to order the sequence reads from individual fragments as well as to differentiate between haplotypes. For example, during the partitioning of individual fragments, parental polynucleotide fragments may separated into different partitions.
With an increase in the number of partitions, the likelihood of a fragment from both a maternal and paternal haplotype contained in the same partition becomes negligibly small. Thus, sequence reads from fragments in the same partition may be assembled and ordered.
VIII. Polynucleotide Phasing
This disclosure also provides methods and compositions to prepare polynucleotide fragments in such a manner that may enable phasing or linkage information to be generated. Such information may allow for the detection of linked genetic variations in sequences, including genetic variations (e.g., SNPs, mutations, indels, copy number variations, transversions, translocations, inversions, etc.) that are separated by long stretches of polynucleotides. The term “indel” refers to a mutation resulting in a colocalized insertion and deletion and a net gain or loss in nucleotides. A “microindel” is an indel that results in a net gain or loss of 1 to 50 nucleotides. These variations may exist in either a cis or trans relationship. In a cis relationship, two or more genetic variations exist in the same polynucleotide or strand. In a trans relationship, two or more genetic variations exist on multiple polynucleotide molecules or strands.
Methods provided herein may be used to determine polynucleotide phasing. For example, a polynucleotide sample (e.g., a polynucleotide that spans a given locus or loci) may be partitioned such that at most one molecule of polynucleotide is present per partition (e.g., microwell). The polynucleotide may then be fragmented, barcoded, and sequenced. The sequences may be examined for genetic variation. The detection of genetic variations in the same sequence tagged with two different bar codes may indicate that the two genetic variations are derived from two separate strands of DNA, reflecting a trans relationship. Conversely, the detection of two different genetic variations tagged with the same bar codes may indicate that the two genetic variations are from the same strand of DNA, reflecting a cis relationship.
Phase information may be important for the characterization of a polynucleotide fragment, particularly if the polynucleotide fragment is derived from a subject at risk of, having, or suspected of a having a particular disease or disorder (e.g., hereditary recessive disease such as cystic fibrosis, cancer, etc.). The information may be able to distinguish between the following possibilities: (1) two genetic variations within the same gene on the same strand of DNA and (2) two genetic variations within the same gene but located on separate strands of DNA. Possibility (1) may indicate that one copy of the gene is normal and the individual is free of the disease, while possibility (2) may indicate that the individual has or will develop the disease, particularly if the two genetic variations are damaging to the function of the gene when present within the same gene copy. Similarly, the phasing information may also be able to distinguish between the following possibilities: (1) two genetic variations, each within a different gene on the same strand of DNA and (2) two genetic variations, each within a different gene but located on separate strands of DNA.
IX. Sequencing Polynucleotides from Small Numbers of Cells
Methods provided herein may also be used to prepare polynucleotide contained within cells in a manner that enables cell-specific information to be obtained. The methods enable detection of genetic variations (e.g., SNPs, mutations, indels, copy number variations, transversions, translocations, inversions, etc.) from very small samples, such as from samples comprising about 10-100 cells. In some cases, about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 cells may be used in the methods described herein. In some cases, at least about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 cells may be used in the methods described herein. In other cases, at most about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 cells may be used in the methods described herein.
In an example, a method comprises partitioning a cellular sample (or crude cell extract) such that at most one cell (or extract of one cell) is present per partition, lysing the cells, fragmenting the polynucleotides contained within the cells by any of the methods described herein, attaching the fragmented polynucleotides to barcodes, pooling, and sequencing.
As described elsewhere herein, the barcodes and other reagents may be contained within a microcapsule. These microcapsules may be loaded into a partition (e.g., a microwell) before, after, or concurrently with the loading of the cell, such that each cell is contacted with a different microcapsule. This technique may be used to attach a unique barcode to polynucleotides obtained from each cell. The resulting tagged polynucleotides may then be pooled and sequenced, and the barcodes may be used to trace the origin of the polynucleotides. For example, polynucleotides with identical barcodes may be determined to originate from the same cell, while polynucleotides with different barcodes may be determined to originate from different cells.
The methods described herein may be used to detect the distribution of oncogenic mutations across a population of cancerous tumor cells. For example, some tumor cells may have a mutation, or amplification, of an oncogene (e.g., HER2, BRAF, EGFR, KRAS) in both alleles (homozygous), others may have a mutation in one allele (heterozygous), and still others may have no mutation (wild-type). The methods described herein may be used to detect these differences, and also to quantify the relative numbers of homozygous, heterozygous, and wild-type cells. Such information may be used, for example, to stage a particular cancer and/or to monitor the progression of the cancer and its treatment over time.
In some examples, this disclosure provides methods of identifying mutations in two different oncogenes (e.g., KRAS and EGFR). If the same cell comprises genes with both mutations, this may indicate a more aggressive form of cancer. In contrast, if the mutations are located in two different cells, this may indicate that the cancer is more benign, or less advanced.
X. Analysis of Gene Expression
Methods of the disclosure may be applicable to processing samples for the detection of changes in gene expression. A sample may comprise a cell, mRNA, or cDNA reverse transcribed from mRNA. The sample may be a pooled sample, comprising extracts from several different cells or tissues, or a sample comprising extracts from a single cell or tissue.
Cells may be placed directly into an partition (e.g., a microwell) and lysed. After lysis, the methods of the invention may be used to fragment and barcode the polynucleotides of the cell for sequencing. Polynucleotides may also be extracted from cells prior to introducing them into a partition used in a method of the invention. Reverse transcription of mRNA may be performed in a partition described herein, or outside of such a partition. Sequencing cDNA may provide an indication of the abundance of a particular transcript in a particular cell over time, or after exposure to a particular condition.
The methods presented throughout this disclosure provide several advantages over current polynucleotide processing methods. First, inter-operator variability is greatly reduced. Second, the methods may be carried out in microfluidic devices, which have a low cost and can be easily fabricated. Third, the controlled fragmentation of the target polynucleotides allows the user to produce polynucleotide fragments with a defined and appropriate length. This aids in partitioning the polynucleotides and also reduces the amount of sequence information loss due to the present of overly-large fragments. The methods and systems also provide a facile workflow that maintains the integrity of the processed polynucleotide. Additionally, the use of restriction enzymes enables the user to create DNA overhangs (“sticky ends”) that may be designed for compatibility with adapters and/or barcodes.
This example demonstrates a method for the generation of non-overlapping DNA fragments suitable for DNA sequencing and other downstream applications. An implementation of this method is schematically illustrated in
With reference to
The partitioned fragments are then further fragmented, to generate a plurality of non-overlapping second polynucleotide fragments 105. Referring again to the left-hand side of
The second set polynucleotide fragments are barcoded, and the barcoded sequences are pooled. Referring to the lower left-hand side of
With continued reference to
With continued reference to
The fourth polynucleotides fragments are barcoded, and the barcoded sequences are pooled. Referring to the lower right-hand side of
The example above describes sequencing the barcoded second fragments separately from the barcoded fourth fragments. The barcoded second fragments and the barcoded fourth fragments may also be combined, and the combined sample may be sequenced. One or more steps of the process may be carried out in a device. The steps carried out in a device may be carried out in the same device or in different devices.
After sequencing, sequence contigs are assembled and the overlapping sequences between the second fragments and the fourth fragments are used to assemble the sequence of the genome.
Example 2
A simulation was performed to evaluate the size distribution of fragments generated by a 6Mer cutter (StuI), a 4Mer cutter (CviQI), and two to seven 4Mer cutters. Random 1Mbp DNA sequences were generated in silico and cuts were simulated based on the occurrence of the recognition sites for each of the restriction enzymes within the random sequences.
As shown in
Next, simulated cuts were made in a random 1Mbp DNA sequence using combinations of one to seven different 4Mer cutters. The 4Mer cutters were: (A) CviQI (G/TAC); (B) BfaI (C/TAG); (C) HinPlI (G/CGC); (D) CviAII (C/ATG); (E) TaqαI (T/CGA); (F) MseI (T/TAA); and (G) MspI (C/CGG). The results of these simulations are shown in
The number of enzymes used to cut a sequence can be chosen so that a particular fraction of a target nucleotide (e.g., a genomic) sequence within 100 nucleotides of a restriction enzyme is achieved. For example, the fraction of a random genome within 100 nucleotides of a restriction site for a 4Mer cutter is equal to 1-0.44x, where x is the number of independent 4Mer cutters. Similarly, the fraction of a random genome within 100 nucleotides of a restriction site for a 5Mer cutter is equal to 1-0.25x, where x is the number of independent 5Mer cutters. For a 6Mer cutter, the fraction of a random genome within 100 nucleotides of a restriction site is equal to 1-0.95x, where x is the number of independent 6Mer cutters.
Table 1 shows the percentage of sequences with a length greater than 100 nucleotides for each of the seven enzymatic treatments described above. These sequences are considered those likely to result in missing data. Increasing the number of enzymes decreases the percentage of sequences greater than 100 nucleotides. The number of enzymes and their restriction site recognition length may be chosen in order to minimize the loss of sequence information from sequences greater than 100 nucleotides from a restriction site while also minimizing the generation of sequences less than 50 nucleotides, which are undesirable because the underutilize the read length capacity of sequencing instruments. The presence of these fragments may be minimized or avoided by selecting restriction enzymes that cut more rarely but at the potential price of reduced sequencing coverage of the DNA (i.e., more fragments may have bases >100 bases from a restriction site). These fragments may also be physically removed by a size selection step. Since these fragments are small and some fraction of the bases represented in the small fragments may be covered in larger fragments from other enzymes, the effect on coverage would likely be minimal.
The exemplary 4Mer cutter methods presented herein are optimized to provide fragments compatible with current DNA sequencing technology, which may achieve accurate read lengths up to about 100 nucleotides from the terminus of a fragment. One of ordinary skill in the art will readily recognize that other restriction enzymes (e.g., 5Mer cutters, 6Mer cutters, etc.) would be suitable for DNA sequencing technologies capable of accurately reading larger fragments of DNA (e.g., 300-400, or more nucleotides). The methods presented in this disclosure are, of course generalizable, and may be used to obtain DNA fragments of any size distribution compatible with present or future sequencing technology.
As described elsewhere herein, many downstream applications of the polynucleotide processing methods provided herein may utilize polynucleotide barcodes. An adapter may be used to provide compatible ends for the attachment of a barcode to a polynucleotide fragment (e.g., by ligation or PCR). In these cases, the desired products may be, for example:
However, the reaction described above may also result in several unwanted side products, including multimers produced by self-ligation of the fragmented genomic DNA and adapters (or other molecules, such as barcodes, which are not shown). For the sake of simplicity,
One unwanted side product is a multimer of genomic DNA fragments. This may occur, for example, if genomic DNA fragments with compatible ends are ligated to each other after cutting. In
As indicated in
The enzymes are chosen such that the desired product (i.e., the genomic DNA fragment with adapters on each end) does not contain a recognition site for either enzyme. Therefore, the product will not be re-cut by any enzyme contained within the partition. This process increases the yield of the desired product, while minimizing the number of unwanted side products and reducing the amount of starting material required to produce a desired amount of a product. As described in this disclosure, a pair of enzymes may be chosen so that one enzyme recognizes one undesirable side-product and regenerates a starting material and another recognizes another undesirable side product and regenerates another starting material, but neither enzyme recognizes the desired product. This can be done for an unlimited number of side products.
In general, one strategy for selecting such pairs is to choose two enzymes that create identical (or similar, ligatable) termini after cutting, but have recognition sequences of different lengths.
The exemplary embodiment shown in
Similarly, the embodiment shown in
Pseudo-complimentary nucleotides that preferentially bind natural nucleotides over themselves (e.g., Biochemistry (1996) 35, 11170-11176; Nucleic Acids Research (1996) 15, 2470-2475), may also be used to minimize or avoid the formation of certain multimers, for example adapter-adapter multimers and barcode-barcode multimers. If adapters and/or barcodes (and/or other polynucleotides are synthesized using pseudo-complimentary nucleotides, they will prefer to hybridize with naturally occurring polynucleotide fragments (e.g., genomic DNA fragments) rather than themselves, therefore leading to a higher yield of the desired product.
As described throughout this disclosure, the polynucleotide processing methods described herein may involve the treatment of partitioned polynucleotides with a variety of reagents. These reagents may include, for example, restriction enzymes, ligases, phosphatases, kinases, barcodes, adapters, or any other reagent useful in polynucleotide processing or in a downstream application, such as sequencing.
The capsule is dispensed into a partition (e.g., a microwell). A target polynucleotide and a ligase are then added to the partition. The capsule is made to release its contents by exposure to a stimulus, such as a change in temperature, a solvent, or stirring. The restriction enzyme fragments the target polynucleotide and the ligase attaches the barcode reagents to the target polynucleotide fragments generated by the restriction enzyme.
The restriction digestion and ligation may proceed according to any of the methods described herein, for example by non-overlapping fragmentation techniques, by pseudo-random fragmentation methods, and/or by pairing of restriction enzymes to recycle unwanted side products into new starting products (e.g., target polynucleotide fragments and barcodes). Adapters may also be included within the microcapsule. The barcodes shown in
The right-hand side of
This application is a continuation of U.S. application Ser. No. 15/588,519, filed May 5, 2017, which is a continuation of U.S. patent application Ser. No. 15/376,582, filed Dec. 12, 2016, which is a continuation-in-part of U.S. patent application Ser. No. 14/104,650, filed on Dec. 12, 2013, now U.S. Pat. No. 9,567,631, which claims priority to U.S. Provisional Application No. 61/737,374, filed on Dec. 14, 2012; U.S. patent application Ser. No. 15/376,582 is also a continuation-in-part of U.S. patent application Ser. No. 14/250,701, filed on Apr. 11, 2014, which is a continuation of U.S. patent application Ser. No. 14/175,973, filed on Feb. 7, 2014, now U.S. Pat. No. 9,388,465, which claims priority to U.S. Provisional Application No. 61/844,804, filed on Jul. 10, 2013, U.S. Provisional Application No. 61/840,403, filed on Jun. 27, 2013, U.S. Provisional Application No. 61/800,223, filed on Mar. 15, 2013, and U.S. Provisional Application No. 61/762,435, filed on Feb. 8, 2013, each of which is entirely incorporated herein by reference for all purposes.
| Number | Name | Date | Kind |
|---|---|---|---|
| 2797149 | Skeggs | Jun 1957 | A |
| 3047367 | Kessler | Jul 1962 | A |
| 3479141 | William et al. | Nov 1969 | A |
| 4124638 | Hansen | Nov 1978 | A |
| 4253846 | Smythe et al. | Mar 1981 | A |
| 4582802 | Zimmerman et al. | Apr 1986 | A |
| 5137829 | Nag et al. | Aug 1992 | A |
| 5149625 | Church et al. | Sep 1992 | A |
| 5185099 | Delpuech et al. | Feb 1993 | A |
| 5202231 | Drmanac et al. | Apr 1993 | A |
| 5270183 | Corbett et al. | Dec 1993 | A |
| 5413924 | Kosak et al. | May 1995 | A |
| 5418149 | Gelfand et al. | May 1995 | A |
| 5436130 | Mathies et al. | Jul 1995 | A |
| 5478893 | Ghosh et al. | Dec 1995 | A |
| 5489523 | Mathur | Feb 1996 | A |
| 5512131 | Kumar et al. | Apr 1996 | A |
| 5558071 | Ward et al. | Sep 1996 | A |
| 5585069 | Zanzucchi et al. | Dec 1996 | A |
| 5587128 | Wilding et al. | Dec 1996 | A |
| 5605793 | Stemmer | Feb 1997 | A |
| 5618711 | Gelfand et al. | Apr 1997 | A |
| 5658548 | Padhye et al. | Aug 1997 | A |
| 5695940 | Drmanac et al. | Dec 1997 | A |
| 5700642 | Monforte et al. | Dec 1997 | A |
| 5705628 | Hawkins | Jan 1998 | A |
| 5708153 | Dower et al. | Jan 1998 | A |
| 5736330 | Fulton | Apr 1998 | A |
| 5739036 | Parris | Apr 1998 | A |
| 5744311 | Fraiser et al. | Apr 1998 | A |
| 5756334 | Perler et al. | May 1998 | A |
| 5830663 | Embleton et al. | Nov 1998 | A |
| 5834197 | Parton | Nov 1998 | A |
| 5842787 | Kopf-Sill et al. | Dec 1998 | A |
| 5846719 | Brenner et al. | Dec 1998 | A |
| 5846727 | Soper et al. | Dec 1998 | A |
| 5851769 | Gray et al. | Dec 1998 | A |
| 5856174 | Lipshutz et al. | Jan 1999 | A |
| 5872010 | Karger et al. | Feb 1999 | A |
| 5897783 | Howe et al. | Apr 1999 | A |
| 5900481 | Lough et al. | May 1999 | A |
| 5942609 | Hunkapiller et al. | Aug 1999 | A |
| 5958703 | Dower et al. | Sep 1999 | A |
| 5965443 | Reznikoff et al. | Oct 1999 | A |
| 5994056 | Higuchi | Nov 1999 | A |
| 5997636 | Gamarnik et al. | Dec 1999 | A |
| 6033880 | Haff et al. | Mar 2000 | A |
| 6046003 | Mandecki | Apr 2000 | A |
| 6051377 | Mandecki | Apr 2000 | A |
| 6057107 | Fulton | May 2000 | A |
| 6057149 | Burns et al. | May 2000 | A |
| 6103537 | Ullman et al. | Aug 2000 | A |
| 6110678 | Weisburg et al. | Aug 2000 | A |
| 6123798 | Gandhi et al. | Sep 2000 | A |
| 6133436 | Koester et al. | Oct 2000 | A |
| 6143496 | Brown et al. | Nov 2000 | A |
| 6159717 | Savakis et al. | Dec 2000 | A |
| 6171850 | Nagle et al. | Jan 2001 | B1 |
| 6172218 | Brenner | Jan 2001 | B1 |
| 6207384 | Mekalanos et al. | Mar 2001 | B1 |
| 6258571 | Chumakov et al. | Jul 2001 | B1 |
| 6265552 | Schatz | Jul 2001 | B1 |
| 6291243 | Fogarty et al. | Sep 2001 | B1 |
| 6294385 | Goryshin et al. | Sep 2001 | B1 |
| 6296020 | McNeely et al. | Oct 2001 | B1 |
| 6297006 | Drmanac et al. | Oct 2001 | B1 |
| 6297017 | Schmidt et al. | Oct 2001 | B1 |
| 6303343 | Kopf-Sill | Oct 2001 | B1 |
| 6306590 | Mehta et al. | Oct 2001 | B1 |
| 6327410 | Walt et al. | Dec 2001 | B1 |
| 6355198 | Kim et al. | Mar 2002 | B1 |
| 6361950 | Mandecki | Mar 2002 | B1 |
| 6372813 | Johnson et al. | Apr 2002 | B1 |
| 6379929 | Burns et al. | Apr 2002 | B1 |
| 6406848 | Bridgham et al. | Jun 2002 | B1 |
| 6409832 | Weigl et al. | Jun 2002 | B2 |
| 6432290 | Harrison et al. | Aug 2002 | B1 |
| 6432360 | Church | Aug 2002 | B1 |
| 6485944 | Church et al. | Nov 2002 | B1 |
| 6492118 | Abrams et al. | Dec 2002 | B1 |
| 6511803 | Church et al. | Jan 2003 | B1 |
| 6524456 | Ramsey et al. | Feb 2003 | B1 |
| 6569631 | Pantoliano et al. | May 2003 | B1 |
| 6579851 | Goeke et al. | Jun 2003 | B2 |
| 6586176 | Trnovsky et al. | Jul 2003 | B1 |
| 6593113 | Tenkanen et al. | Jul 2003 | B1 |
| 6613752 | Kay et al. | Sep 2003 | B2 |
| 6632606 | Ullman et al. | Oct 2003 | B1 |
| 6632655 | Mehta et al. | Oct 2003 | B1 |
| 6670133 | Knapp et al. | Dec 2003 | B2 |
| 6723513 | Lexow | Apr 2004 | B2 |
| 6767731 | Hannah | Jul 2004 | B2 |
| 6800298 | Burdick et al. | Oct 2004 | B1 |
| 6806052 | Bridgham et al. | Oct 2004 | B2 |
| 6806058 | Jesperson et al. | Oct 2004 | B2 |
| 6859570 | Walt et al. | Feb 2005 | B2 |
| 6880576 | Karp et al. | Apr 2005 | B2 |
| 6884788 | Bulpitt et al. | Apr 2005 | B2 |
| 6913935 | Thomas | Jul 2005 | B1 |
| 6929859 | Chandler et al. | Aug 2005 | B2 |
| 6969488 | Bridgham et al. | Nov 2005 | B2 |
| 6974669 | Mirkin et al. | Dec 2005 | B2 |
| 7041481 | Anderson et al. | May 2006 | B2 |
| 7115400 | Adessi et al. | Oct 2006 | B1 |
| 7129091 | Ismagilov et al. | Oct 2006 | B2 |
| 7138267 | Jendrisak et al. | Nov 2006 | B1 |
| 7211654 | Gao et al. | May 2007 | B2 |
| 7262056 | Wooddell et al. | Aug 2007 | B2 |
| 7268167 | Higuchi et al. | Sep 2007 | B2 |
| 7282370 | Bridgham et al. | Oct 2007 | B2 |
| 7294503 | Quake et al. | Nov 2007 | B2 |
| 7297485 | Bornarth et al. | Nov 2007 | B2 |
| 7316903 | Yanagihara et al. | Jan 2008 | B2 |
| 7323305 | Leamon et al. | Jan 2008 | B2 |
| 7329493 | Chou et al. | Feb 2008 | B2 |
| 7425431 | Church et al. | Sep 2008 | B2 |
| 7536928 | Kazuno | May 2009 | B2 |
| 7544473 | Brenner | Jun 2009 | B2 |
| 7604938 | Takahashi et al. | Oct 2009 | B2 |
| 7608434 | Reznikoff et al. | Oct 2009 | B2 |
| 7608451 | Cooper et al. | Oct 2009 | B2 |
| 7622280 | Holliger et al. | Nov 2009 | B2 |
| 7638276 | Griffiths et al. | Dec 2009 | B2 |
| 7645596 | Williams et al. | Jan 2010 | B2 |
| 7666664 | Sarofim et al. | Feb 2010 | B2 |
| 7700325 | Cantor et al. | Apr 2010 | B2 |
| 7708949 | Stone et al. | May 2010 | B2 |
| 7709197 | Drmanac | May 2010 | B2 |
| 7745178 | Dong | Jun 2010 | B2 |
| 7745218 | Kim et al. | Jun 2010 | B2 |
| 7776927 | Chu et al. | Aug 2010 | B2 |
| RE41780 | Anderson et al. | Sep 2010 | E |
| 7799553 | Mathies et al. | Sep 2010 | B2 |
| 7842457 | Berka et al. | Nov 2010 | B2 |
| 7901891 | Drmanac | Mar 2011 | B2 |
| 7910354 | Drmanac et al. | Mar 2011 | B2 |
| 7943671 | Herminghaus et al. | May 2011 | B2 |
| 7947477 | Schroeder et al. | May 2011 | B2 |
| 7960104 | Drmanac et al. | Jun 2011 | B2 |
| 7968287 | Griffiths et al. | Jun 2011 | B2 |
| 7972778 | Brown et al. | Jul 2011 | B2 |
| 8003312 | Krutzik et al. | Aug 2011 | B2 |
| 8008018 | Quake et al. | Aug 2011 | B2 |
| 8053192 | Bignell et al. | Nov 2011 | B2 |
| 8067159 | Brown et al. | Nov 2011 | B2 |
| 8101346 | Takahama | Jan 2012 | B2 |
| 8124404 | Alphey | Feb 2012 | B2 |
| 8133719 | Drmanac et al. | Mar 2012 | B2 |
| 8137563 | Ma et al. | Mar 2012 | B2 |
| 8168385 | Brenner | May 2012 | B2 |
| 8252539 | Quake et al. | Aug 2012 | B2 |
| 8268564 | Roth et al. | Sep 2012 | B2 |
| 8273573 | Ismagilov et al. | Sep 2012 | B2 |
| 8278071 | Brown et al. | Oct 2012 | B2 |
| 8298767 | Brenner et al. | Oct 2012 | B2 |
| 8304193 | Ismagilov et al. | Nov 2012 | B2 |
| 8318433 | Brenner | Nov 2012 | B2 |
| 8318460 | Cantor et al. | Nov 2012 | B2 |
| 8329407 | Ismagilov et al. | Dec 2012 | B2 |
| 8337778 | Stone et al. | Dec 2012 | B2 |
| 8361299 | Sabin et al. | Jan 2013 | B2 |
| 8420386 | Ivics et al. | Apr 2013 | B2 |
| 8461129 | Bolduc et al. | Jun 2013 | B2 |
| 8563274 | Brenner et al. | Oct 2013 | B2 |
| 8592150 | Drmanac et al. | Nov 2013 | B2 |
| 8598328 | Koga et al. | Dec 2013 | B2 |
| 8603749 | Gillevet | Dec 2013 | B2 |
| 8679756 | Brenner et al. | Mar 2014 | B1 |
| 8748094 | Weitz et al. | Jun 2014 | B2 |
| 8748102 | Berka et al. | Jun 2014 | B2 |
| 8765380 | Berka et al. | Jul 2014 | B2 |
| 8822148 | Ismagliov et al. | Sep 2014 | B2 |
| 8829171 | Steemers et al. | Sep 2014 | B2 |
| 8835358 | Fodor et al. | Sep 2014 | B2 |
| 8846883 | Brown et al. | Sep 2014 | B2 |
| 8871444 | Griffiths et al. | Oct 2014 | B2 |
| 8889083 | Ismagilov et al. | Nov 2014 | B2 |
| 8927218 | Forsyth | Jan 2015 | B2 |
| 8975302 | Light et al. | Mar 2015 | B2 |
| 8986286 | Tanghoj et al. | Mar 2015 | B2 |
| 8986628 | Stone et al. | Mar 2015 | B2 |
| 9005935 | Belyaev | Apr 2015 | B2 |
| 9012370 | Hong | Apr 2015 | B2 |
| 9012390 | Holtze et al. | Apr 2015 | B2 |
| 9017948 | Agresti et al. | Apr 2015 | B2 |
| 9029083 | Griffiths et al. | May 2015 | B2 |
| 9029085 | Agresti et al. | May 2015 | B2 |
| 9040256 | Grunenwald et al. | May 2015 | B2 |
| 9068210 | Agresti et al. | Jun 2015 | B2 |
| 9074251 | Steemers et al. | Jul 2015 | B2 |
| 9080211 | Grunenwald et al. | Jul 2015 | B2 |
| 9102980 | Brenner et al. | Aug 2015 | B2 |
| 9133009 | Baroud et al. | Sep 2015 | B2 |
| 9150916 | Christen et al. | Oct 2015 | B2 |
| 9175295 | Kaminaka et al. | Nov 2015 | B2 |
| 9238206 | Rotem et al. | Jan 2016 | B2 |
| 9238671 | Goryshin et al. | Jan 2016 | B2 |
| 9249460 | Pushkarev et al. | Feb 2016 | B2 |
| 9273349 | Nguyen et al. | Mar 2016 | B2 |
| 9290808 | Fodor et al. | Mar 2016 | B2 |
| 9328382 | Drmanac et al. | May 2016 | B2 |
| 9347059 | Saxonov | May 2016 | B2 |
| 9388465 | Hindson et al. | Jul 2016 | B2 |
| 9410201 | Hindson et al. | Aug 2016 | B2 |
| 9436088 | Seul et al. | Sep 2016 | B2 |
| 9486757 | Romanowsky et al. | Nov 2016 | B2 |
| 9498761 | Holtze et al. | Nov 2016 | B2 |
| 9567631 | Hindson et al. | Feb 2017 | B2 |
| 9574226 | Gormley et al. | Feb 2017 | B2 |
| 9637799 | Fan et al. | May 2017 | B2 |
| 9644204 | Hindson et al. | May 2017 | B2 |
| 9689024 | Hindson et al. | Jun 2017 | B2 |
| 9694361 | Bharadwaj et al. | Jul 2017 | B2 |
| 9695468 | Hindson et al. | Jul 2017 | B2 |
| 9701998 | Hindson et al. | Jul 2017 | B2 |
| 9824068 | Wong et al. | Nov 2017 | B2 |
| 9856530 | Hindson et al. | Jan 2018 | B2 |
| 9946577 | Stafford et al. | Apr 2018 | B1 |
| 9951386 | Hindson et al. | Apr 2018 | B2 |
| 9957558 | Leamon et al. | May 2018 | B2 |
| 9975122 | Masquelier et al. | May 2018 | B2 |
| 10011872 | Belgrader et al. | Jul 2018 | B1 |
| 10017759 | Kaper et al. | Jul 2018 | B2 |
| 10030267 | Hindson et al. | Jul 2018 | B2 |
| 10041116 | Hindson et al. | Aug 2018 | B2 |
| 10053723 | Hindson et al. | Aug 2018 | B2 |
| 10059989 | Giresi et al. | Aug 2018 | B2 |
| 10071377 | Bharadwaj et al. | Sep 2018 | B2 |
| 10119167 | Srinivasan et al. | Nov 2018 | B2 |
| 10137449 | Bharadwaj et al. | Nov 2018 | B2 |
| 10144950 | Nolan | Dec 2018 | B2 |
| 10150117 | Bharadwaj et al. | Dec 2018 | B2 |
| 10150963 | Hindson et al. | Dec 2018 | B2 |
| 10150964 | Hindson et al. | Dec 2018 | B2 |
| 10150995 | Giresi et al. | Dec 2018 | B1 |
| 10161007 | Abate et al. | Dec 2018 | B2 |
| 10174310 | Nolan | Jan 2019 | B2 |
| 10208343 | Hindson et al. | Feb 2019 | B2 |
| 10221436 | Hardenbol et al. | Mar 2019 | B2 |
| 10221442 | Hindson et al. | Mar 2019 | B2 |
| 10227648 | Hindson et al. | Mar 2019 | B2 |
| 10253364 | Hindson et al. | Apr 2019 | B2 |
| 10273541 | Hindson et al. | Apr 2019 | B2 |
| 10323279 | Hindson et al. | Jun 2019 | B2 |
| 10337061 | Hindson et al. | Jul 2019 | B2 |
| 10344329 | Hindson et al. | Jul 2019 | B2 |
| 10347365 | Wong et al. | Jul 2019 | B2 |
| 10357771 | Bharadwaj et al. | Jul 2019 | B2 |
| 10400280 | Hindson et al. | Sep 2019 | B2 |
| 10428326 | Belhocine et al. | Oct 2019 | B2 |
| 10450607 | Hindson et al. | Oct 2019 | B2 |
| 10457986 | Hindson et al. | Oct 2019 | B2 |
| 10480028 | Hindson et al. | Nov 2019 | B2 |
| 20010020588 | Adourian et al. | Sep 2001 | A1 |
| 20010036669 | Jedrzejewski et al. | Nov 2001 | A1 |
| 20010041357 | Fouillet et al. | Nov 2001 | A1 |
| 20010044109 | Mandecki | Nov 2001 | A1 |
| 20010048900 | Bardell et al. | Dec 2001 | A1 |
| 20010051348 | Lee | Dec 2001 | A1 |
| 20010053519 | Fodor et al. | Dec 2001 | A1 |
| 20020001856 | Chow et al. | Jan 2002 | A1 |
| 20020005354 | Spence et al. | Jan 2002 | A1 |
| 20020034737 | Drmanac | Mar 2002 | A1 |
| 20020043463 | Shenderov | Apr 2002 | A1 |
| 20020051971 | Stuelpnagel et al. | May 2002 | A1 |
| 20020051992 | Bridgham et al. | May 2002 | A1 |
| 20020058332 | Quake et al. | May 2002 | A1 |
| 20020065609 | Ashby et al. | May 2002 | A1 |
| 20020068278 | Giese et al. | Jun 2002 | A1 |
| 20020089100 | Kawasaki | Jul 2002 | A1 |
| 20020092767 | Bjornson et al. | Jul 2002 | A1 |
| 20020113009 | O'Connor et al. | Aug 2002 | A1 |
| 20020119455 | Chan et al. | Aug 2002 | A1 |
| 20020119536 | Stern | Aug 2002 | A1 |
| 20020119544 | Yan et al. | Aug 2002 | A1 |
| 20020131147 | Paolini et al. | Sep 2002 | A1 |
| 20020160518 | Hayenga et al. | Oct 2002 | A1 |
| 20020164820 | Brown | Nov 2002 | A1 |
| 20020166582 | O'Connor et al. | Nov 2002 | A1 |
| 20020172965 | Kamb et al. | Nov 2002 | A1 |
| 20020175079 | Christel et al. | Nov 2002 | A1 |
| 20020179849 | Maher et al. | Dec 2002 | A1 |
| 20030005967 | Karp | Jan 2003 | A1 |
| 20030007898 | Bohm et al. | Jan 2003 | A1 |
| 20030008285 | Fischer | Jan 2003 | A1 |
| 20030008323 | Ravkin et al. | Jan 2003 | A1 |
| 20030022231 | Wangh et al. | Jan 2003 | A1 |
| 20030027203 | Fields | Feb 2003 | A1 |
| 20030027214 | Kamb | Feb 2003 | A1 |
| 20030027221 | Scott et al. | Feb 2003 | A1 |
| 20030028981 | Chandler et al. | Feb 2003 | A1 |
| 20030032141 | Nguyen et al. | Feb 2003 | A1 |
| 20030036206 | Chien et al. | Feb 2003 | A1 |
| 20030039978 | Hannah | Feb 2003 | A1 |
| 20030044777 | Beattie | Mar 2003 | A1 |
| 20030044836 | Levine et al. | Mar 2003 | A1 |
| 20030075446 | Culbertson et al. | Apr 2003 | A1 |
| 20030082587 | Seul et al. | May 2003 | A1 |
| 20030089605 | Timperman | May 2003 | A1 |
| 20030104466 | Knapp et al. | Jun 2003 | A1 |
| 20030108897 | Drmanac | Jun 2003 | A1 |
| 20030124509 | Kenis et al. | Jul 2003 | A1 |
| 20030149307 | Hai et al. | Aug 2003 | A1 |
| 20030170698 | Gascoyne et al. | Sep 2003 | A1 |
| 20030182068 | Battersby et al. | Sep 2003 | A1 |
| 20030207260 | Trnovsky et al. | Nov 2003 | A1 |
| 20030215862 | Parce et al. | Nov 2003 | A1 |
| 20040040851 | Karger et al. | Mar 2004 | A1 |
| 20040063138 | McGinnis et al. | Apr 2004 | A1 |
| 20040081962 | Chen et al. | Apr 2004 | A1 |
| 20040101680 | Barber et al. | May 2004 | A1 |
| 20040101880 | Rozwadowski et al. | May 2004 | A1 |
| 20040132122 | Banerjee et al. | Jul 2004 | A1 |
| 20040214175 | McKernan et al. | Oct 2004 | A9 |
| 20040224331 | Cantor et al. | Nov 2004 | A1 |
| 20040258701 | Dominowski et al. | Dec 2004 | A1 |
| 20050019839 | Jespersen et al. | Jan 2005 | A1 |
| 20050037397 | Mirkin et al. | Feb 2005 | A1 |
| 20050042625 | Schmidt et al. | Feb 2005 | A1 |
| 20050079510 | Berka et al. | Apr 2005 | A1 |
| 20050130188 | Walt et al. | Jun 2005 | A1 |
| 20050136417 | Cole et al. | Jun 2005 | A1 |
| 20050172476 | Stone et al. | Aug 2005 | A1 |
| 20050181379 | Su et al. | Aug 2005 | A1 |
| 20050202429 | Trau et al. | Sep 2005 | A1 |
| 20050202489 | Cho et al. | Sep 2005 | A1 |
| 20050221339 | Griffiths et al. | Oct 2005 | A1 |
| 20050244850 | Huang et al. | Nov 2005 | A1 |
| 20050272159 | Ismagilov et al. | Dec 2005 | A1 |
| 20050287572 | Mathies et al. | Dec 2005 | A1 |
| 20060002890 | Hersel et al. | Jan 2006 | A1 |
| 20060008799 | Cai et al. | Jan 2006 | A1 |
| 20060020371 | Ham et al. | Jan 2006 | A1 |
| 20060040382 | Heffron et al. | Feb 2006 | A1 |
| 20060073487 | Oliver et al. | Apr 2006 | A1 |
| 20060078888 | Griffiths et al. | Apr 2006 | A1 |
| 20060153924 | Griffiths et al. | Jul 2006 | A1 |
| 20060163385 | Link et al. | Jul 2006 | A1 |
| 20060177832 | Brenner | Aug 2006 | A1 |
| 20060177833 | Brenner | Aug 2006 | A1 |
| 20060199193 | Koo et al. | Sep 2006 | A1 |
| 20060240506 | Kushmaro et al. | Oct 2006 | A1 |
| 20060257893 | Takahashi et al. | Nov 2006 | A1 |
| 20060263888 | Fritz et al. | Nov 2006 | A1 |
| 20060275782 | Gunderson et al. | Dec 2006 | A1 |
| 20060286570 | Rowlen et al. | Dec 2006 | A1 |
| 20060292583 | Schneider et al. | Dec 2006 | A1 |
| 20070003442 | Link et al. | Jan 2007 | A1 |
| 20070009954 | Wang et al. | Jan 2007 | A1 |
| 20070020617 | Trnovsky et al. | Jan 2007 | A1 |
| 20070020640 | McCloskey et al. | Jan 2007 | A1 |
| 20070026401 | Hofmann et al. | Feb 2007 | A1 |
| 20070031829 | Yasuno et al. | Feb 2007 | A1 |
| 20070042400 | Choi et al. | Feb 2007 | A1 |
| 20070042419 | Barany et al. | Feb 2007 | A1 |
| 20070054119 | Garstecki et al. | Mar 2007 | A1 |
| 20070072208 | Drmanac | Mar 2007 | A1 |
| 20070077572 | Tawfik et al. | Apr 2007 | A1 |
| 20070092914 | Griffiths et al. | Apr 2007 | A1 |
| 20070099208 | Drmanac et al. | May 2007 | A1 |
| 20070111241 | Cereb et al. | May 2007 | A1 |
| 20070134277 | Chen et al. | Jun 2007 | A1 |
| 20070141584 | Roberts et al. | Jun 2007 | A1 |
| 20070154903 | Marla et al. | Jul 2007 | A1 |
| 20070160503 | Sethu et al. | Jul 2007 | A1 |
| 20070172873 | Brenner et al. | Jul 2007 | A1 |
| 20070190543 | Livak | Aug 2007 | A1 |
| 20070195127 | Ahn et al. | Aug 2007 | A1 |
| 20070207060 | Zou et al. | Sep 2007 | A1 |
| 20070228588 | Noritomi et al. | Oct 2007 | A1 |
| 20070231823 | McKernan et al. | Oct 2007 | A1 |
| 20070238113 | Kanda et al. | Oct 2007 | A1 |
| 20070259357 | Brenner | Nov 2007 | A1 |
| 20070264320 | Lee et al. | Nov 2007 | A1 |
| 20080003142 | Link et al. | Jan 2008 | A1 |
| 20080004436 | Tawfik et al. | Jan 2008 | A1 |
| 20080014589 | Link et al. | Jan 2008 | A1 |
| 20080124726 | Monforte | May 2008 | A1 |
| 20080138878 | Kubu et al. | Jun 2008 | A1 |
| 20080213766 | Brown et al. | Sep 2008 | A1 |
| 20080228268 | Shannon et al. | Sep 2008 | A1 |
| 20080241820 | Krutzik et al. | Oct 2008 | A1 |
| 20080242560 | Gunderson et al. | Oct 2008 | A1 |
| 20080268431 | Choy et al. | Oct 2008 | A1 |
| 20080268450 | Nam et al. | Oct 2008 | A1 |
| 20080268507 | Xu et al. | Oct 2008 | A1 |
| 20080299595 | Wong et al. | Dec 2008 | A1 |
| 20090005252 | Drmanac et al. | Jan 2009 | A1 |
| 20090011943 | Drmanac et al. | Jan 2009 | A1 |
| 20090012187 | Chu et al. | Jan 2009 | A1 |
| 20090025277 | Takanashi | Jan 2009 | A1 |
| 20090035770 | Mathies et al. | Feb 2009 | A1 |
| 20090048124 | Leamon et al. | Feb 2009 | A1 |
| 20090053169 | Castillo et al. | Feb 2009 | A1 |
| 20090062129 | McKernan et al. | Mar 2009 | A1 |
| 20090068170 | Weitz et al. | Mar 2009 | A1 |
| 20090098555 | Roth et al. | Apr 2009 | A1 |
| 20090099041 | Church et al. | Apr 2009 | A1 |
| 20090105959 | Braverman et al. | Apr 2009 | A1 |
| 20090118488 | Drmanac et al. | May 2009 | A1 |
| 20090134027 | Jary | May 2009 | A1 |
| 20090137404 | Drmanac et al. | May 2009 | A1 |
| 20090137414 | Drmanac et al. | May 2009 | A1 |
| 20090143244 | Bridgham et al. | Jun 2009 | A1 |
| 20090148961 | Luchini et al. | Jun 2009 | A1 |
| 20090155780 | Xiao et al. | Jun 2009 | A1 |
| 20090155781 | Drmanac et al. | Jun 2009 | A1 |
| 20090197248 | Griffiths et al. | Aug 2009 | A1 |
| 20090197772 | Griffiths et al. | Aug 2009 | A1 |
| 20090202984 | Cantor | Aug 2009 | A1 |
| 20090203531 | Kurn | Aug 2009 | A1 |
| 20090264299 | Drmanac et al. | Oct 2009 | A1 |
| 20090286687 | Dressman et al. | Nov 2009 | A1 |
| 20090325260 | Otto et al. | Dec 2009 | A1 |
| 20100021973 | Makarov et al. | Jan 2010 | A1 |
| 20100021984 | Edd et al. | Jan 2010 | A1 |
| 20100022414 | Link et al. | Jan 2010 | A1 |
| 20100035254 | Williams | Feb 2010 | A1 |
| 20100062494 | Church et al. | Mar 2010 | A1 |
| 20100069263 | Shendure et al. | Mar 2010 | A1 |
| 20100086914 | Bentley et al. | Apr 2010 | A1 |
| 20100105112 | Holtze et al. | Apr 2010 | A1 |
| 20100113296 | Myerson | May 2010 | A1 |
| 20100120098 | Grunenwald et al. | May 2010 | A1 |
| 20100130369 | Shenderov et al. | May 2010 | A1 |
| 20100136544 | Agresti et al. | Jun 2010 | A1 |
| 20100137163 | Link et al. | Jun 2010 | A1 |
| 20100173394 | Colston, Jr. et al. | Jul 2010 | A1 |
| 20100187705 | Lee et al. | Jul 2010 | A1 |
| 20100203647 | Hang et al. | Aug 2010 | A1 |
| 20100210479 | Griffiths et al. | Aug 2010 | A1 |
| 20100216153 | Lapidus et al. | Aug 2010 | A1 |
| 20100248237 | Froehlich et al. | Sep 2010 | A1 |
| 20100248991 | Roesler et al. | Sep 2010 | A1 |
| 20100304982 | Hinz et al. | Dec 2010 | A1 |
| 20110000560 | Miller et al. | Jan 2011 | A1 |
| 20110008775 | Gao et al. | Jan 2011 | A1 |
| 20110028412 | Cappello et al. | Feb 2011 | A1 |
| 20110033548 | Lai et al. | Feb 2011 | A1 |
| 20110033854 | Drmanac et al. | Feb 2011 | A1 |
| 20110053798 | Hindson et al. | Mar 2011 | A1 |
| 20110059556 | Strey et al. | Mar 2011 | A1 |
| 20110071053 | Drmanac et al. | Mar 2011 | A1 |
| 20110086780 | Colston, Jr. et al. | Apr 2011 | A1 |
| 20110092376 | Colston, Jr. et al. | Apr 2011 | A1 |
| 20110092392 | Colston, Jr. et al. | Apr 2011 | A1 |
| 20110160078 | Fodor et al. | Jun 2011 | A1 |
| 20110166034 | Kwong et al. | Jul 2011 | A1 |
| 20110195496 | Muraguchi et al. | Aug 2011 | A1 |
| 20110201526 | Berka et al. | Aug 2011 | A1 |
| 20110212090 | Pedersen et al. | Sep 2011 | A1 |
| 20110217736 | Hindson | Sep 2011 | A1 |
| 20110218123 | Weitz et al. | Sep 2011 | A1 |
| 20110257889 | Klammer et al. | Oct 2011 | A1 |
| 20110263457 | Krutzik et al. | Oct 2011 | A1 |
| 20110267457 | Weitz et al. | Nov 2011 | A1 |
| 20110281736 | Drmanac et al. | Nov 2011 | A1 |
| 20110281738 | Drmanac et al. | Nov 2011 | A1 |
| 20110287435 | Grunenwald et al. | Nov 2011 | A1 |
| 20110293701 | Bratzler et al. | Dec 2011 | A1 |
| 20110305761 | Shum et al. | Dec 2011 | A1 |
| 20110306141 | Bronchetti et al. | Dec 2011 | A1 |
| 20110319281 | Drmanac | Dec 2011 | A1 |
| 20120000777 | Garrell et al. | Jan 2012 | A1 |
| 20120003657 | Myllykangas et al. | Jan 2012 | A1 |
| 20120010091 | Linnarson et al. | Jan 2012 | A1 |
| 20120010098 | Griffiths et al. | Jan 2012 | A1 |
| 20120010107 | Griffiths et al. | Jan 2012 | A1 |
| 20120014977 | Furihata et al. | Jan 2012 | A1 |
| 20120015382 | Weitz et al. | Jan 2012 | A1 |
| 20120015822 | Weitz et al. | Jan 2012 | A1 |
| 20120041727 | Mishra et al. | Feb 2012 | A1 |
| 20120071331 | Casbon et al. | Mar 2012 | A1 |
| 20120121481 | Romanowsky et al. | May 2012 | A1 |
| 20120132288 | Weitz et al. | May 2012 | A1 |
| 20120135893 | Drmanac et al. | May 2012 | A1 |
| 20120165219 | Van Der Zaag et al. | Jun 2012 | A1 |
| 20120172259 | Rigatti et al. | Jul 2012 | A1 |
| 20120184449 | Hixson et al. | Jul 2012 | A1 |
| 20120190032 | Ness et al. | Jul 2012 | A1 |
| 20120190037 | Durin et al. | Jul 2012 | A1 |
| 20120196288 | Beer et al. | Aug 2012 | A1 |
| 20120208705 | Steemers et al. | Aug 2012 | A1 |
| 20120208724 | Steemers et al. | Aug 2012 | A1 |
| 20120211084 | Weitz et al. | Aug 2012 | A1 |
| 20120220494 | Samuels et al. | Aug 2012 | A1 |
| 20120220497 | Jacobson et al. | Aug 2012 | A1 |
| 20120222748 | Weitz et al. | Sep 2012 | A1 |
| 20120230338 | Ganeshalingam et al. | Sep 2012 | A1 |
| 20120231972 | Golyshin et al. | Sep 2012 | A1 |
| 20120252012 | Armougom et al. | Oct 2012 | A1 |
| 20120253689 | Rogan et al. | Oct 2012 | A1 |
| 20120289428 | Duffy et al. | Nov 2012 | A1 |
| 20120295819 | Leamon et al. | Nov 2012 | A1 |
| 20120297493 | Cooper et al. | Nov 2012 | A1 |
| 20120309002 | Link | Dec 2012 | A1 |
| 20120316074 | Saxonov | Dec 2012 | A1 |
| 20130017978 | Kavanagh et al. | Jan 2013 | A1 |
| 20130018970 | Woundy et al. | Jan 2013 | A1 |
| 20130022682 | Lee et al. | Jan 2013 | A1 |
| 20130028812 | Prieto et al. | Jan 2013 | A1 |
| 20130041004 | Drager et al. | Feb 2013 | A1 |
| 20130046030 | Rotem et al. | Feb 2013 | A1 |
| 20130059310 | Brenner et al. | Mar 2013 | A1 |
| 20130078638 | Berka et al. | Mar 2013 | A1 |
| 20130079231 | Pushkarev et al. | Mar 2013 | A1 |
| 20130079251 | Boles | Mar 2013 | A1 |
| 20130084243 | Goetsch et al. | Apr 2013 | A1 |
| 20130096073 | Sidelman | Apr 2013 | A1 |
| 20130109575 | Kleinschmidt et al. | May 2013 | A1 |
| 20130109576 | Shuber et al. | May 2013 | A1 |
| 20130109596 | Peterson et al. | May 2013 | A1 |
| 20130121893 | Delamarche et al. | May 2013 | A1 |
| 20130130919 | Chen et al. | May 2013 | A1 |
| 20130157870 | Pushkarev et al. | Jun 2013 | A1 |
| 20130157899 | Adler, Jr. et al. | Jun 2013 | A1 |
| 20130178368 | Griffiths et al. | Jul 2013 | A1 |
| 20130189700 | So et al. | Jul 2013 | A1 |
| 20130203605 | Shendure et al. | Aug 2013 | A1 |
| 20130203675 | Desimone et al. | Aug 2013 | A1 |
| 20130210639 | Link et al. | Aug 2013 | A1 |
| 20130210991 | Fonnum et al. | Aug 2013 | A1 |
| 20130211055 | Raines et al. | Aug 2013 | A1 |
| 20130225418 | Watson | Aug 2013 | A1 |
| 20130225623 | Buxbaum et al. | Aug 2013 | A1 |
| 20130268206 | Porreca et al. | Oct 2013 | A1 |
| 20130273640 | Krishnan et al. | Oct 2013 | A1 |
| 20130274117 | Church et al. | Oct 2013 | A1 |
| 20130296173 | Callow et al. | Nov 2013 | A1 |
| 20130311106 | White et al. | Nov 2013 | A1 |
| 20130343317 | Etemad et al. | Dec 2013 | A1 |
| 20130344508 | Schwartz et al. | Dec 2013 | A1 |
| 20140030350 | Ashrafi et al. | Jan 2014 | A1 |
| 20140037514 | Stone et al. | Feb 2014 | A1 |
| 20140038178 | Otto et al. | Feb 2014 | A1 |
| 20140057799 | Johnson et al. | Feb 2014 | A1 |
| 20140065234 | Shum et al. | Mar 2014 | A1 |
| 20140080717 | Li et al. | Mar 2014 | A1 |
| 20140093916 | Belyaev et al. | Apr 2014 | A1 |
| 20140120529 | Andersen et al. | May 2014 | A1 |
| 20140155274 | Xie et al. | Jun 2014 | A1 |
| 20140155295 | Hindson et al. | Jun 2014 | A1 |
| 20140194323 | Gillevet | Jul 2014 | A1 |
| 20140199331 | Robillard et al. | Jul 2014 | A1 |
| 20140199730 | Agresti et al. | Jul 2014 | A1 |
| 20140199731 | Agresti et al. | Jul 2014 | A1 |
| 20140200166 | Van Rooyen et al. | Jul 2014 | A1 |
| 20140206073 | Park et al. | Jul 2014 | A1 |
| 20140206554 | Hindson et al. | Jul 2014 | A1 |
| 20140214334 | Plattner et al. | Jul 2014 | A1 |
| 20140227684 | Hindson et al. | Aug 2014 | A1 |
| 20140227706 | Kato et al. | Aug 2014 | A1 |
| 20140228255 | Hindson et al. | Aug 2014 | A1 |
| 20140235506 | Hindson et al. | Aug 2014 | A1 |
| 20140242664 | Zhang et al. | Aug 2014 | A1 |
| 20140274740 | Srinivasan et al. | Sep 2014 | A1 |
| 20140287963 | Hindson et al. | Sep 2014 | A1 |
| 20140302503 | Lowe et al. | Oct 2014 | A1 |
| 20140315725 | Faham et al. | Oct 2014 | A1 |
| 20140315755 | Chen et al. | Oct 2014 | A1 |
| 20140323316 | Drmanac et al. | Oct 2014 | A1 |
| 20140357500 | Vigneault et al. | Dec 2014 | A1 |
| 20140357530 | Zhang et al. | Dec 2014 | A1 |
| 20140378322 | Hindson et al. | Dec 2014 | A1 |
| 20140378345 | Hindson et al. | Dec 2014 | A1 |
| 20140378349 | Hindson et al. | Dec 2014 | A1 |
| 20140378350 | Hindson et al. | Dec 2014 | A1 |
| 20150005188 | Levner et al. | Jan 2015 | A1 |
| 20150005199 | Hindson et al. | Jan 2015 | A1 |
| 20150005200 | Hindson et al. | Jan 2015 | A1 |
| 20150011430 | Saxonov | Jan 2015 | A1 |
| 20150011432 | Saxonov | Jan 2015 | A1 |
| 20150031037 | Li et al. | Jan 2015 | A1 |
| 20150057163 | Rotem et al. | Feb 2015 | A1 |
| 20150066385 | Schnall-Levin et al. | Mar 2015 | A1 |
| 20150072396 | Gee et al. | Mar 2015 | A1 |
| 20150072899 | Ward et al. | Mar 2015 | A1 |
| 20150111256 | Church et al. | Apr 2015 | A1 |
| 20150111788 | Fernandez et al. | Apr 2015 | A1 |
| 20150119280 | Srinivas et al. | Apr 2015 | A1 |
| 20150125904 | Ting et al. | May 2015 | A1 |
| 20150133344 | Shendure et al. | May 2015 | A1 |
| 20150211056 | Um et al. | Jul 2015 | A1 |
| 20150218633 | Hindson et al. | Aug 2015 | A1 |
| 20150220532 | Wong | Aug 2015 | A1 |
| 20150224466 | Hindson et al. | Aug 2015 | A1 |
| 20150225777 | Hindson et al. | Aug 2015 | A1 |
| 20150225778 | Hindson et al. | Aug 2015 | A1 |
| 20150225786 | Litterst et al. | Aug 2015 | A1 |
| 20150232942 | Abate et al. | Aug 2015 | A1 |
| 20150247182 | Faham et al. | Sep 2015 | A1 |
| 20150258543 | Baroud et al. | Sep 2015 | A1 |
| 20150259736 | Steemers et al. | Sep 2015 | A1 |
| 20150267191 | Steelman et al. | Sep 2015 | A1 |
| 20150267246 | Baroud et al. | Sep 2015 | A1 |
| 20150291942 | Gloeckner et al. | Oct 2015 | A1 |
| 20150292988 | Bharadwaj et al. | Oct 2015 | A1 |
| 20150298091 | Weitz et al. | Oct 2015 | A1 |
| 20150299772 | Zhang | Oct 2015 | A1 |
| 20150299784 | Fan et al. | Oct 2015 | A1 |
| 20150329617 | Winther et al. | Nov 2015 | A1 |
| 20150329852 | Nolan et al. | Nov 2015 | A1 |
| 20150329891 | Tan et al. | Nov 2015 | A1 |
| 20150337298 | Xi et al. | Nov 2015 | A1 |
| 20150353999 | Agresti et al. | Dec 2015 | A1 |
| 20150361418 | Reed et al. | Dec 2015 | A1 |
| 20150368638 | Steemers et al. | Dec 2015 | A1 |
| 20150368694 | Pan et al. | Dec 2015 | A1 |
| 20150376605 | Jarosz et al. | Dec 2015 | A1 |
| 20150376608 | Kaper et al. | Dec 2015 | A1 |
| 20150376609 | Hindson et al. | Dec 2015 | A1 |
| 20150376700 | Schnall-Levin et al. | Dec 2015 | A1 |
| 20150379196 | Schnall-Levin et al. | Dec 2015 | A1 |
| 20160008778 | Weitz et al. | Jan 2016 | A1 |
| 20160024558 | Hardenbol | Jan 2016 | A1 |
| 20160025726 | Altin et al. | Jan 2016 | A1 |
| 20160032282 | Vigneault et al. | Feb 2016 | A1 |
| 20160034093 | Xie et al. | Feb 2016 | A1 |
| 20160053253 | Salathia et al. | Feb 2016 | A1 |
| 20160060621 | Agresti et al. | Mar 2016 | A1 |
| 20160060691 | Giresi et al. | Mar 2016 | A1 |
| 20160115474 | Jelinek et al. | Apr 2016 | A1 |
| 20160122753 | Mikkelsen et al. | May 2016 | A1 |
| 20160122817 | Jarosz et al. | May 2016 | A1 |
| 20160145683 | Fan et al. | May 2016 | A1 |
| 20160153005 | Zhang et al. | Jun 2016 | A1 |
| 20160160235 | Solodushko et al. | Jun 2016 | A1 |
| 20160177359 | Ukanis et al. | Jun 2016 | A1 |
| 20160177375 | Abate et al. | Jun 2016 | A1 |
| 20160194699 | Borodina et al. | Jul 2016 | A1 |
| 20160201125 | Samuels et al. | Jul 2016 | A1 |
| 20160203196 | Schnall-Levin et al. | Jul 2016 | A1 |
| 20160208323 | Bernstein et al. | Jul 2016 | A1 |
| 20160231324 | Zhao et al. | Aug 2016 | A1 |
| 20160232291 | Kyriazopoulou-Panagiotopoulou et al. | Aug 2016 | A1 |
| 20160244742 | Linnarsson et al. | Aug 2016 | A1 |
| 20160244809 | Belgrader et al. | Aug 2016 | A1 |
| 20160244811 | Edwards | Aug 2016 | A1 |
| 20160244825 | Vigneault et al. | Aug 2016 | A1 |
| 20160251697 | Nolan et al. | Sep 2016 | A1 |
| 20160257984 | Hardenbol et al. | Sep 2016 | A1 |
| 20160281160 | Jarosz et al. | Sep 2016 | A1 |
| 20160289769 | Schwartz et al. | Oct 2016 | A1 |
| 20160304860 | Hindson et al. | Oct 2016 | A1 |
| 20160314242 | Schnall-Levin et al. | Oct 2016 | A1 |
| 20160326583 | Johnson et al. | Nov 2016 | A1 |
| 20160348093 | Price et al. | Dec 2016 | A1 |
| 20160350478 | Chin et al. | Dec 2016 | A1 |
| 20160376663 | Brown | Dec 2016 | A1 |
| 20170009274 | Abate et al. | Jan 2017 | A1 |
| 20170016041 | Greenfield et al. | Jan 2017 | A1 |
| 20170114390 | Hindson et al. | Apr 2017 | A1 |
| 20170145476 | Ryvkin et al. | May 2017 | A1 |
| 20170159109 | Zheng et al. | Jun 2017 | A1 |
| 20170183701 | Agresti et al. | Jun 2017 | A1 |
| 20170211127 | Mikkelsen et al. | Jul 2017 | A1 |
| 20170235876 | Jaffe et al. | Aug 2017 | A1 |
| 20170247757 | Hindson et al. | Aug 2017 | A1 |
| 20170260584 | Zheng et al. | Sep 2017 | A1 |
| 20170268056 | Vigneault et al. | Sep 2017 | A1 |
| 20170321252 | Hindson et al. | Nov 2017 | A1 |
| 20170335385 | Hindson et al. | Nov 2017 | A1 |
| 20170342404 | Hindson et al. | Nov 2017 | A1 |
| 20170343545 | Hadrup et al. | Nov 2017 | A1 |
| 20170348691 | Bharadwaj et al. | Dec 2017 | A1 |
| 20170356027 | Hindson et al. | Dec 2017 | A1 |
| 20170362587 | Hindson et al. | Dec 2017 | A1 |
| 20180008984 | Bharadwaj et al. | Jan 2018 | A1 |
| 20180015472 | Bharadwaj et al. | Jan 2018 | A1 |
| 20180015473 | Bharadwaj et al. | Jan 2018 | A1 |
| 20180016634 | Hindson et al. | Jan 2018 | A1 |
| 20180030512 | Hindson et al. | Feb 2018 | A1 |
| 20180030515 | Regev et al. | Feb 2018 | A1 |
| 20180057868 | Walder et al. | Mar 2018 | A1 |
| 20180071695 | Weitz et al. | Mar 2018 | A1 |
| 20180080021 | Reuter et al. | Mar 2018 | A1 |
| 20180087050 | Zheng et al. | Mar 2018 | A1 |
| 20180088112 | Fan et al. | Mar 2018 | A1 |
| 20180112253 | Hindson et al. | Apr 2018 | A1 |
| 20180179580 | Hindson et al. | Jun 2018 | A1 |
| 20180179591 | Belgrader et al. | Jun 2018 | A1 |
| 20180180601 | Pedersen et al. | Jun 2018 | A1 |
| 20180195060 | Wang et al. | Jul 2018 | A1 |
| 20180195112 | Lebofsky et al. | Jul 2018 | A1 |
| 20180196781 | Wong et al. | Jul 2018 | A1 |
| 20180216162 | Belhocine et al. | Aug 2018 | A1 |
| 20180237951 | Bock et al. | Aug 2018 | A1 |
| 20180258466 | Hindson et al. | Sep 2018 | A1 |
| 20180258482 | Hindson et al. | Sep 2018 | A1 |
| 20180265928 | Schnall-Levin et al. | Sep 2018 | A1 |
| 20180267036 | Fan et al. | Sep 2018 | A1 |
| 20180273933 | Gunderson et al. | Sep 2018 | A1 |
| 20180282804 | Hindson et al. | Oct 2018 | A1 |
| 20180305685 | Li et al. | Oct 2018 | A1 |
| 20180312822 | Lee et al. | Nov 2018 | A1 |
| 20180312873 | Zheng | Nov 2018 | A1 |
| 20180327839 | Hindson et al. | Nov 2018 | A1 |
| 20180334670 | Bharadwaj et al. | Nov 2018 | A1 |
| 20180335424 | Chen et al. | Nov 2018 | A1 |
| 20180340169 | Belhocine et al. | Nov 2018 | A1 |
| 20180340170 | Belhocine et al. | Nov 2018 | A1 |
| 20180340171 | Belhocine et al. | Nov 2018 | A1 |
| 20180340172 | Belhocine et al. | Nov 2018 | A1 |
| 20180340939 | Gaublomme et al. | Nov 2018 | A1 |
| 20180346979 | Hindson et al. | Dec 2018 | A1 |
| 20180363029 | Hindson et al. | Dec 2018 | A1 |
| 20180371538 | Blauwkamp et al. | Dec 2018 | A1 |
| 20180371540 | Hindson et al. | Dec 2018 | A1 |
| 20180371545 | Wong et al. | Dec 2018 | A1 |
| 20180376609 | Ju et al. | Dec 2018 | A1 |
| 20190002967 | Chen et al. | Jan 2019 | A1 |
| 20190024166 | Hindson et al. | Jan 2019 | A1 |
| 20190032129 | Hindson et al. | Jan 2019 | A1 |
| 20190032130 | Giresi et al. | Jan 2019 | A1 |
| 20190040382 | Steemers et al. | Feb 2019 | A1 |
| 20190040464 | Giresi et al. | Feb 2019 | A1 |
| 20190060890 | Bharadwaj et al. | Feb 2019 | A1 |
| 20190060904 | Bharadwaj et al. | Feb 2019 | A1 |
| 20190060905 | Bharadwaj et al. | Feb 2019 | A1 |
| 20190064173 | Bharadwaj et al. | Feb 2019 | A1 |
| 20190071656 | Chang et al. | Mar 2019 | A1 |
| 20190078150 | Chen et al. | Mar 2019 | A1 |
| 20190085391 | Hindson et al. | Mar 2019 | A1 |
| 20190127731 | McDermott | May 2019 | A1 |
| 20190136316 | Hindson et al. | May 2019 | A1 |
| 20190136317 | Hindson et al. | May 2019 | A1 |
| 20190136319 | Hindson et al. | May 2019 | A1 |
| 20190153436 | Belhocine et al. | May 2019 | A1 |
| 20190153532 | Bharadwaj et al. | May 2019 | A1 |
| 20190169666 | Hardenbol et al. | Jun 2019 | A1 |
| 20190169700 | Abate et al. | Jun 2019 | A1 |
| 20190176152 | Bharadwaj et al. | Jun 2019 | A1 |
| 20190177800 | Boutet et al. | Jun 2019 | A1 |
| 20190203291 | Hindson et al. | Jul 2019 | A1 |
| 20190241965 | Abate et al. | Aug 2019 | A1 |
| 20190249226 | Bent et al. | Aug 2019 | A1 |
| 20190276817 | Hindson et al. | Sep 2019 | A1 |
| 20190330701 | Abate et al. | Oct 2019 | A1 |
| Number | Date | Country |
|---|---|---|
| 102292455 | Dec 2011 | CN |
| 102050953 | Nov 2012 | CN |
| 103202812 | Jul 2013 | CN |
| 0249007 | Dec 1987 | EP |
| 0271281 | Jun 1988 | EP |
| 0637996 | Jul 1997 | EP |
| 1019496 | Sep 2004 | EP |
| 1672064 | Jun 2006 | EP |
| 1482036 | Oct 2007 | EP |
| 1841879 | Oct 2007 | EP |
| 1944368 | Jul 2008 | EP |
| 1594980 | Nov 2009 | EP |
| 1967592 | Apr 2010 | EP |
| 2258846 | Dec 2010 | EP |
| 2145955 | Feb 2012 | EP |
| 1905828 | Aug 2012 | EP |
| 2136786 | Oct 2012 | EP |
| 1908832 | Dec 2012 | EP |
| 2540389 | Jan 2013 | EP |
| 2635679 | Sep 2013 | EP |
| 2752664 | Jul 2014 | EP |
| 3013957 | Sep 2018 | EP |
| 2097692 | May 1985 | GB |
| 2485850 | May 2012 | GB |
| S5949832 | Mar 1984 | JP |
| S60227826 | Nov 1985 | JP |
| 2006507921 | Mar 2006 | JP |
| 2006289250 | Oct 2006 | JP |
| 2007015990 | Jan 2007 | JP |
| 2007268350 | Oct 2007 | JP |
| 2009513948 | Apr 2009 | JP |
| 2009208074 | Sep 2009 | JP |
| 2012131798 | Jul 2012 | JP |
| 2012522517 | Sep 2012 | JP |
| 2321638 | Apr 2008 | RU |
| WO-8402000 | May 1984 | WO |
| WO-9301498 | Jan 1993 | WO |
| WO-9418218 | Aug 1994 | WO |
| WO-9419101 | Sep 1994 | WO |
| WO-9423699 | Oct 1994 | WO |
| WO-9530782 | Nov 1995 | WO |
| WO-9629629 | Sep 1996 | WO |
| WO-9641011 | Dec 1996 | WO |
| WO-9802237 | Jan 1998 | WO |
| WO-9852691 | Nov 1998 | WO |
| WO-9909217 | Feb 1999 | WO |
| WO-9942597 | Aug 1999 | WO |
| WO-9952708 | Oct 1999 | WO |
| WO-0008212 | Feb 2000 | WO |
| WO-0023181 | Apr 2000 | WO |
| WO-0026412 | May 2000 | WO |
| WO-0034527 | Jun 2000 | WO |
| WO-0043766 | Jul 2000 | WO |
| WO-0070095 | Nov 2000 | WO |
| WO-0102850 | Jan 2001 | WO |
| WO-0114589 | Mar 2001 | WO |
| WO-0189787 | Nov 2001 | WO |
| WO-0190418 | Nov 2001 | WO |
| WO-0127610 | Mar 2002 | WO |
| WO-0231203 | Apr 2002 | WO |
| WO-02086148 | Oct 2002 | WO |
| WO-0218949 | Jan 2003 | WO |
| WO-03062462 | Jul 2003 | WO |
| WO-2004002627 | Jan 2004 | WO |
| WO-2004010106 | Jan 2004 | WO |
| WO-2004061083 | Jul 2004 | WO |
| WO-2004065617 | Aug 2004 | WO |
| WO-2004069849 | Aug 2004 | WO |
| WO-2004091763 | Oct 2004 | WO |
| WO-2004102204 | Nov 2004 | WO |
| WO-2004103565 | Dec 2004 | WO |
| WO-2004105734 | Dec 2004 | WO |
| WO-2005002730 | Jan 2005 | WO |
| WO-2005021151 | Mar 2005 | WO |
| WO-2005023331 | Mar 2005 | WO |
| WO-2005040406 | May 2005 | WO |
| WO-2005049787 | Jun 2005 | WO |
| WO-2005082098 | Sep 2005 | WO |
| WO-2006030993 | Mar 2006 | WO |
| WO-2006071770 | Jul 2006 | WO |
| WO-2006078841 | Jul 2006 | WO |
| WO-2006086210 | Aug 2006 | WO |
| WO-2006096571 | Sep 2006 | WO |
| WO-2007001448 | Jan 2007 | WO |
| WO-2007002490 | Jan 2007 | WO |
| WO-2007012638 | Feb 2007 | WO |
| WO-2007018601 | Feb 2007 | WO |
| WO-2007024840 | Mar 2007 | WO |
| WO-2007081385 | Jul 2007 | WO |
| WO-2007081387 | Jul 2007 | WO |
| WO-2007084192 | Jul 2007 | WO |
| WO-2007089541 | Aug 2007 | WO |
| WO-2007093819 | Aug 2007 | WO |
| WO-2007111937 | Oct 2007 | WO |
| WO-2007114794 | Oct 2007 | WO |
| WO-2007121489 | Oct 2007 | WO |
| WO-2007133710 | Nov 2007 | WO |
| WO-2007138178 | Dec 2007 | WO |
| WO-2007139766 | Dec 2007 | WO |
| WO-2007140015 | Dec 2007 | WO |
| WO-2007147079 | Dec 2007 | WO |
| WO-2007149432 | Dec 2007 | WO |
| WO-2008021123 | Feb 2008 | WO |
| WO-2008091792 | Jul 2008 | WO |
| WO-2008102057 | Aug 2008 | WO |
| WO-2008109176 | Sep 2008 | WO |
| WO-2008121342 | Oct 2008 | WO |
| WO-2008061193 | Nov 2008 | WO |
| WO-2008134153 | Nov 2008 | WO |
| WO-2008135512 | Nov 2008 | WO |
| WO-2008150432 | Dec 2008 | WO |
| WO-2008135512 | Jan 2009 | WO |
| WO-2009005680 | Jan 2009 | WO |
| WO-2009011808 | Jan 2009 | WO |
| WO-2009015296 | Jan 2009 | WO |
| WO-2009023821 | Feb 2009 | WO |
| WO-2009048532 | Apr 2009 | WO |
| WO-2009061372 | May 2009 | WO |
| WO-2009085215 | Jul 2009 | WO |
| WO-2009147386 | Dec 2009 | WO |
| WO-2010004018 | Jan 2010 | WO |
| WO-2010009735 | Jan 2010 | WO |
| WO-2010033200 | Mar 2010 | WO |
| WO-2010048605 | Apr 2010 | WO |
| WO-2010104604 | Sep 2010 | WO |
| WO-2010115154 | Oct 2010 | WO |
| WO-2010127304 | Nov 2010 | WO |
| WO-2010148039 | Dec 2010 | WO |
| WO-2010151776 | Dec 2010 | WO |
| WO-2010117620 | Feb 2011 | WO |
| WO-2011028539 | Mar 2011 | WO |
| WO-2011047870 | Apr 2011 | WO |
| WO-2011056546 | May 2011 | WO |
| WO-2011066476 | Jun 2011 | WO |
| WO-2011074960 | Jun 2011 | WO |
| WO-2011106314 | Sep 2011 | WO |
| WO-2011140627 | Nov 2011 | WO |
| WO-2011156529 | Dec 2011 | WO |
| WO-2012012037 | Jan 2012 | WO |
| WO-2012019765 | Feb 2012 | WO |
| WO-2011140510 | Mar 2012 | WO |
| WO-2012047889 | Apr 2012 | WO |
| WO-2012048340 | Apr 2012 | WO |
| WO-2012048341 | Apr 2012 | WO |
| WO-2012055929 | May 2012 | WO |
| WO-2012061832 | May 2012 | WO |
| WO-2012083225 | Jun 2012 | WO |
| WO-2012087736 | Jun 2012 | WO |
| WO-2012100216 | Jul 2012 | WO |
| WO-2012106546 | Aug 2012 | WO |
| WO-2012112804 | Aug 2012 | WO |
| WO-2012112970 | Aug 2012 | WO |
| WO-2012116250 | Aug 2012 | WO |
| WO-2012116331 | Aug 2012 | WO |
| WO-2012136734 | Oct 2012 | WO |
| WO-2012142531 | Oct 2012 | WO |
| WO-2012142611 | Oct 2012 | WO |
| WO-2012148497 | Nov 2012 | WO |
| WO-2012149042 | Nov 2012 | WO |
| WO-2012150317 | Nov 2012 | WO |
| WO-2012166425 | Dec 2012 | WO |
| WO-2013019751 | Feb 2013 | WO |
| WO-2013035114 | Mar 2013 | WO |
| WO-2013036929 | Mar 2013 | WO |
| WO-2013055955 | Apr 2013 | WO |
| WO-2013096643 | Jun 2013 | WO |
| WO-2013122996 | Aug 2013 | WO |
| WO-2013123125 | Aug 2013 | WO |
| WO-2013126741 | Aug 2013 | WO |
| WO-2013134261 | Sep 2013 | WO |
| WO-2013150083 | Oct 2013 | WO |
| WO-2013177220 | Nov 2013 | WO |
| WO-2013188872 | Dec 2013 | WO |
| WO-2014018460 | Jan 2014 | WO |
| WO-2014028378 | Feb 2014 | WO |
| WO-2014028537 | Feb 2014 | WO |
| WO-2014053854 | Apr 2014 | WO |
| WO-2014071361 | May 2014 | WO |
| WO-2014072703 | May 2014 | WO |
| WO-2014074611 | May 2014 | WO |
| WO-2014093676 | Jun 2014 | WO |
| WO-2014108810 | Jul 2014 | WO |
| WO-2014140309 | Sep 2014 | WO |
| WO-2014144495 | Sep 2014 | WO |
| WO-2014145047 | Sep 2014 | WO |
| WO-2014150931 | Sep 2014 | WO |
| WO-2014182835 | Nov 2014 | WO |
| WO-2014189957 | Nov 2014 | WO |
| WO-2014200767 | Dec 2014 | WO |
| WO-2014210353 | Dec 2014 | WO |
| WO-2015031691 | Mar 2015 | WO |
| WO-2015044428 | Apr 2015 | WO |
| WO-2014210353 | Jul 2015 | WO |
| WO-2015157567 | Oct 2015 | WO |
| WO-2015164212 | Oct 2015 | WO |
| WO-2015185067 | Dec 2015 | WO |
| WO-2015188839 | Dec 2015 | WO |
| WO-2015200891 | Dec 2015 | WO |
| WO-2015200893 | Dec 2015 | WO |
| WO-2016040476 | Mar 2016 | WO |
| WO-2016033251 | Apr 2016 | WO |
| WO-2016061517 | Apr 2016 | WO |
| WO-2016100976 | Jun 2016 | WO |
| WO-2016126871 | Aug 2016 | WO |
| WO-2016130578 | Aug 2016 | WO |
| WO-2016138496 | Sep 2016 | WO |
| WO-2016149661 | Sep 2016 | WO |
| WO-2016168584 | Oct 2016 | WO |
| WO-2016170126 | Oct 2016 | WO |
| WO-2016187256 | Nov 2016 | WO |
| WO-2016187717 | Dec 2016 | WO |
| WO-2016191618 | Dec 2016 | WO |
| WO-2016207647 | Dec 2016 | WO |
| WO-2016207653 | Dec 2016 | WO |
| WO-2016207661 | Dec 2016 | WO |
| WO-2017015075 | Jan 2017 | WO |
| WO-2017025594 | Feb 2017 | WO |
| WO-2017034970 | Mar 2017 | WO |
| WO-2017053905 | Mar 2017 | WO |
| WO-2017075265 | May 2017 | WO |
| WO-2017075294 | May 2017 | WO |
| WO-2017079593 | May 2017 | WO |
| WO-2017096158 | Jun 2017 | WO |
| WO-2017117358 | Jul 2017 | WO |
| WO-2017145476 | Aug 2017 | WO |
| WO-2017151828 | Sep 2017 | WO |
| WO-2017156336 | Sep 2017 | WO |
| WO-2017180420 | Oct 2017 | WO |
| WO-2017197343 | Feb 2018 | WO |
| WO-2018031631 | Feb 2018 | WO |
| WO-2018039338 | Mar 2018 | WO |
| WO-2018039969 | Mar 2018 | WO |
| WO-2018045186 | Mar 2018 | WO |
| WO-2018058073 | Mar 2018 | WO |
| WO-2018103025 | Jun 2018 | WO |
| WO-2018119301 | Jun 2018 | WO |
| WO-2018119447 | Jun 2018 | WO |
| WO-2018125982 | Jul 2018 | WO |
| WO-2018129368 | Jul 2018 | WO |
| WO-2018132635 | Jul 2018 | WO |
| WO-2018119447 | Aug 2018 | WO |
| WO-2018172726 | Sep 2018 | WO |
| WO-2018174827 | Sep 2018 | WO |
| WO-2018191701 | Oct 2018 | WO |
| WO-2018213643 | Nov 2018 | WO |
| WO-2018226546 | Dec 2018 | WO |
| WO-2018236615 | Dec 2018 | WO |
| WO-2019028166 | Feb 2019 | WO |
| WO-2019040637 | Feb 2019 | WO |
| WO-2019071039 | Apr 2019 | WO |
| WO-2019083852 | May 2019 | WO |
| WO-2019084043 | May 2019 | WO |
| WO-2019084165 | May 2019 | WO |
| WO-2019084328 | May 2019 | WO |
| WO-2019099751 | May 2019 | WO |
| WO-2019108851 | Jun 2019 | WO |
| WO-2019113235 | Jun 2019 | WO |
| WO-2019118355 | Jun 2019 | WO |
| WO-2019126789 | Jun 2019 | WO |
| WO-2019134633 | Jul 2019 | WO |
| WO-2019148042 | Aug 2019 | WO |
| WO-2019152108 | Aug 2019 | WO |
| WO-2019157529 | Aug 2019 | WO |
| WO-2019165318 | Aug 2019 | WO |
| WO-2019169028 | Sep 2019 | WO |
| WO-2019169347 | Sep 2019 | WO |
| Entry |
|---|
| Adamson et al., “Production of arrays of chemically distinct nanolitre plugs via repeated splitting in microfluidic devices”, Lab Chip 6(9): 1178-1186 (Sep. 2006). |
| Brenner, et al. In vitro cloning of complex mixtures of DNA on microbeads: physical separation of differentially expressed cDNAs. Proc Natl Acad Sci U S A. Feb. 15, 2000;97(4):1665-70. |
| Chang et al. Droplet-based microfluidic platform platform for heterogeneous enzymatic assays, 2013, Lab Chip, 13, 1817-1822 (Year: 2013). |
| Co-pending U.S. Appl. No. 15/887,711, filed Feb. 2, 2018. |
| Co-pending U.S. Appl. No. 15/887,947, filed Feb. 2, 2018. |
| Fu, et al. A Microfabricated Fluorescence-Activated Cell Sorter. Nature Biotechnology.1999; 17:1109-1111. |
| JPK “Determining the elastic modulus of biological samples using atomic force microscopy” (https://www.jpk.com/ app-technotes-img/AFM/pdf/jpk-app-elastic-modulus.14-1.pdf) 2009, pp. 1-9 (Year: 2009). |
| Kolodeziejczyk et al., “The technology and biology of single-cell RNA sequencing”, Molecular Cell, vol. 58 (May 21, 2015). |
| Lasken, et al. (1996) Archaebacterial DNA Polymerases Tightly Bind Uracil-containing DNA. The Journal of Biological Chemistry, 271(30):17692-17696 (Year: 1996). |
| Lee et al. Alginate: Properties and biomedical applications. Prog Polym Sci 37(1):106-126 (2012). |
| Miller-Stephenson Chemicals 157 FS Series catalog, www.miller-stephenon.com. |
| Morimoto, et al. Monodisperse semi-permeable microcapsules for continuous observation of cells. 2009. Lab Chip 9(15):2217-2223. |
| Narayanan, J. et al. “Determination of agarose gel pore size: Absorbance measurements vis a vis other techniques” Journal of Physics: Conference Series 28 (2006) 83-86 (Year: 2006). |
| Sakaguchi, et al. (1996) Cautionary Note on the Use of dUMP-Containing PCR Primers with Pfu and VentR. Biotechniques, 21(3): 369-370 (Year: 1996). |
| Spormann Laboratory, Polymerase Chain Reaction (PCR), Alfred Spormann Laboratory, 2009, 1-3. (Year: 2009). |
| ThermoFisher, Protocols, M-270 Streptavidin, ThermoFisherScientific, 2007, 1-5. (Year: 2007). |
| Xia and Whitesides, Soft Lithography, Angew. Chem. Int. Ed. 37:550-575 (1998). |
| Zhou, Y. et al. “Development of an enzyme activity screening system for p-glucosidase-displaying yeasts using calcium alginate micro-beads and flow sorting” Appl Microbiol Biotechnol (2009) 84:375-382 (Year: 2009). |
| Abate, et al. Beating Poisson encapsulation statistics using close-packed ordering. Lab Chip. Sep. 21, 2009;9(18):2628-31. doi: 10.1039/b909386a. Epub Jul. 28, 2009. |
| Abate, et al. High-throughput injection with microfluidics using picoinjectors. Proc Natl Acad Sci U S A. Nov. 9, 2010;107(45):19163-6. doi: 10.1073/pNas.1006888107. Epub Oct. 20, 2010. |
| Abate et al., Valve-based flow focusing for drop formation. Appl Phys Lett. 2009;94. 3 pages. |
| Adey, et al. Rapid, low-input, low-bias construction of shotgun fragment libraries by high-density in vitro transposition. Genome Biology 11:R119 (2010). |
| Agresti, et al. Selection of ribozymes that catalyse multiple-turnover Diels-Alder cycloadditions by using in vitro compartmentalization. Proc Natl Acad Sci U S A. Nov. 8, 2005;102(45):16170-5. Epub Oct. 31, 2005. |
| Ahern, “Biochemical, Reagents Kits Offer Scientists Good Return on Investment” The Scientist (1995) 9(15):1-7. |
| Aitman, et al. Copy number polymorphism in Fcgr3 predisposes to glomerulonephritis in rats and humans. Nature. Feb. 16, 2006;439(7078):851-5. |
| Akselband, “Enrichment of slow-growing marine microorganisms from mixed cultures using gel microdrop (GMD) growth assay and fluorescence-activated cell sorting”, J. Exp. Marine Bioi., 329: 196-205 (2006). |
| Akselband, “Rapid mycobacteria drug susceptibility testing using gel microdrop (GMD) growth assay and flow cytometry”, J. Microbiol. Methods, 62:181-197 (2005). |
| Altemos et al., “Genomic Characterization of Large Heterochromatic Gaps in the Human Genome Assembly,” PLOS Computational Biology, May 15, 2014, vol. 10, Issue 5, 14 pages. |
| Amini, S. et al. “Haplotype-resolved whole-genome sequencing by contiguity-preserving transposition and combinatorial indexing” Nature Genetics (2014) 46:1343-1349 doi:10.1038/ng.3119. |
| Anna, S.L., et al., “Formation of dispersions using “flow focusing” in microchannels,” Applied Physics Letters, vol. 82, No. 3, pp. 364-366 (2003). |
| Anonymous, “Oligo(dT)25 cellulose beads” NEB (2012) Retrieved from the Internet:https://www.neb.com/˜/media/Catalog/A11-Products/286CA51268E24DE1B06F1CB288698B54/Datacards%20or%Manuals/S1408Datasheet-Lot0011205.pdf. |
| Anonymous, “Oligotex Handbook” Qiagen (2012) XP055314680, Retrieved from the Internet: URL:http://www.qiagen.com/de/resources/download.apsx?id=f9fald98-d54d-47e7-a20b-8b0cb8975009&lang=en. |
| Anonymous: “Viscosity-Basic concepts” (2004) XP055314117, Retrieved from the Internet: URL:http://lhtc.epfl.ch/webdav/site/lhtc/shared/import/migration/2 VISCOSITY.pdf. |
| Attia, et al. Micro-injection moulding of polymer microfluidic devices. Microfluidics and nanofluidics. 2009; 7(1):1-28. |
| Balikova, et al. Autosomal-dominant microtia linked to five tandem copies of a copy-number-variable region at chromosome 4p16. Am J Hum Genet. Jan. 2008;82(1):181-7. doi: 10.1016/j.ajhg.2007.08.001. |
| Bansal et al. An MCMC algorithm for haplotype assembly from whole-genome sequence data,†(2008) Genome Res 18:1336-1346. |
| Bansal et al. “HapCUT: an efficient and accurate algorithm for the haplotype assembly problem,” Bioinformatics (2008) 24:i153-i159. |
| Baret, et al. Fluorescence-activated droplet sorting (FADS): efficient microfluidic cell sorting based on enzymatic activity. Lab Chip. Jul. 7, 2009;9(13):1850-8. doi: 10.1039/b902504a. Epub Apr. 23, 2009. |
| BD. BD Rhapsody™ Single-Cell Analysis System: Analyze hundreds of genes across tens of thousands of single cells in parallel. BD, Becton, Dickinson and Company. BDGM1012 Rev. 1. 2017. 8 pages. |
| Bedtools: General Usage,†http://bedtools.readthedocs.io/en/latest/content/generalusage.html; Retrieved from the Internet Jul. 8, 2016. |
| Bentley et al. “Accurate whole human genome sequencing using reversible terminator chemistry,” (2008) Nature 456:53-59. |
| Bentzen, et al. Large-scale detection of antigen-specific T cells using peptide-MHC-I multimers labeled with DNA barcodes. Nat Biotechnol. Oct. 2016;34(10):1037-1045. doi: 10.1038/nbt.3662. Epub Aug. 29, 2016. |
| Berkum, et al. Hi-C: a method to study the three-dimensional architecture of genomes. J Vis Exp. May 6, 2010;(39). pii: 1869. doi: 10.3791/1869. |
| Biles et al., Low-fidelity Pyrococcus furiosis DNA polymerase mutants useful in error-prone PCR. Nucl. Acids Res. 32(22):e176 2004. |
| Bodi, K. et al. “Comparison of Commercially Available Target Enrichment Methods for Next-Generation Sequencing” J Biomolecular Techniques (2013) 24:73-86. |
| Boone, et al. Plastic advances microfluidic devices. The devices debuted in silicon and glass, but plastic fabrication may make them hugely successful in biotechnology application. Analytical Chemistry. Feb. 2002; 78A-86A. |
| Boulanger, et al, “Massively parallel haplotyping on microscopic beads for the high-throughput phase analysis of single molecules”, PLoS One, vol. 7:1-10, 2012. |
| Braeckmans et al., Scanning the Code. Modern Drug Discovery. 2003:28-32. |
| Bransky, et al. A microfluidic droplet generator based on a piezoelectric actuator. Lab Chip. Feb. 21, 2009;9(4):516-20. doi: 10.1039/b814810d. Epub Nov. 20, 2008. |
| Bray, “The JavaScript Object Notation (JSON) Data Interchange Format,” Mar. 2014, retrieved from the Internet Feb. 15, 2015; https://tools.ietf.org/html/rfc7159. |
| Briggs, et al. “Tumor-infiltrating immune repertoires captures by single-cell barcoding in emulsion” with Supplementary material. bioRxiv 134841; doi: https://doi.org/10.1101/134841. Posted May 5, 2017. |
| Brouzes, et al. Droplet microfluidic technology for single-cell high-throughput screening. Proc Natl Acad Sci U S A. Aug. 25, 2009;106(34):14195-200. doi: 10.1073/pnas.0903542106. Epub Jul. 15, 2009. |
| Brown, K., Targeted Sequencing Using Droplet-Based Microfluidics, RainDance Technologies, 2009, 1-18. |
| Browning, et al. Haplotype phasing: existing methods and new developments. Nat Rev Genet. Sep. 16, 2011;12(10):703-14. doi: 10.1038/nrg3054. Review. |
| Browning, S.R. et al. “Haplotype Phasing: Existing Methods and New Developments” NaRevGenet (Sep. 16, 2011) 12(10):703-714. |
| Buchman GW, et al. Selective RNA amplification: a novel method using dUMP-containing primers and uracil DNA glycosylase. PCR Methods Appl. Aug. 1993; 3(1):28-31. |
| Buenrostro, et al. ATAC-seq: A Method for Assaying Chromatin Accessibility Genome-Wide. Curr Protoc Mol Biol.; 109: 21.29.1-21.29.9. doi:10.1002/0471142727.mb2129s109. |
| Buenrostro, et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature. Jul. 23, 2015;523(7561):486-90. doi: 10.1038/nature14590. Epub Jun. 17, 2015. |
| Buenrostro, et al. “Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position.” Nat Methods. Dec. 2013;10(12):1213-8. doi: 10.1038/nmeth.2688. Epub Oct. 6, 2013. |
| Burns, et al. An Integrated Nanoliter DNA Analysis Device. Science. Oct. 16, 1998;282(5388):484-7. |
| Burns, et al. Microfabricated structures for integrated DNA analysis. Proc Natl Acad Sci U S A. May 28, 1996; 93(11): 5556-5561. |
| Burns, et al. The intensification of rapid reactions in multiphase systems using slug flow in capillaries. Lab Chip. Sep. 2001;1(1):10-5. Epub Aug. 9, 2001. |
| Cappuzzo, et al. Increased HER2 gene copy number is associated with response to gefitinib therapy in epidermal growth factor receptor-positive non-small-cell lung cancer patients. J Clin Oncol. Aug. 1, 2005;23(22):5007-18. |
| Carroll, “The selection of high-producing cell lines using flow cytometry and cell sorting”, Exp. Op. Bioi. Therp., 4:11 1821-1829 (2004). |
| Caruccio N., Preparation of Next-Generation Sequencing Libraries Using Nextera Technology: Simultaneous DNA Fragmentation and Adaptor Tagging by In Vitro Transposition. Ch. 17 Methods in Microbiology 733:241 (2011). |
| Casbon, et al, “Reflex: intramolecular barcoding of long-range PCR products for sequencing multiple pooled DNAs”, Nucleic Acids Res., pp. 1-6, 2013. |
| Chaudhary “A rapid method of cloning functional variable-region antibody genes in Escherichia coli as single-chain immunotoxins” Proc. Natl. Acad. Sci USA 87: 1066-1070 (Feb. 1990). |
| Chechetkin et al., Sequencing by hybridization with the generic 6-mer oligonucleotide microarray: an advanced scheme for data processing. J Biomol Struct Dyn. Aug. 2000;18(1):83-101. |
| Chen et al. BreakDancer: an algorithm for high-resolution mapping of genomic structural variation,†Nature Methods (2009) 6(9):677-681. |
| Chen, et al. Chemical transfection of cells in picoliter aqueous droplets in fluorocarbon oil. Anal Chem. Nov. 15, 2011;83(22):8816-20. doi: 10.1021/ac2022794. Epub Oct. 17, 2011. |
| Choi et al. “Identification of novel isoforms of the EML4-ALK transforming gene in non— small cell lung cancer,†Cancer Res (2008) 68:4971-4976. |
| Choi, et al. Identification of novel isoforms of the EML4-ALK transforming gene in non-small cell lung cancer. Cancer Res. Jul. 1, 2008;68(13):4971-6. doi: 10.1158/0008-5472.CAN-07-6158. |
| Chokkalingam, et al. Probing cellular heterogeneity in cytokine-secreting immune cells using droplet-based microfluidics. Lab Chip. Dec. 21, 2013;13(24):4740-4. doi: 10.1039/c3lc50945a. |
| Chou, et al. Disposable Microdevices for DNA Analysis and Cell Sorting. Proc. Solid-State Sensor and Actuator Workshop, Hilton Head, SC. Jun. 8-11, 1998; 11-14. |
| Christian, et al. Targeting DNA double-strand breaks with TAL effector nucleases. Genetics. 2010;186:757-761. |
| Christiansen et al. “The Covalent Eukaryotic Topoisomerase I-DNA Intermediate Catalyzes pH-dependent Hydrolysis and Alcoholysis” J Biol Chem (Apr. 14, 1994) 269(15):11367-11373. |
| Chu, et al. Controllable monodisperse multiple emulsions. Angew Chem Int Ed Engl. 2007;46(47):8970-4. |
| Chung, et al. Structural and molecular interrogation of intact biological systems. Nature. May 16, 2013;497(7449):332-7. doi: 10.1038/nature12107. Epub Apr. 10, 2013. |
| Clark, et al. Single-cell epigenomics: powerful new methods for understanding gene regulation and cell identity. Genome Biol. Apr. 18, 2016;17:72. doi: 10.1186/s13059-016-0944-x. |
| Clausell-Tormos et al., “Droplet-based microfluidic platforms for the encapsulation and screening of mammalian cells and multicellular organisms”, Chem. Biol. 15:427-437 (2008). |
| Cleary et al. Joint variant and de novo mutation identification on pedigrees from highthroughput sequencing data,†J Comput Biol (2014) 21:405-419. |
| Cong, et al. Multiplex genome engineering using CRISPR/CAS systems. Science. Feb. 15, 2013;339(6121):819-23. doi: 10.1126/science.1231143. Epub Jan. 3, 2013. |
| Cook, et al. Copy-number variations associated with neuropsychiatric conditions. Nature. Oct. 16, 2008;455(7215):919-23. doi: 10.1038/nature07458. |
| Co-pending U.S. Appl. No. 15/440,772, filed Feb. 23, 2017. |
| Co-pending U.S. Appl. No. 15/449,741, filed Mar. 3, 2017. |
| Co-pending U.S. Appl. No. 15/687,856, filed Aug. 28, 2017. |
| Co-pending U.S. Appl. No. 15/717,840, filed Sep. 27, 2017. |
| Co-pending U.S. Appl. No. 15/717,847, filed Sep. 27, 2017. |
| Co-pending U.S. Appl. No. 15/717,871, filed Sep. 27, 2017. |
| Co-pending U.S. Appl. No. 15/718,764, filed Sep. 28, 2017. |
| Co-pending U.S. Appl. No. 15/718,893, filed Sep. 28, 2017. |
| Co-pending U.S. Appl. No. 15/719,459, filed Sep. 28, 2017. |
| Co-pending U.S. Appl. No. 15/720,085, filed Sep. 29, 2017. |
| Co-pending U.S. Appl. No. 15/825,740, filed Nov. 29, 2017. |
| Co-pending U.S. Appl. No. 15/831,726, filed Dec. 5, 2017. |
| Co-pending U.S. Appl. No. 15/831,847, filed Dec. 5, 2017. |
| Co-pending U.S. Appl. No. 15/832,183, filed Dec. 5, 2017. |
| Co-pending U.S. Appl. No. 15/832,547, filed Dec. 5, 2017. |
| Co-pending U.S. Appl. No. 15/842,550, filed Dec. 14, 2017. |
| Co-pending U.S. Appl. No. 15/842,687, filed Dec. 14, 2017. |
| Co-pending U.S. Appl. No. 15/842,713, filed Dec. 14, 2017. |
| Co-pending U.S. Appl. No. 15/847,659, filed Dec. 19, 2017. |
| Co-pending U.S. Appl. No. 15/847,752, filed Dec. 19, 2017. |
| Co-pending U.S. Appl. No. 15/848,714, filed Dec. 20, 2017. |
| Co-pending U.S. Appl. No. 15/872,499, filed Jan. 16, 2018. |
| Coufal, et al. L1 retrotransposition in human neural progenitor cells. Nature. Aug. 27, 2009;460(7259):1127-31. doi: 10.1038/nature08248. Epub Aug. 5, 2009. |
| Curcio. Improved Techniques for High-Throughput Molecular Diagnostics. PhD Thesis. 2002. |
| Cusanovich; et al., “Multiplex single-cell profiling of chromatin accessibility by combinatorial cellular indexing. Sciencexpress, May 7, 2014, p. 1-9.” |
| Cusanovich, et al. Supplementary materials for Multiplex single-cell profiling of chromatin accessibility by combinatorial cellular indexing. Science. May 22, 2015;348(6237):910-4. doi: 10.1126/science.aab1601. Epub May 7, 2015. |
| Damean, et al. Simultaneous measurement of reactions in microdroplets filled by concentration gradients. Lab Chip. Jun. 21, 2009;9(12):1707-13. doi: 10.1039/b821021g. Epub Mar. 19, 2009. |
| Dangla, et al. Droplet microfluidics driven by gradients of confinement. Proc Natl Acad Sci U S A. Jan. 15, 2013; 110(3): 853-858. Published online Jan. 2, 2013. doi: 10.1073/pnas.1209186110. |
| De Bruin et al., UBS Investment Research. Q-Series®: DNA Sequencing. UBS Securities LLC. Jul. 12, 2007. 15 pages. |
| Dekker, et al. Capturing chromosome conformation. Science. Feb. 15, 2002;295(5558):1306-11. |
| Demirci, et al. Single cell epitaxy by acoustic picolitre droplets. Lab Chip. Sep. 2007;7(9):1139-45. Epub Jul. 10, 2007. |
| Dey, et al. Integrated genome and transcriptome sequencing of the same cell. Dey, Siddharth S. et al. “Integrated Genome and Transcriptome Sequencing from the Same Cell.” Nature biotechnology 33.3 (2015): 285-289. PMC. Web. Dec. 18, 2017. |
| Doerr, “The smallest bioreactor”, Nature Methods, 2:5 326 (2005). |
| Doshi, et al. Red blood cell-mimicking synthetic biomaterial particles. Proceedings of the National Academy of Sciences 106.51 (2009): 21495-21499. |
| Dowding, et al. Oil core/polymer shell microcapsules by internal phase separation from emulsion droplets. II: controlling the release profile of active molecules. Langmuir. Jun. 7, 2005;21(12):5278-84. |
| Draper, et al. Compartmentalization of electrophoretically separated analytes in a multiphase microfluidic platform. Anal Chem. Jul. 3, 2012;84(13):5801-8. doi: 10.1021/ac301141x. Epub Jun. 13, 2012. |
| Dressler, et al. Droplet-based microfluidics enabling impact on drug discovery. J Biomol Screen. Apr. 2014;19(4):483-96. doi: 10.1177/1087057113510401. Epub Nov. 15, 2013. |
| Dressman et al. Supplementary Information pp. 1-2 of article published 2003, PNAS 100(15:8817-22). |
| Dressman et al. Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations. Proc. Natl. Acad. Sci. 2003. 100(15):8817-8822. |
| Drmanac et al., Sequencing by hybridization (SBH): advantages, achievements, and opportunities. Adv Biochem Eng Biotechnol. 2002;77 :75-101. |
| Droplet Based Sequencing (slides) dated (Mar. 12, 2008). |
| Eastburn, et al. Ultrahigh-throughput mammalian single-cell reverse-transcriptase polymerase chain reaction in microfluidic droplets. Anal Chem. Aug. 20, 2013;85(16):8016-21. doi: 10.1021/ac402057q. Epub Aug. 8, 2013. |
| Eid et al. Real-time sequencing form single polymerase molecules,†Science (2009) 323:133-138. |
| Esser-Kahn, et al. Triggered release from polymer capsules. Macromolecules. 2011; 44:5539-5553. |
| Fabi, et al. Correlation of efficacy between EGFR gene copy number and lapatinib/capecitabine therapy in HER2-positive metastatic breast cancer. J. Clin. Oncol. 2010; 28:15S. 2010 ASCO Meeting abstract Jun. 14, 2010:1059. |
| Fan, et al. Noninvasive diagnosis of fetal aneuploidy by shotgun sequencing DNA from maternal blood. Proc Natl Acad Sci U S A. Oct. 21, 2008;105(42)16266-71. doi: 10.1073/pnas.0808319105. Epub Oct. 6, 2008. |
| Fan, et al. Whole-genome molecular haplotyping of single cells. Nature Biotechnology, vol. 29, No. 1. Jan. 1, 2011. pp. 51-57. |
| Fang, et al. Fluoride-cleavable biotinylation phosphoramidite for 5′-end-labeling and affinity purification of synthetic oligonucleotides. Nucleic Acids Res. Jan. 15, 2003;31(2):708-15. |
| Fisher, et al. A scalable, fully automated process for construction of sequence-ready human exome targeted capture libraries. Genome Biol. 2011;12(1):R1. doi: 10.1186/gb-2011-12-1-r1. Epub Jan. 4, 2011. |
| Frampton, G.M. et al. “Development and validation of a clinical cancer genomic profiling test based on massively parallel DNA sequencing” Nature Biotechnology (2013) 31(11):1023-1031. doi:10.1038/nbr.2696. |
| Fredrickson, et al. Macro-to-micro interfaces for microfluidic devices. Lab Chip. Dec. 2004;4(6):526-33. Epub Nov. 10, 2004. |
| Freiberg, et al. Polymer microspheres for controlled drug release. Int J Pharm. Sep. 10, 2004;282(1-2):1-18. |
| Fulton et al., Advanced multiplexed analysis with the FlowMetrix system. Clin Chem. Sep. 1997;43(9): 1749-56. |
| Garstecki, et al. Formation of monodisperse bubbles in a microfluidic flow-focusing device. Applied Physics Letters. 2004; 85(13):2649-2651. DOI: 10.1063/1.1796526. |
| Gartner, et al. The Microfluidic Toolbox—examples for fluidic interfaces and standardization concepts. Proc. SPIE 4982, Microfluidics, BioMEMS, and Medical Microsystems, (Jan. 17, 2003); doi: 10.1117/12.479566. |
| Gericke, et al. Functional cellulose beads: preparation, characterization, and applications. Chemical reviews 113.7 (2013): 4812-4836. |
| Ghadessy, et al. Directed evolution of polymerase function by compartmentalized self-replication. Proc Natl Acad Sci U S A. Apr. 10, 2001;98(8):4552-7. Epub Mar. 27, 2001. |
| Gonzalez, et al. The influence of CCL3L1 gene-containing segmental duplications on HIV-1/AIDS susceptibility. Science. Mar. 4, 2005;307(5714):1434-40. Epub Jan. 6, 2005. |
| Gordon et al. Consed: A Graphical Tool for Sequence Finishing,†Genome Research (1998) 8:198-202. |
| Granieri, Lucia. Droplet-based microfluidics and engineering of tissue plasminogen activator for biomedical applications. Ph.D. Thesis, Nov. 13, 2009 (131 pages). |
| Grasland-Mongrain, et al. Droplet coalescence in microfluidic devices. Jan.-Jul. 2003. 31 pages. http://www.eleves.ens.fr/home/grasland/rapports/stage4.pdf. |
| Guo, et al. Droplet microfluidics for high-throughput biological assays. Lab Chip. Jun. 21, 2012;12(12):2146-55. doi: 10.1039/c2lc21147e. Epub Feb. 9, 2012. |
| Gyarmati, et al. Reversible disulphide formation in polymer networks: a versatile functional group from synthesis to applications. European Polymer Journal. 2013; 49:1268-1286. |
| Hamilton, A.J. “microRNA in erythrocytes” Biochem. Soc. Trans. (2010) 38, 229-231. |
| Han, X. et al. “CRISPR-Cas9 delivery to hard-to-transfect cells via membrane deformation” Science Advances (2015) 1(7): E1500454 (8 pages). |
| Hashimshony, et al. CEL-Seq: Single-Cell RNA-Seq by Multiplexed Linear Amplification. Cell Rep. Sep. 27, 2012;2(3):666-73. doi: 10.1016/j.celrep.2012.08.003. Epub Aug. 30, 2012. |
| He, “Selective Encapsulation of Single Cells and Subcellular Organelles into Picoliter- and Femtoliter-Volume Droplets” Anal. Chem 77: 1539-1544 (2005). |
| He, J. et al. “Genotyping-by-sequencing (GBS), an ultimate marker-assisted selections (MAS) tool to accelerate plant breeding” Frontiers in Plant Sci (Sep. 30, 2014) 5:1-8. |
| Heng et al. “Fast and accurate long-read alignment with Burrows-Wheeler transform,” Bioinformatics (2010) 25(14): 1754-1760. |
| Hiatt, et al. Parallel, tag-directed assembly of locally derived short sequence reads. Nat Methods. Feb. 2010;7(2):119-22. doi: 10.1038/nmeth.1416. Epub Jan. 17, 2010. |
| Hirsch et al. (2002) “Easily reversible desthiobiotin binding to streptavidin, avidin, and other biotin-binding proteins: uses for protein labeling, detection, and isolation.” Analytical of Biochemistry 308(2):343-357. |
| Hjerten, et al. General methods to render macroporous stationary phases nonporous and deformable, exemplified with agarose and silica beads and their use in high-performance ion-exchange and hydrophobic-interaction chromatography of proteins. Chromatographia 31.1-2 (1991): 85-94. |
| Holtze, et al. Biocompatible surfactants for water-in-fluorocarbon emulsions. Lab Chip. Oct. 2008;8(10):1632-9. doi: 10.1039/b806706f. Epub Sep. 2, 2008. |
| Hosokawa, et al. Massively parallel whole genome amplification for single-cell sequencing using droplet microfluidics. Scientific Reports 7, Article No. 5199 (2017). |
| Hosono S, et al. Unbiased whole-genome amplification directly from clinical samples. Genome Res. May 2003; 13(5):954-64. Epub Apr. 14, 2003. |
| Huang et al. EagleView: A genome assembly viewer for next-generationsequencing technologies,†Genome Research (2008) 18:1538-1543. |
| Huebner, “Quantitative detection of protein expression in single cells using droplet microfluidics”, Chem. Commun. 1218-1220 (2007). |
| Hug, et al. Measurement of the number of molecules of a single mRNA species in a complex mRNA preparation. J Theor Biol. Apr. 21, 2003;221(4):615-24. |
| Illumina, Inc. An Introduction to Next-Generation Sequencing Technology. Feb. 28, 2012. |
| Illumina Nextera Enrichment Sample Preparation Guide. Feb. 2013. |
| Illumina TruSeq Custom Enrichment Kit Data Sheet. (c) 2014. |
| Imburgio, et al, “Studies of promoter recognition and start site selection by T7 RNA polymerase using a comprehensive collection of promoter variants”, Biochemistry., 39:10419-30, 2000. |
| Ioannidis, N. Manufacturing of agarose-based chromatographic adsorbents with controlled pore and particle sizes. A thesis submitted to The University of Birmingham for the degree of Doctor of Philosophy. 2009. |
| Jena, et al. Cyclic olefin copolymer based microfluidic devices for biochip applications: Ultraviolet surface grafting using 2-methacryloyloxyethyl phosphorylcholine. Biomicrofluidics. Mar. 2012;6(1):12822-1282212. doi: 10.1063/1.3682098. Epub Mar. 15, 2012. |
| Jung, et al. Micro machining of injection mold inserts for fluidic channel of polymeric biochips. Sensors. 2007; 7(8):1643-1654. |
| Kamperman, et al. Centering Single Cells in Microgels via Delayed Crosslinking Supports Long-Term 3D Culture by Preventing Cell Escape. Small. Jun. 2017;13(22). doi: 10.1002/smll.201603711. Epub Apr. 28, 2017. |
| Kanehisa et al. KEGG: Kyoto Encyclopedia of Genes and Genomes,†Nucleic Acids Research (2000) 28:27-30. |
| Kaper, et al. Supporting Information for “Whole-genome haplotyping by dilution, amplification, and sequencing.” Proc Natl Acad Sci U S A. Apr. 2, 2013;110(14):5552-7. doi: 10.1073/pnas.1218696110. Epub Mar. 18, 2013. |
| Kaper, et al. Whole-genome haplotyping by dilution, amplification, and sequencing. Proc Natl Acad Sci U S A. Apr. 2, 2013;110(14):5552-7. doi: 10.1073/pnas.1218696110. Epub Mar. 18, 2013. |
| Karmakar, et al. Organocatalytic removal of formaldehyde adducts from RNA and DNA bases. Nat Chem. Sep. 2015;7(9):752-8. doi: 10.1038/nchem.2307. Epub Aug. 3, 2015. |
| Katsura, et al. Indirect micromanipulation of single molecules in water-in-oil emulsion. Electrophoresis. Jan. 2001;22(2):289-93. |
| Kebschull, et al. High-Throughput Mapping of Single-Neuron Projections by Sequencing of Barcoded RNA. Neuron. Sep. 7, 2016;91(5):975-87. doi: 10.1016/j.neuron.2016.07.036. Epub Aug. 18, 2016. |
| Kenis, et al. Microfabrication Inside Capillaries Using Multiphase Laminar Flow Patterning. Science. Jul. 2, 1999;285(5424):83-5. |
| Khomiakova et al., Analysis of perfect and mismatched DNA duplexes by a generic hexanucleotide microchip. Mol Biol(Mosk). Jul.-Aug. 2003;37(4):726-41. Russian. Abstract only. |
| Kim et al., Albumin loaded microsphere of amphiphilic poly( ethylene glycol)/poly(a-ester) multiblock copolymer. Eu. J. Pharm. Sci. 2004;23:245-51. Available online Sep. 27, 2004. |
| Kim, et al. Fabrication of monodisperse gel shells and functional microgels in microfluidic devices. Angew Chem Int Ed Engl. 2007;46(11):1819-22. |
| Kim et al. “HapEdit: an accuracy assessment viewer for haplotype assembly using massively parallel DNA-sequencing technologies,” Nucleic Acids Research (2011) pp. 1-5. |
| Kim, et al. Rapid prototyping of microfluidic systems using a PDMS/polymer tape composite. Lab Chip. May 7, 2009;9(9):1290-3. doi: 10.1039/b818389a. Epub Feb. 10, 2009. |
| Kirkness et al. Sequencing of isolated sperm cells for direct haplotyping of a human genome,†Genome Res (2013) 23:826-832. |
| Kirkness et al. “Sequencing of isolated sperm cells for direct haplotyping of a human genome,” Genome Res (2013) 23:826-832. |
| Kitzman et al. “Haplotype-resolved genome sequencing of a Gujarati Indian individual.” Nat Biotechnol (2011) 29:59-63. |
| Kitzman, et al. Noninvasive whole-genome sequencing of a human fetus. Sci Transl Med. Jun. 6, 2012;4(137):137ra76. doi: 10.1126/scitranslmed.3004323. |
| Kivioj, et al., “Counting Absolute Numbers of Molecules Using Unique Molecular Identifiers”, Nature Methods 9, 72-74 (2012). |
| Klein, et al. Droplet barcoding for single-cell transcriptomics applied to embryonic stem cells. Cell. May 21, 2015;161(5):1187-201. doi: 10.1016/j.cell.2015.04.044. |
| Knight, et al. Subtle chromosomal rearrangements in children with unexplained mental retardation. Lancet. Nov. 13, 1999;354(9191):1676-81. |
| Kobayashi, et al. Effect of slot aspect ratio on droplet formation from silicon straight-through microchannels. J Colloid Interface Sci. Nov. 1, 2004;279(1):277-80. |
| Korlach et al., Methods in Enzymology, Real-Time DNA Sequencing from Single Polymerase Molecules, (2010) 472:431-455. |
| Koster et al., “Drop-based microfluidic devices for encapsulation of single cells”, Lab on a Chip The Royal Soc. of Chern. 8: 1110-1115 (2008). |
| Kozarewa, et al, “96-plex molecular barcoding for the Illumina Genome Analyzer”, Methods Mol Biol., 733:279-98, 2011. |
| Kozarewa, et al. “Amplification-free Illumina sequencing-library preparation facilitates improved mapping and assembly of GC-biased genomes”, Nat Methods., 6: 291-5, 2009. |
| Kutyavin, et al. Oligonucleotides containing 2-aminoadenine and 2-thiothymine act as selectively binding complementary agents. Biochemistry. Aug. 27, 1996;35(34):11170-6. |
| Kwok, et al, “Single-molecule analysis for molecular haplotyping”, Hum Mutat., 23:442-6, 2004. |
| Lagally, et al. Single-Molecular DNA Amplification and Analysis in an Integrated Microfluidic Device. Anal Chem. Feb. 1, 2001;73(3):565-70. |
| Lagus, et al. A review of the theory, methods and recent applications of high-throughput single-cell droplet microfluidics. J. Phys. D: Appl. Phys. (2013) 46:114005. (21 pages). |
| Laird et al, Hairpin-bisulfite PCR: Assessing epigenetic methylation patterns on complementary strands of individual DNA molecules, 2004, PNAS, 101, 204-209. |
| Lake, et al. “Integrative Single-Cell Analysis by Transcriptional and Epigenetic States in Human Adult Brain”. Apr. 19, 2017. doi: https://doi.org/10.1101/128520. |
| Lan, et al. “Single-cell genome sequencing at ultra-high-throughput with microfluidic droplet barcoding” with Supplementary Material. Nat Biotechnol. May 29, 2017. doi: 10.1038/nbt.3880. [Epub ahead of print]. |
| Layer et al. LUMPY: A probabilistic framework for structural variant discovery,†Genome Biology (2014) 15(6):R84. |
| Lee, et al. ACT-PRESTO: Rapid and consistent tissue clearing and labeling method for 3-dimensional (3D) imaging. Sci Rep. Jan. 11, 2016;6:18631. doi: 10.1038/srep18631. |
| Lee, et al., “Highly multiplexed subcellular RNA sequencing in situ. Science. Mar. 21, 2014;343(6177):1360-3. doi: 10.1126/science.1250212. Epub Feb. 27, 2014.” |
| Lee, J-H. et al. “Fluorescent in situ sequencing (FISSEQ) of RNA for gene expression profiling in intact cells and tissues” Nature Protocols (Feb. 12, 2015) 10(3):442-458. |
| Lennon; et al., “Lennon et al. A scalable, fully automated process for construction of sequence-ready barcoded libraries for 454. Genome Biology 11:R15 (2010).” |
| Li, et al. A single-cell-based platform for copy number variation profiling through digital counting of amplified genomic DNA fragments. ACS Appl Mater Interfaces. Mar. 24, 2017. doi: 10.1021/acsami.7b03146. [Epub ahead of print]. |
| Li, et al. Step-emulsification in a microfluidic device. Lab Chip. Feb. 21, 2015;15(4):1023-31. doi: 10.1039/c4lc01289e. |
| Li, Y., et al., “PEGylated PLGA Nanoparticles as protein carriers: synthesis, preparation and biodistribution in rats,” Journal of Controlled Release, vol. 71, pp. 203-211 (2001). |
| Lienemann, et al. Single cell-laden protease-sensitive microniches for long-term culture in 3D. Lab Chip. Feb. 14, 2017;17(4):727-737. doi: 10.1039/c61c01444e. |
| Linch, et al. Bone marrow processing and cryopreservation. Journal of Clinical Pathology; Feb. 1982, vol. 35, No. 2; pp. 186-190. |
| Lippert et al. Algorithmic strategies for the single nucleotide polymorphism haplotype assembly problem,†Brief. Bionform (2002) 3:23-31. |
| Liu, et al. Preparation of uniform-sized PLA microcapsules by combining Shirasu porous glass membrane emulsification technique and multiple emulsion-solvent evaporation method. J Control Release. Mar. 2, 2005;103(1):31-43. Epub Dec. 21, 2004. |
| Liu, et al. Smart thermo-triggered squirting capsules for Nanoparticle delivery. Soft Matter. 2010; 6(16):3759-3763. |
| Lo, et al. On the design of clone-based haplotyping. Genome Biol. 2013;14(9):R100. |
| Loscertales, I.G., et al., “Micro/Nano Encapsulation via Electrified Coaxial Liquid Jets,” Science, vol. 295, pp. 1695-1698 (2002). |
| Love, “A microengraving method for rapid selection of single cells producing antigen-specific antibodies”, Nature Biotech, 24:6 703 (Jun. 2006). |
| Lowe, Adam J. Norbornenes and [n]polynorbornanes as molecular scaffolds for anion recognition. Ph.D. Thesis (May 2010). (361 pages). |
| Lundin, et al, “Hierarchical molecular tagging to resolve long continuous sequences by massively parallel sequencing”, Sci Rep., 3:1186, 2003. |
| Lupski. Genomic rearrangements and sporadic disease. Nat Genet. Jul. 2007;39(7 Suppl):S43-7. |
| Maan, et al. Spontaneous droplet formation techniques for monodisperse emulsions preparation—Perspectives for food applications. Journal of Food Engineering. vol. 107, Issues 3-4, Dec. 2011, pp. 334-346. |
| MacAulay; et al., “G&T-seq: parallel sequencing of single-cell genomes and transcriptomes. Nature Methods, 2015, p. 1-7.” |
| MacAulay, et al. Single-Cell Multiomics: Multiple Measurements from Single Cells. Trends in Genetics 33.2 (2017): 155-168. PMC. Web. Dec. 18, 2017. |
| Macosko, et al. Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets. Cell. May 21, 2015;161(5):1202-14. doi: 10.1016/j.cell.2015.05.002. |
| Mair, et al. Injection molded microfluidic chips featuring integrated interconnects. Lab Chip. Oct. 2006;6(10):1346-54. Epub Jul. 31, 2006. |
| Makino, et al. Preparation of hydrogel microcapsules: Effects of preparation conditions upon membrane properties. Colloids and Surfaces B: Biointerfaces. Nov. 1998; 12(2), 97-104. |
| Man. Monolithic Structures for Integrated Microfluidic Analysis. PhD Thesis. 2001. |
| Marcus. Gene method offers diagnostic hope. The Wall Street Journal. Jul. 11, 2012. |
| Margulies et al. Genome sequencing in microfabricated high-density picoliter reactors,†Nature (2005) 437:376-380. |
| Maricic T, et al. Optimization of 454 sequencing library preparation from small amounts of DNA permits sequence determination of both DNA strands. Biotechniques. Jan. 2009; 46(1):51-2, 54-7. |
| Matochko, et al. Uniform amplification of phage display libraries in monodisperse emulsions. Methods. Sep. 2012;58(1):18-27. doi: 10.1016/j.ymeth.2012.07.012. Epub Jul. 20, 2012. |
| Mazutis, et al. Selective droplet coalescence using microfluidic systems. Lab Chip. Apr. 24, 2012;12(10):1800-6. doi: 10.1039/c2lc40121e. Epub Mar. 27, 2012. |
| McKenna, Aaron et al. “The Genome Analysis Toolkit: A MapReduce Framework for Analyzing next-Generation DNA Sequencing Data.” Genome Research 20.9 (2010): 1297-1303. PMC. Web. Feb. 2, 2017. |
| Merriman, et al. Progress in ion torrent semiconductor chip based sequencing. Electrophoresis. Dec. 2012;33(23):3397-417. doi: 10.1002/elps.201200424. |
| Microfluidic ChipShop. Microfluidic product catalogue. Mar. 2005. |
| Microfluidic ChipShop. Microfluidic product catalogue. Oct. 2009. |
| Miller et al. “Assembly Algorithms for next-generation sequencing data,” Genomics, 95 (2010), pp. 315-327. |
| Miller JC, et al. An improved zinc-finger nuclease architecture for highly specific genome editing. Nat. Biotechnol. 2007;25:778-785. |
| MiRNA (http://www.exiqon.com/what-are-microRNAs) accessed Oct. 19, 2017. |
| Mirzabekov, “DNA Sequencing by Hybridization—a Megasequencing Method and a Diagnostic Tool?” Trends in Biotechnology 12(1): 27-32 (1994). |
| Moore, et al. Behavior of capillary valves in centrifugal microfluidic devices prepared by three-dimensional printing. Microfluidics and Nanofluidics. 2011; 10(4):877-888. |
| Morgan, et al. Chapter 12: Human microbiome analysis. PLoS Comput Biol. 2012;8(12):e1002808. doi: 10.1371/journal.pcbi.1002808. Epub Dec. 27, 2012. |
| Mouritzen et al., Single nucleotide polymorphism genotyping using locked nucleic acid (LNa). Expert Rev Mol Diagn. Jan. 2003;3(1):27-38. |
| Mozhanova, A.A. et al. “Local elastic properties of biological materials studied by SFM” (2003) XP055314108, Retrieved from the Internet: URL:http://www.ntmdt.com/data/media/files/publications/2003/08.08_a.a.mozhanova_n.i.n_english.pdf. |
| Muotri, et al. L1 retrotransposition in neurons is modulated by MeCP2. Nature. Nov. 18, 2010;468(7322):443-6. doi: 10.1038/nature09544. |
| Myllykangas et al. “Efficient targeted resequencing of human germline and cancer genomes by oligonucleotide-selective sequencing,” Nat Biotechnol, (2011) 29:1024-1027. |
| Myllykangas et al., Targeted Sequencing Library Preparation by Genomic DNA Circularization, BMC Biotechnology, 2011, 11(122), 1-12. |
| Nagano, et al. Single-cell Hi-C reveals cell-to-cell variability in chromosome structure. Nature. Oct. 3, 2013;502(7469):59-64. doi: 10.1038/nature12593. Epub Sep. 25, 2013. |
| Nagashima, et al. Preparation of monodisperse poly (acrylamide-co-acrylic acid) hydrogel microspheres by a membrane emulsification technique and their size-dependent surface properties. Colloids and Surfaces B: Biointerfaces. Jun. 15, 1998; 11(1-2), 47-56. |
| Navin. The first five years of single-cell cancer genomics and beyond. Genome Res. Oct. 2015;25(10):1499-507. doi: 10.1101/gr.191098.115. |
| Nguyen, et al. In situ hybridization to chromosomes stabilized in gel microdrops. Cytometry. 1995; 21:111-119. |
| Nisisako, et al. Droplet formation in a microchannel network. Lab Chip. Feb. 2002;2(1):24-6. Epub Jan. 18, 2002. |
| Nisisako, T. et al. “Droplet Formation in a Microchannel on PMMA Plate” Abstract. 2001 Kluwer Academic Publishers. p. 137-138. |
| Novak, et al. Single cell multiplex gene detection and sequencing using microfluidicallygenerated agarose emulsions. Angew Chem Int Ed Engl. Jan. 10, 2011;50(2):390-5. doi: 10.1002/anie.201006089. |
| Oberholzer, et al. Polymerase chain reaction in liposomes. Chem Biol. Oct. 1995;2(10):677-82. |
| Ogawa, et al. Production and characterization of O/W emulsions containing cationic droplets stabilized by lecithin-chitosan membranes. J Agric Food Chem. Apr. 23, 2003;51(9):2806-12. |
| Okushima, S., et al,. “Controlled Production ofMonodisperse Double Emulsions by Two-Step Droplet Breakup in Microfluidic Devices,” Langmuir, vol. 20, pp. 9905-9908 (2004). |
| Oligotex Handbook. For purification of poly A+ RNA from total RNA and directly from cultured cells or tissues as well as purification of polyadenylated in vitro transcripts. Jun. 2012. |
| Orakdogen, N. “Novel responsive poly(N,N-dimethylaminoethyl methacrylate) gel beads: preparation, mechanical properties and pH-dependent swelling behavior” J Polym Res (2012) 19:9914. |
| Oyola, et al, “Optimizing Illumina next-generation sequencing library preparation for extremely AT-biased genomes”, BMC Genomics.,13:1, 2012. |
| Pantel, et al. Detection methods of circulating tumor cells. J Thorac Dis. Oct. 2012;4(5):446-7. doi: 10.3978/j.issn.2072-1439.2012.08.15. |
| Patel, et al. Single-cell RNA-seq highlights intratumoral heterogeneity in primary glioblastoma. Science. Jun. 20, 2014;344(6190):1396-401. doi: 10.1126/science.1254257. Epub Jun. 12, 2014. |
| Perez, C., et al., “Poly(lactic acid)-poly(ethylene glycol) Nanoparticles as new carriers for the delivery of plasmid DNA,” Journal of Controlled Release, vol. 75, pp. 211-224 (2001). |
| Perrott, Jimmy. Optimization and Improvement of Emulsion PCR for the Ion Torrent Next-Generation Sequencing Platform. (2011) Thesis. |
| Peters, et al. Accurate whole-genome sequencing and haplotyping from 10 to 20 human cells. Nature. Jul. 11, 2012;487(7406):190-5. doi: 10.1038/Nature11236. |
| Picot, J. et al. “A biomimetic microfluidic chip to study the circulation and mechanical retention of red blood cells in the spleen” Am J Hematology (Jan. 12, 2015) 90(4):339-345. |
| Pinto, et al. Functional impact of global rare copy number variation in autism spectrum disorders. Nature. Jul. 15, 2010;466(7304):368-72. doi: 10.1038/nature09146. Epub Jun. 9, 2010. |
| Plunkett, et al. Chymotrypsin responsive hydrogel: application of a disulfide exchange protocol for the preparation of methacrylamide containing peptides. Biomacromolecules. Mar.-Apr. 2005;6(2):632-7. |
| “Portable Water Filters” (http://www.portablewaterfilters.org/water-filter-guide/particle-contaminant-size-chart-microns/) 2015, accessed Oct. 19, 2017. |
| Porteus MH, Baltimore D. Chimeric nucleases stimulate gene targeting in human cells. Science. 2003;300:763. |
| Pott, et al. Single-cell ATAC-seq: strength in numbers. Genome Biol. Aug. 21, 2015;16:172. doi: 10.1186/s13059-015-0737-7. |
| Preissl, et al. Single nucleus analysis of the chromatin landscape in mouse forebrain development. Posted Jul. 4, 2017. bioRxiv 159137; doi: https://doi.org/10.1101/159137. |
| Pushkarev et al. Single-molecule sequencing of an individual human genome,†Nature Biotech (2009) 17:847-850. |
| Rakszewska, A. et al. “One drop at a time: toward droplet microfluidics as a versatile tool for single-cell analysis” NPG Asia Materials (2014) 6(10):e133 (12 pages). |
| Ram, et al. Strategy for microbiome analysis using 16S rRNA gene sequence analysis on the Illumina sequencing platform. Syst Biol Reprod Med. Jun. 2011;57(3):162-70. doi: 10.3109/19396368.2011.555598. Epub Mar. 1, 2011. |
| Ramsey, J.M. “The burgeoning power of the shrinking laboratory” Nature Biotech (1999) 17:1061-1062. |
| Ramskold et al. (2012) “Full-length mRNA-Seq from single-cell levels of RNA and individual circulating tumor cells” Nature Biotechnology 30(8):777-782. |
| Ran et al. Genome engineering using the CRISPR-Cas9 system. Nature Protocols 8:2281-2308 (2013). |
| Reis, A. et al. “CRISPR/Cas9 and Targeted Genome Editing: A New Era in Molecular Biology” (2014) XP002766825: URL:https://ww.neb.com/tools-and-resources/feabture-articles/crispr-cas9-and-targeted-genome-editing-a-new-era-in-molecular-biology. |
| Reisner, et al, “Single-molecule denaturation mapping of DNA in nanofluidic channels”, Proc Natl Acad Sci U.S.A., 107: 13294-9, 2010. |
| Repp et al. “Genotyping by Multiplex Polymerase Chain Reaction for Detection of Endemic Hepatitis B Virus Transmission” J Clinical Microbiology (1993) 31:1095-1102. |
| Richardson, et al. Novel inhibition of archaeal family-D DNA polymerase by uracil. Nucleic acids research 41.7 (2013): 4207-4218. |
| Roche. Using Multiplex Identifier (MID) Adaptors for the GS FLX Titanium Chemistry Basic MID Set Genome Sequencer FLX System, Technical Bulletin 004-2009, (Apr. 1, 2009) pp. 1-7. URL:http://454.com/downloads/my454/documentation/technical-bulletins/TCB-09004 UsingMultiplexIdentifierAdaptorsForTheGSFLXTitaniumSeriesChemistry-BasicMIDSet.pdf. |
| Roche. Using Multiplex Identifier (MID) Adaptors for the GS FLX Titanium Chemistry Extended MID Set Genome Sequencer FLX System, Technical Bulletin 005-2009, (Apr. 1, 2009) pp. 1-7. URL:http://454.com/downloads/my454/documentation/technical-bulletins/TCB-09005UsingMultiplexIdentifierAdaptorsForTheGSFLXTitaniumChemistry-ExtendedMIDSet.pdf. |
| Rodrigue, S. et al. “Whole genome amplification and de novo assembly of single bacterial cells” PLoS One. Sep. 2, 2009;4(9):e6864. doi: 10.1371/journal.pone.0006864. |
| Rogozin, et al. A highly conserved family of inactivated archaeal B family DNA polymerases. Biol Direct. Aug. 6, 2008;3:32. doi: 10.1186/1745-6150-3-32. |
| Ropers. New perspectives for the elucidation of genetic disorders. Am J Hum Genet. Aug. 2007;81(2):199-207. Epub Jun. 29, 2007. |
| Rotem, et al. High-Throughput Single-Cell Labeling (Hi-SCL) for RNA-Seq Using Drop-Based Microfluidics. PLoS One. May 22, 2015;10(5):e0116328. doi: 10.1371/journal.pone.0116328.eCollection 2015. |
| Rotem, et al. Single Cell Chip-Seq Using Drop-Based Microfluidics. Abstract #50. Frontiers of Single Cell Analysis, Stanford University Sep. 5-7, 2013. |
| Rotem, et al. Single-cell ChIP-seq reveals cell subpopulations defined by chromatin state. Nat Biotechnol. Nov. 2015;33(11):1165-72. doi: 10.1038/nbt.3383. Epub Oct. 12, 2015. |
| Ryan, “Rapid assay for mycobacterial growth and antibiotic susceptibility using gel microdrop and encapsulation”, J. Clinical Microbial., 33:7 1720-1726 (1995). |
| Sahin, et al. Microfluidic EDGE emulsification: the importance of interface interactions on droplet formation and pressure stability. Sci Rep. May 27, 2016;6:26407. doi: 10.1038/srep26407. |
| Sander JD, et al. Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA). Nat. Methods. 2011;8:67-69. |
| Schirinzi et al., Combinatorial sequencing-by-hybridization: Analysis of the NF1 gene. Genet Test. 2006 Spring;10(1):8-17. |
| Schmeider, et al. Fast identification and removal of sequence contamination from genomic and metagenomic datasets. PLoS One. Mar. 9, 2011;6(3):e17288. doi: 10.1371/journal.pone.0017288. |
| Schmitt, “Bead-based multiplex genotyping of human papillomaviruses”, J. Clinical Microbial., 44:2 504-512 (2006). |
| Schubert, et al. Microemulsifying fluorinated oils with mixtures of fluorinated and hydrogenated surfactants. Colloids and Surfaces A; Physicochemical and Engineering Aspects, 84(1994) 97-106. |
| Schwartz, et al., “Capturing native long-range contiguity by in situ library construction and optical sequencing”, PNAS (Nov. 2012), 109(46)18749-18754. |
| Sebat, et al. Strong association of de novo copy number mutations with autism. Science. Apr. 20, 2007;316(5823):445-9. Epub Mar. 15, 2007. |
| Seiffert, et al. Smart microgel capsules from macromolecular precursors. J Am Chem Soc. May 12, 2010;132(18):6606-9. doi: 10.1021/ja102156h. |
| Shah, “Fabrication of mono disperse thermosensitive microgels and gel capsules in micro fluidic devices”, Soft Matter, 4:2303-2309 (2008). |
| Shahi, et al. Abseq: Ultrahigh-throughput single cell protein profiling with droplet microfluidic barcoding. Sci Rep. 2017; 7: 44447. Published online Mar. 14, 2017. doi: 10.1038/srep44447. |
| Shendure et al. Accurate Multiplex Polony Sequencing of an Evolved bacterial Genome. Science (2005) 309:1728-1732. |
| Shimkus, et al. A chemically cleavable biotinylated nucleotide: usefulness in the recovery of protein-DNA complexes from avidin affinity columns. Proc Natl Acad Sci U S A. May 1985;82(9):2593-7. |
| Shlien, et al. Copy number variations and cancer. Genome Med. Jun. 16, 2009;1(6):62. doi: 10.1186/gm62. |
| Shlien, et al. Excessive genomic DNA copy number variation in the Li-Fraumeni cancer predisposition syndrome. Proc Natl Acad Sci U S A. Aug. 12, 2008;105(32):11264-9. doi: 10.1073/pnas.0802970105. Epub Aug. 6, 2008. |
| Shuttleworth, et al. Recognition of the pro-mutagenic base uracil by family B DNA polymerases from archaea. J Mol Biol. Mar. 26, 2004;337(3):621-34. |
| Sigma. Streptavidin-agarose (S1638) product information sheet. www.sigma-aldrich.com. |
| Simeonov et al., Single nucleotide polymorphism genotyping using short, fluorescently labeled locked nucleic acid (LNA) probes and fluorescence polarization detection. Nucleic Acids Res. Sep. 1, 2002;30(17):e91. |
| Skerra A. Phosphorothioate primers improve the amplification of DNA sequences by DNA polymerases with proofreading activity. Nucleic Acids Res. Jul. 25, 1992; 20(14):3551-4. |
| Song, et al. Reactions in droplets in microfluidic channels. Angew Chem Int Ed Engl. Nov. 13, 2006;45(44):7336-56. |
| Sorokin et al., Discrimination between perfect and mismatched duplexes with oligonucleotide gel microchips: role of thermodynamic and kinetic effects during hybridization. J Biomol Struct Dyn. Jun. 2005;22(6):725-34. |
| SSH Tunnel—Local and Remote Port Forwarding Explained With Examples,†Trackets Blog, http://blog.trackets.com/2014/05/17/ssh-tunnel-local-and-remote-port-forwarding-explained with-examples.html; Retrieved from the Internet Jul. 7, 2016. |
| Stoeckius, et al. Large-scale simultaneous measurement of epitopes and transcriptomes in single cells. bioRxiv 113068; doi: https://doi.org/10.1101/113068. |
| Stoeckius, et al. Simultaneous epitope and transcriptome measurement in single cells. Nature methods. Jul. 31, 2017. Supplemental Materials. |
| Su, et al., Microfluidics-Based Biochips: Technology Issues, Implementation Platforms, and Design-Automation Challenges. IEEE Transactions on Computer-Aided Design of Integrated Circuits and Systems. 2006;25(2):211-23. (Feb. 2006). |
| Sun et al., Progress in research and application of liquid-phase chip technology. Chinese Journal Experimental Surgery. May 2005;22(5):639-40. |
| Susaki, et al. Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis. Cell. Apr. 24, 2014;157(3):726-39. doi: 10.1016/j.ce11.2014.03.042. Epub Apr. 17, 2014. |
| Syed, et al. Nature Methods 2 pgs (Nov. 2009). |
| Tawfik, D.S., et al., “Man-made cell-like compartments for molecular evolution,” Nature Biotechnology, vol. 16, pp. 652-656 (1998). |
| Tayyab, S. et al. “Size exclusion chromatography and size exclusion HPLC of proteins” Biochem Ed, Pergamon, (1991) 19(3):149-152. |
| Tewhey, et al. Microdroplet-based PCR amplification for large-scale targeted sequencing. Nat Biotechnol. Nov. 2009;27(11):1025-31. doi: 10.1038/nbt.1583. Epub Nov. 1, 2009. |
| Tewhey et al., Supplementary Materials, Nature Biotechnology, 2009, 27(11), 1-22. |
| Tewhey et al. The importance of phase information for human genomics,†Nat Rev Genet (2011) 12:215-223. |
| The SAM/BAM Format Specificatio Working Group, “Sequence Allignment/ Map Format Specification,” Dec. 28, 2014. |
| Theberge, et al. Microdroplets in microfluidics: an evolving platform for discoveries in chemistry and biology. Angew Chem Int Ed Engl. Aug. 9, 2010;49(34):5846-68. doi: 10.1002/anie.200906653. |
| Thorsen, et al. Dynamic pattern formation in a vesicle-generating microfluidic device. Physical Review Letters. American Physical Society. 2001; 86(18):4163-4166. |
| Tomer, et al. Advanced CLARITY for rapid and high-resolution imaging of intact tissues. Nat Protoc. Jul. 2014;9(7):1682-97. doi: 10.1038/nprot.2014.123. Epub Jun. 19, 2014. |
| Tonelli, et al. Perfluoropolyether functional oligomers: unusual reactivity in organic chemistry. Journal of fluorine chemistry. 2002; 118(1)107-121. |
| Tubeleviciute, et al. Compartmentalized self-replication (CSR) selection of Thermococcus litoralis Sh1B DNa polymerase for diminished uracil binding. Protein Eng Des Sel. Aug. 2010;23(8):589-97. doi: 10.1093/protein/gzq032. Epub May 31, 2010. |
| Turner, et al. Assaying chromosomal inversions by single-molecule haplotyping. Nat Methods. Jun. 2006;3(6):439-45. |
| Turner, et al, “High-throughput haplotype determination over long distances by haplotype fusion PCR and ligation haplotyping”, Nat Protoc., 4:1771-83, 2009. |
| Turner, et al. Methods for genomic partitioning. Annu Rev Genomics Hum Genet. 2009;10:263-84. doi: 10.1146/annurev-genom-082908-150112. Review. |
| Umbanhowar, P.B., et al., “Monodisperse Emulsion Generation via Drop Break Off in a Coflowing Stream,” Langmuir, vol. 16, pp. 347-351 (2000). |
| Ushijima et al, Detection and interpretation of altered methylation patterns in cancer cells, 2005, Nature reviews, 5, 223-231. |
| Van Dijke, et al. Effect of viscosities of dispersed and continuous phases in microchannel oil-in-water emulsification. Microfluid Nanofluid (2010) 9: 77. https://doi.org/10.1007/s10404-009-0521-7. |
| Van Nieuwerburgh, et al, “Illumina mate-paired DNA sequencing-library preparation using Cre-Lox recombination”, Nucleic Acids Res., 40:1-8, 2012. |
| Wagner, et al. Biocompatible fluorinated polyglycerols for droplet microfluidics as an alternative to PEG-based copolymer surfactants. Lab Chip. Jan. 7, 2016;16(1):65-9. doi: 10.1039/c51c00823a. Epub Dec. 2, 2015. |
| Wang, et al. A novel thermo-induced self-bursting microcapsule with magnetic-targeting property. Chemphyschem. Oct. 5, 2009;10(14):2405-9. |
| Wang, et al. Digital karyotyping. Proc Natl Acad Sci U S A. Dec. 10, 2002;99(25):16156-61. Epub Dec. 2, 2002. |
| Wang et al., “Self-Formed Adaptor PCR: a Simple and Efficient Method for Chromosome Walking”, Applied and Environmental Microbiology (Aug. 2007), 73(15):5048-5051. |
| Wang et al., Single nucleotide polymorphism discrimination assisted by improved base stacking hybridization using oligonucleotide microarrays. Biotechniques. 2003;35:300-08. |
| Ward, et al. Microfluidic flow focusing: Drop size and scaling in pressure versus flow-rate-driven pumping. Electrophoresis. Oct. 2005;26(19):3716-24. |
| Weaver, “Rapid clonal growth measurements at the single-cell level: gel microdroplets and flow cytometry”, Biotechnology, 9:873-877 (1991). |
| Weigl, et al. Microfluidic Diffusion-Based Separation and Detection. Science. 1999; pp. 346-347. |
| Wesolowska, et al. Cost-effective multiplexing before capture allows screening of 25 000 clinically relevant SNPs in childhood acute lymphoblastic leukemia. Leukemia. Jun. 2011;25(6):1001-6. doi: 10.1038/leu.2011.32. Epub Mar. 18, 2011. |
| Wheeler et al., “Database resources of the National Center for Biotechnology Information,” Nucleic Acids Res. (2007) 35 (Database issue): D5-12. |
| Whitesides, “Soft lithography in biology and biochemistry”, Annual Review of Biomedical Engineering, 3:335-373 (2001). |
| Williams et al., Amplification of complex gene libraries by emulsion PCR, Nature Methods 3(7):545-550 (2006). |
| Wiseman, R.W. et al. “Major histocompatibility complex genotyping with massively parallel pyrosequencing” Nature Medicine (Oct. 11, 2009) 15(11):1322-1326. |
| Woo, et al. G/C-modified oligodeoxynucleotides with selective complementarity: synthesis and hybridization properties. Nucleic Acids Res. Jul. 1, 1996;24(13):2470-5. |
| Wood AJ, et al. Targeted genome editing across species using ZFNs and TALENs. Science. 2011;333:307. |
| Xi, et al. New library construction method for single-cell genomes. PLoS One. Jul. 19, 2017;12(7):e0181163. doi: 10.1371/journal.pone.0181163. eCollection 2017. |
| Xia and Whitesides, Soft Lithography, Ann. Rev. Mat. Sci. 28:153-184 (1998). |
| Xiao, et al, “Determination of haplotypes from single DNA molecules: a method for single-molecule barcoding”, Hum Mutat., 28:913-21, 2007. |
| Yamamoto, et al. Chemical modification of Ce(IV)/EDTA-base artificial restriction DNA cutter for versatile manipulation of double-stranded DNA. Nucleic Acids Research. 2007; 35(7):e53. |
| Yan, Pu et al. “Rapid one-step construction of hairpin RNA” Biochem and Biophys Res Comm (Jun. 12, 2009) 383(4):464-468. |
| Zeng, et al. High-performance single cell genetic analysis using microfluidic emulsion generator arrays. Anal Chem. Apr. 15, 2010;82(8):3183-90. doi: 10.1021/ac902683t. |
| Zerbino, Daniel, “Velvet Manual—version 1.1,” Aug. 15, 2008, pp. 1-22. |
| Zerbino et al. “Velvet: Algorithms for de novo short read assembly using de Bruijn graphs,” Genome Research (2008) 18:821-829. |
| Zhang, “Combinatorial marking of cells and organelles with reconstituted fluorescent proteins”, Cell, 119:137-144 (Oct. 1, 2004). |
| Zhang, et al. Degradable disulfide core-cross-linked micelles as a drug delivery system prepared from vinyl functionalized nucleosides via the RAFT process. Biomacromolecules. Nov. 2008;9(11):3321-31. doi: 10.1021/bm800867n. Epub Oct. 9, 2008. |
| Zhang F, et al. Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. Nat. Biotechnol. 2011;29:149-153. |
| Zhang. Genomics of inherited bone marrow failure and myelodysplasia. Dissertation [online]. University of Washington. 2015 [Retrieved on May 3, 2017]. |
| Zhao, J., et al., “Preparation of hemoglobin-loaded Nano-sized particles with porous structure as oxygen carriers,” Biomaterials, vol. 28, pp. 1414-1422 (2007). |
| Zheng, et al. Massively parallel digital transcriptional profiling of single cells. Nat Commun. Jan. 16, 2017;8:14049. doi: 10.1038/ncomms14049. |
| Zheng, X.Y. et al. “Haplotyping germline and cancer genomes with high-throughput linked-read sequencing” Nature Biotech (Feb. 1, 2016) 34(3):303-311. |
| Zhu et al. Hydrogel Droplet Microfluidics for High-Throughput Single Molecule/Cell Analysis. Accounts of Chemical Research Article ASAP. DOI: 10.1021/acs.accounts.6b00370. |
| Zhu, et al. Synthesis and self-assembly of highly incompatible polybutadienepoly(hexafluoropropoylene oxide) diblock copolymers. Journal of Polymer Science Part B: Polymer Physics. 2005; 43(24):3685-3694. |
| Zimmermann et at., Microscale production of hybridomas by hypo-osmolar electrofusion. Hum⋅ Antibodies Hybridomas. Jan. 1992;3(1 ): 14-8. |
| Zong, et al. Genome-wide detection of single-nucleotide and copy-number variations of a single human cell. Science. Dec. 21, 2012;338(6114):1622-6. doi: 10.1126/science.1229164. |
| Banchelli, et al. Phospholipid membranes decorated by cholesterol-based oligonucleotides as soft hybrid nanostructures. J Phys Chem B. Sep. 4, 2008;112(35):10942-52. doi: 10.1021/jp802415t. Epub Aug. 9, 2008. |
| Bentley, et al. 2008. Supplementary Information. pp. 1-55 Nature. Nov. 6, 2008; 456(7218):53-9. |
| Cejas, P. et al. “Chromatin immunoprecipitation from fixed clinical tissues reveals tumor-specific enhancer profiles” Nature Med (2016) 22(6):685-691. |
| Co-pending U.S. Appl. No. 16/294,769, filed Mar. 6, 2019. |
| Fanielli, M. et al. “Pathology tissue-chromatin immunoprecipitation, coupled with high-throughput sequencing, allows the epigenetic profiling of patient samples” PNAS (2010) 107(50):21535-21540. |
| Hebenstreit. Methods, Challenges and Potentials of Single Cell RNA-seq. Biology (Basel). Nov. 16, 2012;1(3):658-67. doi: 10.3390/biology1030658. |
| Mali, et al. Barcoding cells using cell-surface programmable DNA-binding domains. Nat Methods. May 2013;10(5):403-6. doi: 10.1038/nmeth.2407. Epub Mar. 17, 2013. |
| Mignardi, M. et al. “Oligonucleotide gap-fill ligation for mutation detection and sequencing in situ” Nucl Acids Res (2015) 43(22):e151. |
| Pfeifer, et al. Bivalent cholesterol-based coupling of oligonucleotides to lipid membrane assemblies. J Am Chem Soc. Aug. 25, 2004;126(33):10224-5. |
| Anonymous: “Three Ways to Get Intimate with Epigenetic Marks”. Oct. 24, 2012. Retrieved from Internet: https://epigenie.com/three-ways-to-get-intimate-with-epigenetic-marks/. |
| Co-pending U.S. Appl. No. 16/228,261, filed Dec. 20, 2018. |
| Co-pending U.S. Appl. No. 16/228,362, filed Dec. 20, 2018. |
| Co-pending U.S. Appl. No. 16/230,936, filed Dec. 21, 2018. |
| Co-pending U.S. Appl. No. 16/231,142, filed Dec. 21, 2018. |
| Co-pending U.S. Appl. No. 16/231,185, filed Dec. 21, 2018. |
| Co-pending U.S. Appl. No. 16/242,962, filed Jan. 8, 2019. |
| Co-pending U.S. Appl. No. 16/246,322, filed Jan. 11, 2019. |
| Co-pending U.S. Appl. No. 16/249,688, filed Jan. 16, 2019. |
| Meyer, et al. Targeted high-throughput sequencing of tagged nucleic acid samples. Nucleic Acids Res. 2007;35(15):e97. |
| 10X Genomics. 10x Genomics Chromium™ Single Cell 3′ Solution Utilized for Perturb-seq Approach. Press Release. Dec. 19, 2016. Retrieved from https://www.10xgenomics.com/news/10x-genomics-chromium-single-cell-3-solution-utilized-perturb-seq-approach/. |
| Adamson, et al. A Multiplexed Single-Cell CRISPR Screening Platform Enables Systematic Dissection of the Unfolded Protein Response. Cell. Dec. 15, 2016;167(7):1867-1882.e21. doi: 10.1016/j.cell.2016.11.048. |
| Co-pending U.S. Appl. No. 15/875,899, filed Jan. 19, 2018. |
| Dixit, et al. Perturb-Seq: Dissecting Molecular Circuits with Scalable Single-Cell RNA Profiling of Pooled Genetic Screens. Cell. Dec. 15, 2016;167(7):1853-1866.e17. doi: 10.1016/j.cell.2016.11.038. |
| Adey, et al., “Ultra-low-input, tagmentation-based whole-genome bisulfite sequencing”, Genome Research, 2012, 22 ;6): 1139-1143. |
| Ason et al. DNA sequence bias during Tn5 transposition. Journal of molecular biology 335.5 (2004): 1213-1225. |
| Bjornsson et al., Intra-individual change over time in DNA methylation with familial clustering, JAMA, Jun. 25, 2008, vol. 299 No. 24, pp. 2877-2883. |
| Boyle, et al. “High-resolution genome-wide in vivo footprinting of diverse transcription factors in human cells”, Genome Res. Mar. 2011;21(3):456-64. |
| Buenrostro, et al., “Tranposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position”, Nature Methods, 2013, 10(12): 1213-1218. |
| Co-pending U.S. Appl. No. 16/052,431, filed Aug. 1, 2018. |
| Co-pending U.S. Appl. No. 16/052,486, filed Aug. 1, 2018. |
| Co-pending U.S. Appl. No. 16/056,231, filed Aug. 6, 2018. |
| Gangadharan et al., DNA transposon Hermes insert into DNA in nucleosome-free regions in vivo, Proc nat Ad Sci, Dec. 21, 2010, vol. 107, No. 51, pp. 1966-1972. |
| Green et al. Insertion site preference of Mu, Tn5, and Tn7 transposons. Mobile Dna 3.1 (2012): 3. 0. |
| Haring, et al. Chromatin immunoprecipitation: optimization, quantitative analysis and data normalization. Plant Methods. 2007; 3: 11. |
| Joneja, et al. Linear nicking endonuclease-mediated strand-displacement DNA amplification. Anal Biochem. Jul. 1, 2011;414(1):58-69. doi: 10.1016/j.ab.2011.02.025. Epub Feb. 20, 2011. |
| Knapp, et al. Generating barcoded libraries for multiplex high-throughput sequencing. Methods Mol Biol. 2012;840:155-70. doi: 10.1007/978-1-61779-516-9_19. |
| Lai; et al., ““Characterization and Use of Laser-Based Lysis for Cell Analysis On-Chip”, Journal of the Royal Society, Interface, vol. 5, Supplement 2, pp. S113-S121, Oct. 2008, (Year:2008)”, Journal of the Royal Society, Interface, Oct. 2008, vol. 5, Supplement 2, S113-S121. |
| Park. ChIP—seq: advantages and challenges of a maturing technology. Nature Reviews Genetics vol. 10, pp. 669-680 (2009). |
| “U.S. Appl. No. 61/982,001, filed Apr. 21, 2014 (Year:2014)”. |
| Simon, et al., “Using formaldehyde-assisted isolation of regulatory elements (FAIRE) to isolate active regulatory DNA”, Nature Protocols, 2012, 7(2): 256-267. |
| Smith, et al. Highly-multiplexed barcode sequencing: an efficient method for parallel analysis of pooled samples. Nucleic Acids Research, 38(13): e142 (2010). |
| Song, et al., “DNase-seq: A High-Resolution Technique for Mapping Active Gene Regulatory Elements across the Senome from Mammalian Cells”, Cold Spring Harbor Laboratory Press, 2010, 2010(2), doi:10.1101/pdb.prot5384. |
| Zentner, et al. Surveying the epigenomic landscape, one base at a time. Genome Biol. Oct. 22, 2012;13(10):250. doi: 10.1186/gb4051. |
| Agasti, et al. Photocleavable DNA barcode-antibody conjugates allow sensitive and multiplexed protein analysis in single cells. J Amer Chem Soc ePub, Nov. 2, 2012, vol. 134, No. 45, pp. 18499-18502. |
| Co-pending U.S. Appl. No. 16/033,065, filed Jul. 11, 2018. |
| Co-pending U.S. Appl. No. 16/043,874, filed Jul. 24, 2018. |
| Co-pending U.S. Appl. No. 16/044,374, filed Jul. 24, 2018. |
| Co-pending U.S. Appl. No. 16/107,685, filed Aug. 21, 2018. |
| Co-pending U.S. Appl. No. 16/160,576, filed Oct. 15, 2018. |
| Co-pending U.S. Appl. No. 16/160,719, filed Oct. 15, 2018. |
| Co-pending U.S. Appl. No. 16/165,389, filed Oct. 19, 2018. |
| Co-pending U.S. Appl. No. 16/170,980, filed Oct. 25, 2018. |
| Co-pending U.S. Appl. No. 16/206,168, filed Nov. 30, 2018. |
| Co-pending U.S. Appl. No. 16/212,441, filed Dec. 6, 2018. |
| Delehanty, et al. Peptides for specific intracellular delivery and targeting of nanoparticles: implications for developing nanoparticle-mediated drug delivery. Ther Deliv. Sep. 2010;1(3):411-33. |
| Epicentre., “EZ-Tn5TM Custom Transposome Construction Kits”, http://www.epicentre.com, pp. 1-17, 2012. |
| “How many species of bacteria are there” (wisegeek.com; accessed Jan. 21, 2014). |
| “List of sequenced bacterial genomes” (Wikipedia.com; accessed Jan. 24, 2014). |
| Margulies 2005 Supplementary methods (Year: 2005). |
| “Meyer, et al., From micrograms to picograms: quantitative PCR reduces the material demands of high-throughput sequencing, Nucleic Acids Research, 2008, vol. 36, No. 1, 6 pages”. |
| Thaxton, C.S. et al. “A Bio-Bar-Code Assay Based Upon Dithiothreitol Oligonucleotide Release” Anal Chem (2005) 77:8174-8178. |
| Ullal et al. Cancer Cell Profiling by Barcoding Allows Multiplexed Protein Analysis in Fine-Needle Aspirates. Sci Transl Med. Jan. 15, 2014; 6(219): 219ra9. |
| Zhang, et al. Reconstruction of DNA sequencing by hybridization. Bioinformatics. Jan. 2003;19(1):14-21. |
| Co-pending U.S. Appl. No. 16/196,684, filed Nov. 20, 2018. |
| Jin, et al. Genome-wide detection of DNase I hypersensitive sites in single cells and FFPE tissue samples. Nature. Dec. 3, 2015;528(7580):142-6. doi: 10.1038/nature15740. |
| Anonymous: “TCEP=HCI” Thermo Scientific, Dec. 31, 2013 (Dec. 31, 2013), XP055508461, Retrieved from the Internet: URL:https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0011306_TCEP_HCI_UG.pdf. |
| Caruccio, et al. Nextera Technology for NGS DNA Library Preparation: Simultaneous Fragmentation and Tagging by In Vitro Transposition, Nextera Technology, 2009, 16-3, 1-3. (Year: 2009). |
| Co-pending U.S. Appl. No. 16/045,474, filed Jul. 25, 2018. |
| Co-pending U.S. Appl. No. 16/138,448, filed Sep. 21, 2018. |
| Co-pending U.S. Appl. No. 16/144,832, filed Sep. 27, 2018. |
| Gao et al., Toehold of dsDNA Exchange Affects the Hydrogel Swelling Kinetic of a Polymer-dsDNA Hybrid Hydrogel, Royal Soc. Chem. 7:1741-1746 (Dec. 20, 2010). |
| Greenleaf, et al. Assaying the epigenome in limited numbers of cells. Methods. Jan. 15, 2015;72:51-6. doi: 10.1016/j.ymeth.2014.10.010. Epub Oct. 22, 2014. |
| Hu et al., Shape Controllable Microgel Particles Prepared by Microfluidic Combining External Crosslinking, Biomicrofluidics 6:26502 (May 18, 2012). |
| Lebedev, A. et al. “Hot Start PCR with heat-activatable primers: a novel approach for improved PCR performance” NAR (2008) 36(20):E131-1. |
| McGinnis, et al. Multi-seq: Scalable sample multiplexing for single-cell RNA sequencing using lipid-tagged indices. bioRxiv 387241; doi: https://doi.org/10.1101/387241 . . . . |
| Savva, et al. The structural basis of specific base-excision repair by uracil-DNA glycosylase. Nature. Feb. 9, 1995;373(6514):487-93. |
| Zhang, et al. One-step fabrication of supramolecular microcapsules from microfluidic droplets. Science. Feb. 10, 2012;335(6069):690-4. doi: 10.1126/science.1215416. |
| Co-pending U.S. Appl. No. 15/933,299, filed Mar. 22, 2018. |
| Co-pending U.S. Appl. No. 15/975,468, filed May 9, 2018. |
| Co-pending U.S. Appl. No. 15/980,473, filed May 15, 2018. |
| Co-pending U.S. Appl. No. 15/985,388, filed May 21, 2018. |
| Co-pending U.S. Appl. No. 16/000,803, filed Jun. 5, 2018. |
| Depristo et al. A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nature Genet 43:491-498 (2011). |
| Han, SW et al. “Targeted Sequencing of Cancer-Related Genes in Colorectal Cancer Using Next-Generation Sequencing” PLOS One (2013) 8(5):e64271. |
| Lander, et al. Initial sequencing and analysis of the human genome. Nature, 409 (Feb. 15, 2001): 860-921. |
| Shaikh, et al. A modular microfluidic architecture for integrated biochemical analysis. Proc Natl Acad Sci U S A. Jul. 12, 2005;102(28):9745-50. Epub Jun. 28, 2005. |
| Holmberg, et al. The biotin-streptavidin interaction can be reversibly broken using water at elevated temperatures. Feb. 2, 2005. Electrophoresis, 26:501-510. |
| Invitrogen Dynal. Dynabeads M-280 Streptavidin 2006 product sheet. |
| Morton. Parameters of the human genome. Apr. 23, 1991. Proceedings of the National Academy of Sciences of the United States of America, 88: 7474-7476. |
| National Human Genome Research Institute (NHGRI). The Human Genome Project Completion: Frequently Asked Questions. Last Updated: Oct. 30, 2010. |
| Qiagen. Omniscript Reverse Transcription Handbook. Oct. 2010. |
| Seiffert, et al. Microfluidic fabrication of smart microgels from macromolecular precursors. 2010. Polymer. |
| Wong, et al. Multiplexed Barcoded CRISPR-Cas9 Screening Enabled by CombiGEM. Pnas. Mar. 1, 2016, vol. 113, pp. 2544-2549. |
| Zhu, et al. Reverse transcriptase template switching: a SMART approach for full-length cDNA library construction. Biotechniques. Apr. 2001;30(4):892-7. |
| AH006633.3 (Homo sapiens clone P1 and PAC max interactor 1 (MXI1) gene, complete cds, NCBI Reference Sequence, priority to Jun. 10, 2016, 5 pages) (Year:2016). |
| Ahern, H. The Scientist, vol. 20, pp. 20 and 22. Jul. 1995. |
| Ailenberg, et al. (2000) Controlled Hot Start and Improved Specificity in Carrying Out PCR Utilizing Touch-Up and Loop Incorporated Primers (TULIPS). BioTechniques, 29:1018-1024. (Year: 2000). |
| Epicenter, EZ-Tn5 Transposase, Epicenter, 2012, 1-5. (Year: 2012). |
| Anonymous: “Dynal MPC(TM)-S”, Oct. 13, 2008 (Oct. 13, 2008), XP055603532, Retrieved from the Internet on Jul. 9, 2019; URL:< https://www.veritastk.co.jp/products/pdf/120%2020D.Dynal_MPC-S%28rev005%29.pdf>. |
| Bentolila, et al. Single-step multicolor fluorescence in situ hybridization using semiconductor quantum dot-DNA conjugates. Cell Biochem Biophys. 2006;45(1):59-70. |
| Bystrykh, et al. Generalized DNA barcode design based on Hamming codes. PLoS One. 2012;7(5):e36852. doi: 10.1371/journal.pone.0036852. Epub May 17, 2012. |
| Co-pending U.S. Appl. No. 16/395,090, filed Apr. 25, 2019. |
| Co-pending U.S. Appl. No. 16/419,428, filed May 22, 2019. |
| Co-pending U.S. Appl. No. 16/419,461, filed May 22, 2019. |
| Co-pending U.S. Appl. No. 16/419,555, filed May 22, 2019. |
| Co-pending U.S. Appl. No. 16/419,630, filed May 22, 2019. |
| Co-pending U.S. Appl. No. 16/419,820, filed May 22, 2019. |
| Co-pending U.S. Appl. No. 16/434,089, filed Jun. 6, 2019. |
| Co-pending U.S. Appl. No. 16/435,362, filed Jun. 7, 2019. |
| Co-pending U.S. Appl. No. 16/435,417, filed Jun. 7, 2019. |
| Co-pending U.S. Appl. No. 16/519,863, filed Jul. 23, 2019. |
| Co-pending U.S. Appl. No. 16/680,343, filed Nov. 11, 2019. |
| Definition of “corresponding”, Merriam-Webster Online, downloaded from http://www.merriam-webster.com/dictionary/corresponding (Year: 2019). |
| Dhingra, et al. A complete solution for high throughput single cell targeted multiomic DNA and RNA sequencing for cancer research. Poster. AACR 2019. |
| Ellison et al. Mutations in Active-Site Residues of the Uracil-DNA Glycosytase Encoded by Vaccinia Virus are Incompatible with Virus Viability. J Virology (1996) 70(11):7965-7973. |
| Fu, “A micro fabricated fluorescence-activated cell sorter”, Nature Biotech., 17:1109-1111 (1997). |
| Hamady, et al. Error-correcting barcoded primers for pyrosequencing hundreds of samples in multiplex. Nat Methods. Mar. 2008;5(3):235-7. doi: 10.1038/nmeth.1184. Epub Feb. 10, 2008. |
| Hamady, M. et al. “Error-correcting barcoded primers for pyrosequencing hundreds of samples in multiplex” Nature Methods (2008) 5(3):235-237, Supplementary Data pp. 1-34. |
| Maeda, et al. Development of a DNA barcode tagging method for monitoring dynamic changes in gene expression by using an ultra high-throughput sequencer. Biotechniques. Jul. 2008;45(1):95-7. doi: 10.2144/000112814. |
| Mamedov, I.Z., et al. (2013), Preparing unbiased T-cell receptor and antibody cDNA libraries for the deep next generation sequencing profiling, Front Immunol 4: 456. |
| Zhang, H. et al. “Massively Parallel Single-Molecule and Single-Cell Emulsion Reverse Transcription Polymerase Chain Reaction using Agarose Droplet Microfluidics” Anal Chem (2012) 84:3599-3606, Supporting Information. |
| Zhang, H. et al. Massively Parallel Single-Molecule and Single-Cell Emulsion Reverse Transcription Polymerase Chain Reaction using Agarose Droplet Microfluidics. Anal Chem (2012) 84:3599-3606. |
| Aikawa, et al. Spherical Phospholipid Polymer Hydrogels for Cell Encapsulation Prepared with a Flow-Focusing Microfluidic Channel Device. Langmuir. Jan. 31, 2012;28(4):2145-50. doi: 10.1021/Ia2037586. Epub Dec. 22, 2011. |
| Allazetta, et al. Microfluidic Synthesis of Cell-Type-Specific Artificial Extracellular Matrix Hydrogels. Biomacromolecules. Apr. 8, 2013;14(4):1122-31. doi: 10.1021/bm4000162. Epub Mar. 8, 2013. |
| Bassett, et al. Competitive ligand exchange of crosslinking ions for ionotropic hydrogel formation. J. Mater. Chem. B, 2016,4, 6175-6182. |
| Co-pending PCT/US2019/024418, filed Mar. 27, 2019. |
| Co-pending PCT/US2019/046940, filed Aug. 16, 2019. |
| Co-pending U.S. Appl. No. 16/410,953, filed May 13, 2019. |
| Co-pending U.S. Appl. No. 16/415,617, filed May 17, 2019. |
| Co-pending U.S. Appl. No. 16/434,068, filed Jun. 6, 2019. |
| Co-pending U.S. Appl. No. 16/434,076, filed Jun. 6, 2019. |
| Co-pending U.S. Appl. No. 16/434,084, filed Jun. 6, 2019. |
| Co-pending U.S. Appl. No. 16/434,089, filed Apr. 1, 2019. |
| Co-pending U.S. Appl. No. 16/434,095, filed Jun. 6, 2019. |
| Co-pending U.S. Appl. No. 16/434,099, filed Jun. 6, 2019. |
| Co-pending U.S. Appl. No. 16/434,102, filed Jun. 6, 2019. |
| Co-pending U.S. Appl. No. 16/435,393, filed Jun. 7, 2019. |
| Co-pending U.S. Appl. No. 16/439,568, filed Jun. 12, 2019. |
| Co-pending U.S. Appl. No. 16/439,675, filed Jun. 12, 2019. |
| Co-pending U.S. Appl. No. 16/454,485, filed Jun. 27, 2019. |
| Co-pending U.S. Appl. No. 16/530,930, filed Aug. 2, 2019. |
| Co-pending U.S. Appl. No. 16/575,280, filed Sep. 18, 2019. |
| Co-pending U.S. Appl. No. 16/428,656, filed May 31, 2019. |
| Co-pending U.S. Appl. No. 16/698,740, filed Nov. 27, 2019. |
| Co-pending U.S. Appl. No. 16/717,375, filed Dec. 17, 2019. |
| Ekblom, R. et al. “A field guide to whole-genome sequencing, assembly and annotation” Evolutionary Apps (Jun. 24, 2014) 7(9):1026-1042. |
| Ellison, et al., EGFR Mutation Testing in Lung Ancer: A Review of Available Methods and Their Use for Analysis of Tumour Tissue and Cytology Samples, Journal of Clinical Pathology, 2013, 66:79-89. |
| Farrukh, et al. Bioconjugating Thiols to Poly(acrylamide) Gels for Cell Culture Using Methylsulfonyl Co-monomers. Angew Chem Int Ed Engl. Feb. 5, 2016;55(6):2092-6. doi: 10.1002/anie.201509986. Epub Jan. 6, 2016. |
| Fox, et al. Accuracy of Next Generation Sequencing Platforms. Next Gener Seq Appl. 2014;1. pii: 1000106. |
| Henke, et al. Enzymatic Crosslinking of Polymer Conjugates is Superior over Ionic or UV Crosslinking for the On-Chip Production of Cell-Laden Microgels. Macromol Biosci. Oct. 2016;16(10):1524-1532. doi: 10.1002/mabi.201600174. Epub Jul. 21, 2016. |
| Jarosz, M. et al. “Using 1 ng of DNA to detect haplotype phasing and gene fusions from whole exome sequencing of cancer cell lines” Cancer Res (2015) 75(supp15):4742. |
| Jiang et al. Cell-laden microfluidic microgels for tissue regeneration. Lab Chip 16(23):4482-4506 (Nov. 2016). |
| Kukwikila, et al. Assembly of a biocompatible triazole-linked gene by one-pot click-DNA ligation. Nature Chemistry (2017) doi:10.1038/nchem.2850. |
| Madl, et al. “Bioorthogonal Strategies for Engineering Extracellular matrices”, Madal, Chritopher, Adv. Funct. Master. 2018, vol. 28, 1706046, pp. 1-21. |
| Marquis, et al. Microfluidics-assisted diffusion self-assembly: toward the control of the shape and size of pectin hydrogel microparticles. Biomacromolecules. May 12, 2014;15(5):1568-78. doi: 10.1021/bm401596m. Epub Apr. 8, 2014. |
| McCoy, R. et al. “Illumina TruSeq Synthetic Long-Reads Empower De Novo Assembly and Resolve Complex, Highly-Repetitive Transposable Elements” PLOS (2014) 9(9):e1016689. |
| PCT/IB2010/002243, International Search Report and Written Opinion, dated Feb. 9, 2011, 13pgs. |
| Pelton, et al. (2011) Microgels and Their Synthesis: An Introduction, in Microgel Suspensions: Fundamentals and Applications (eds A. Fernandez-Nieves, H. M. Wyss, J. Mattsson and D. A. Weitz), Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany. doi: 10.1002/9783527632992.ch1. |
| Ritz, A. et al. “Characterization of structural variants with single molecule and hybrid sequencing approaches” Bioinformatics (2014) 30(24):3458-3466. |
| Sahiner. Single step poly(L-Lysine) microgel synthesis, characterization and biocompatibility tests. Polymer, vol. 121, Jul. 14, 2017, pp. 46-54. |
| Seiffert. Microgel capsules tailored by droplet-based microfluidics. Chemphyschem. Feb. 4, 2013;14(2):295-304. doi: 10.1002/cphc.201200749. Epub Dec. 6, 2012. |
| Shelbourne et al., “Fast copper-free click DNA ligation by the ring-strain promoted alkyne-azide cycloaddition reaction,” Chem. Commun., 2011, 47, 6257-6259. |
| Shih, et al. Photoclick Hydrogels Prepared from Functionalized Cyclodextrin and Poly(ethylene glycol) for Drug Delivery and in Situ Cell Encapsulation. Biomacromolecules. Jul. 13, 2015;16(7):1915-23. doi: 10.1021/acs.biomac.5b00471. Epub Jun. 3, 2015. |
| Spitale et al., “Structural imprints in vivo decode RNA regulatory mechanisms,” Nature. Mar. 26, 2015;519(7544):486-90; doi: 10.1038/nature14263. Epub Mar. 18, 2015. |
| Tam, et al. Engineering Cellular Microenvironments with Photo- and Enzymatically Responsive Hydrogels: Toward Biomimetic 3D Cell Culture Models. Acc Chem Res. Apr. 18, 2017;50(4):703-713. doi: 10.1021/acs.accounts.6b00543. Epub Mar. 27, 2017. |
| Uttamapinant, et al. Fast, cell-compatible click chemistry with copper-chelating azides for biomolecular labeling.Angew. Chem. Int. End. Engl., Jun. 11, 2012: 51(24) pp. 5852-5856. |
| Velasco, et al. Microfluidic encapsulation of cells in polymer microgels. Small. Jun. 11, 2012;8(11):1633-42. doi: 10.1002/smll.201102464. Epub Mar. 29, 2012. |
| Voskoboynik, A. et al. The genome sequence of the colonial chordate, Botryllus schlosseri. eLife, 2:e00569 (2013). doi: 10.7554/eLife.00569. Epub Jul. 2, 2013. |
| Zerbino, D.R. “Using the Velvet de novo assembler for short-read sequencing technologies” Curr Protoc Bioinformatics. Sep. 2010;Chapter 11:Unit 11.5. doi: 10.1002/0471250953.bi1105s31. |
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