Patent Grant
8986958
The present invention relates generally to the field of genomic analysis, and more particularly, to methods and kits for making libraries of single-stranded nucleic acid probes comprising predetermined sequences.
The ability to sequence deoxyribonucleic acid (DNA) accurately and rapidly is revolutionizing biology and medicine. The pharmacogenomics challenge is to comprehensively identify the genes and functional polymorphisms associated with the variability in drug response. Screens for numerous genetic markers performed for populations large enough to yield statistically significant data are needed before associations can be made between a given genotype and a particular disease.
The study of complex genomes, and in particular, the search for the genetic basis of disease in humans, requires genotyping on a massive scale, which is demanding in terms of cost, time, and labor. Such costly demands are even greater when the methodology employed involves serial analysis of individual DNA samples, i.e., separate reactions for individual samples. Resequencing of polymorphic areas in the genome that are linked to disease development will contribute greatly to the understanding of diseases, such as cancer, and therapeutic development. Oligonucleotide libraries are the cornerstone of sequence-based gene resequencing and digital profiling strategies. To realize the full commercial potential of various high-throughput sequencing platforms, the cost of generating oligonucleotide libraries must be reduced by a substantial amount. Thus, there is a need for cost-effective methods for preparing populations of high quality oligonucleotide probes with sufficient yield for use in high throughput sequencing platforms and solution based capture methods.
Provided herein are methods for generating a population of single-stranded nucleic acid probes, each probe comprising a predetermined nucleotide sequence, the method comprising: (a) providing a starting population of linear double-stranded nucleic acid precursor molecules each precursor molecule having (i) a probe region having the predetermined sequence which is flanked at a 5′ and a 3′ end by a first and a second restriction enzyme recognition sequence for generating ligation substrates and for ligating a plurality of the double-stranded nucleic acid precursor molecules into head-to-tail concatemers (ii) the 5′ flanking region including the first restriction enzyme recognition sequence and (iii) the 3′ flanking region including the second restriction enzyme recognition sequence; (b) contacting the 5′ and 3′ flanking regions of the linear double-stranded nucleic acid precursor molecules with the first and second restriction enzymes to cleave the first and second restriction enzyme recognition sequences so as to generate the ligation substrates; (c) ligating the ligation substrates together so as to generate a plurality of random head-to-tail concatemers; (d) amplifying the plurality of head-to-tail concatemers; (e) contacting the amplified head-to-tail concatemers with the first and second restriction enzymes so as to release a plurality double-stranded monomer linear precursor molecules; and (f) selectively removing one strand of the double-stranded monomer linear precursor molecules so as to generate a population of single-stranded nucleic acid probes, each probe comprising the predetermined nucleotide sequence.
In one embodiment, the single-stranded nucleic acid probes further comprise a region which hybridizes to a capture nucleic acid molecule.
In yet another embodiment, the selectively removing one strand from the double-stranded monomer linear precursor molecules comprises: (a) contacting the released precursor molecules of step (e) above with alkaline phosphatase; (b) contacting the released precursor molecules of step (e) above with a third restriction enzyme which cleaves the third restriction enzyme recognition sequence; and (c) contacting the released precursor molecules of step (e) above with an exonuclease so as to selectively degrade the one strand of the double-stranded monomer linear precursor molecules.
In one embodiment, the exonuclease is lambda exonuclease.
In another embodiment, the members of the starting population of the linear double-stranded nucleic acid precursor molecules each comprise the same nucleotide sequence in the 5′ flanking region or each comprise the same nucleotide sequence in the 3′ flanking region.
In another embodiment, the 3′ flanking region further comprises a third restriction enzyme recognition sequence.
In another embodiment, the members of the starting population of the linear double-stranded nucleic acid precursor molecules each comprise the same predetermined sequences or different predetermined sequences.
In another embodiment, the ligation substrates of step (b) comprise overhanging nucleic acid ends capable of annealing together.
In another embodiment, the first or second restriction enzyme recognition sequence is cleaved by a type II restriction enzyme.
In another embodiment, the first or second restriction enzyme recognition sequence is cleaved by a Bsm1 enzyme.
In another embodiment, each predetermined nucleotide sequence in the population of linear double-stranded nucleic acid precursor molecules comprise a nucleotide sequence which is at least 95% identical to at least a portion of a sense or anti-sense strand of a target nucleic acid sequence.
In another embodiment, the predetermined sequence hybridizes to one target sequence or hybridizes to different target sequences.
In another embodiment, the predetermined sequences in the population of linear double-stranded nucleic acid precursor molecules hybridize to at least 10 different exon nucleotide sequences.
In another embodiment, the predetermined sequences in the population of linear double-stranded nucleic acid precursor molecules hybridize to at least 1000 different exon nucleotide sequences.
In another embodiment, the predetermined sequences hybridize to the target sequence at an interval of at least every 35 bases across the target sequence.
In another embodiment, the predetermined sequences hybridize to the target sequence of interest at an interval of one base across the target sequence.
In another embodiment, the probe region comprises 20-200 nucleotides.
In another embodiment, the predetermined nucleotide sequence comprises 10-50 nucleotides.
In another embodiment, the region of the single-stranded nucleic acid probe which hybridizes to the capture nucleic acid molecule comprises 10-50 nucleotides.
In another embodiment, the amplifying according to step (d) comprises isothermal amplification.
In another embodiment, the amplifying according to step (d) comprises random amplification primers.
In another embodiment, the random amplification primers each comprise a random 7-mer oligonucleotide and two additional nitroindole residues at the 5′ end.
In another embodiment, the random amplification primers each comprise a random 7-mer oligonucleotide and a phosphorothioate linkage to the 3′ end.
In another embodiment, the capture nucleic acid molecule further comprises a protein binding partner.
In another embodiment, the protein binding partner is biotin.
In another embodiment, each single-stranded nucleic acid probe comprises (i) the predetermined nucleotide sequence having a nucleotide sequence which is at least 95% identical to at least a portion of a sense or an anti-sense strand of a target nucleic acid sequence and (ii) a region which hybridizes to a capture nucleic acid molecule.
Provided herein are also a population of single-stranded nucleic acid probes generated by the disclosed methods.
Provided herein are also methods, wherein the starting population of linear double-stranded nucleic acid precursor molecules is generated by steps comprising: (a) providing a population of a first single-stranded nucleic acid molecule comprising the 5′ flanking region, the probe region which comprises the predetermined sequence, and the capture sequence; (b) providing a population of a second single-stranded nucleic acid molecules comprising the sequence which is complementary to the capture sequence, and the 3′ flanking region; (c) annealing the first and second populations of the single-stranded nucleic acid molecules to form a nucleic acid duplex having overhanging 5′ ends; and (d) conducting a polymerase-dependent strand extension reaction on the overhanging 5′ ends so as to generate the population of double-stranded nucleic acid precursor molecules.
Provided herein are also, methods for enriching a target nucleic acid sequence of interest from a nucleic acid library, comprising: (a) contacting the population of single-stranded nucleic acid probes of the method above with the nucleic acid library having at least one target nucleic acid sequence of interest to form a mixture having unhybridized nucleic acid sequences and duplexes, each duplex having the single-stranded nucleic acid probe hybridized to the target nucleic acid sequence of interest; (b) contacting the duplexes with a population of capture nucleic acid molecules to form complexes having the single-stranded nucleic acid probe hybridized to the target nucleic acid sequence of interest and hybridized to the capture nucleic acid molecule; (c) separating the complex from the mixture; and (d) eluting the target nucleic acid sequence of interest from the complex.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which these inventions belong. All patents, patent applications, published applications, treatises and other publications referred to herein, both supra and infra, are incorporated by reference in their entirety. If a definition and/or description is explicitly or implicitly set forth herein that is contrary to or otherwise inconsistent with any definition set forth in the patents, patent applications, published applications, and other publications that are herein incorporated by reference, the definition and/or description set forth herein prevails over the definition that is incorporated by reference.
The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology and recombinant DNA techniques, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Sambrook, J., and Russell, D. W., 2001, Molecular Cloning: A Laboratory Manual, Third Edition; Ausubel, F. M., et al., eds., 2002, Short Protocols in Molecular Biology, Fifth Edition.
As used herein, the terms “comprising” (and any form or variant of comprising, such as “comprise” and “comprises”), “having” (and any form or variant of having, such as “have” and “has”), “including” (and any form or variant of including, such as “includes” and “include”), or “containing” (and any form or variant of containing, such as “contains” and “contain”), are inclusive or open-ended and do not exclude additional, unrecited additives, components, integers, elements or method steps.
As used herein, the terms “a,” “an,” and “the” and similar referents used herein are to be construed to cover both the singular and the plural unless their usage in context indicates otherwise. Accordingly, the use of the word “a” or “an” when used in the claims or specification, including when used in conjunction with the term “comprising”, may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”
As used herein, the term “nucleic acid molecule” and its variants encompasses both deoxyribonucleotides and ribonucleotides and refers to a polymeric form of nucleotides including two or more nucleotide monomers. The nucleotides can be naturally occurring, artificial, and/or modified nucleotides.
As used herein, an “isolated nucleic acid” and its variants is a nucleic acid molecule that exists in a physical form that is non-identical to any nucleic acid molecule of identical sequence as found in nature; “isolated” does not require, although it does not prohibit, that the nucleic acid so described has itself been physically removed from its native environment. For example, a nucleic acid can be said to be “isolated” when it includes nucleotides and/or internucleoside bonds not found in nature. When, instead, composed of natural nucleosides in phosphodiester linkage, a nucleic acid can be said to be “isolated” when it exists at a purity not found in nature, where purity can be adjudged with respect to the presence of nucleic acids of other sequences, with respect to the presence of proteins, with respect to the presence of lipids, or with respect to the presence of any other component of a biological cell, or when the nucleic acid lacks a sequence that flanks an otherwise identical sequence in an organism's genome, or when the nucleic acid possesses a sequence not identically present in nature. As so defined, “isolated nucleic acid” includes nucleic acids integrated into a host cell chromosome at a heterologous site, recombinant fusions of a native fragment to a heterologous sequence, recombinant vectors present as episomes, or as integrated into a host cell chromosome.
As used herein, “subject” and its variants refers to an organism or to a cell sample, tissue sample, or organ sample derived therefrom, including, for example, cultured cell lines, biopsy, blood sample, or fluid sample containing a cell. For example, an organism may be an animal, including but not limited to, an animal such as a cow, a pig, a mouse, a rat, a chicken, a cat, a dog, etc., and is usually a mammal, such as a human.
As used herein, the term “specifically bind” and its variants refers to two components (e.g., target-specific binding region and target) that are bound (e.g., hybridized, annealed, complexed) to one another sufficiently that the intended capture and enrichment steps can be conducted. As used herein, the term “specific” refers to the selective binding of two components (e.g., target-specific binding region and target) and not generally to other components unintended for binding to the subject components.
As used herein, the term “high stringency hybridization conditions” and its variants means any condition in which hybridization will occur when there is at least 95%, preferably about 97% to 100% nucleotide complementarity (identity) between the nucleic acid sequences of the nucleic acid molecule and its binding partner. However, depending upon the desired purpose, the hybridization conditions may be “medium stringency hybridization,” which can be selected that require less complementarity, such as from about 50% to about 90% (e.g., 60%, 70%, 80%, 85%). The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990)), modified as in Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993)). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (J. Mol. Biol. 215:403-410 (1990)).
As used herein, the term “complementary” and its variants refers to nucleic acid sequences that are capable of base-pairing according to the standard Watson-Crick complementary rules. That is, the larger purines will base pair with the smaller pyrimidines to form combinations of guanine paired with cytosine (G:C) and adenine paired with either thymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA.
As used herein, the term “target” and its variants refers to a nucleic acid molecule or polynucleotide whose presence and/or amount and/or sequence is desired to be determined and which has an affinity for a given target capture probe. Examples of targets include regions of genomic DNA, PCR amplified products derived from RNA or DNA, DNA derived from RNA or DNA, ESTs, cDNA, and mutations, variants or modifications thereof.
As used herein, the term “predetermined nucleic acid sequence” and its variants means that the nucleic acid sequence of a nucleic acid probe is known and was chosen before synthesis of the nucleic acid molecule in accordance with the invention disclosed herein.
As used herein, the term “essentially identical” and its variants as applied to synthesized and/or amplified nucleic acid molecules refers to nucleic acid molecules that are designed to have identical nucleic acid sequences, but that may occasionally contain minor sequence variations in comparison to a desired sequence due to base changes introduced during the nucleic acid molecule synthesis process, amplification process, or due to other processes in the method. As used herein, essentially identical nucleic acid molecules are at least 95% identical to the desired sequence, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identical, or absolutely identical, to the desired sequence.
As used herein, the term “resequencing” and its variants refers to a technique that determines the sequence of a genome of an organism using a reference sequence that has already been determined. It should be understood that resequencing may be performed on both the entire genome/transcriptome of an organism or a portion of the genome/transcriptome large enough to include the genetic change of the organism as a result of selection. Resequencing may be carried out using various sequencing methods, such as any sequencing platform amenable to producing DNA sequencing reads that can be aligned back to a reference genome, and is typically based on highly parallel technologies such as, for example, dideoxy “Sanger” sequencing, pyrosequencing on beads (e.g., as described in U.S. Pat. No. 7,211,390, assigned to 454 Life Sciences Corporation, Branford, Conn.), ligation based sequencing on beads (e.g., Applied Biosystems Inc,/Invitrogen), sequencing on glass slides (e.g., Illumina Genome Analyzer System, based on technology described in WO 98/44151 (Mayer, P., and Farinelli, L.), microarrays, or fluorescently labeled micro-beads.
As used herein, the term “target nucleotide” and its variants refers to a nucleic acid molecule or polynucleotide in a starting population of nucleic acid molecules having a target sequence whose presence and/or amount and/or nucleotide sequence is desired to be determined and which has an affinity for a given target capture probe.
As used herein, the term “target sequence” and its variants refers generally to a nucleic acid sequence on a single strand of nucleic acid. The target sequence may be a portion of a gene, a regulatory sequence, genomic DNA, cDNA, RNA including mRNA and rRNA, or others. The target sequence may be a target sequence from a sample, or a secondary target such as a product of an amplification reaction.
As used herein, the term “processing” and its variants refers generally to a manipulation of a precursor nucleic acid substrate into a processed form of the substrate, such as by cleavage with a restriction endonuclease, modification and/or amplification with DNA polymerases, manipulation of DNA termini (e.g., by adding terminal 5′ phosphates with a polynucleotide kinase or removing 5′ terminal phosphates with a suitable phosphatase), degradation of unwanted DNA strands with exonuclease, and the like.
As used herein, the term “head to tail concatemer” and its variants refers to at least two or more monomeric structures each having a first end and a second end, such as double-stranded nucleic acid molecules, covalently joined in the configuration of the second end of the first monomer joined to the first end of the second monomer.
Other objects, features and advantages of the disclosed compositions, methods, systems and kits will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments, are given by way of illustration only, since various changes and modifications within the spirit and scope of the inventions provided herein will become apparent to those skilled in the art from this detailed description.
Provided herein are compositions, systems, methods and kits for generating a population of single-stranded nucleic acid probes, each probe comprising a predetermined nucleic acid sequence. The methods of the invention are useful in any situation in which it is desirable to make populations of single-stranded nucleic acid molecules (hundreds to thousands, to tens of thousands, to hundreds of thousands, to millions of oligonucleotides), wherein each nucleic acid molecule has a predetermined nucleic acid sequence. For example, the methods may be used to generate a high quantity of a complex population (e.g., library) of single-stranded nucleic acid probes, while maintaining a uniform representation of the individual nucleic acid probes within the population. The applications for pools of oligonucleotides include, but are not limited to, using the oligonucleotides to generate primers for PCR amplification, primers for multiplexing PCR and transcription, probes for SNP (single nucleotide polymorphism) detection, and libraries of nucleic acid probes for genomic analysis, RNA expression analysis, including siRNA and shRNA expression analysis. In some embodiments, the methods according to this aspect of the invention are used to generate a library of target specific probes for solution based capture methods, as described in Examples 1-3 herein.
The methods according to this aspect of the invention comprise (a) providing a starting population of double-stranded nucleic acid precursor molecules, wherein each precursor molecule in the starting population comprises a probe region comprising a predetermined sequence that is flanked on the 5′ end by a 5′ flanking region comprising a first processing site and is flanked on the 3′ end by a 3′ flanking region comprising a second processing site, wherein the first and second processing sites are selected to generate ligation substrates for ligation of a plurality of the double-stranded nucleic acid precursor molecules into head-to-tail concatemers; (b) processing the 5′ and 3′ flanking regions of the double-stranded nucleic acid precursor molecules to generate ligation substrates; (c) ligating the ligation substrates together to generate head-to-tail concatemers; (d) amplifying the head-to-tail concatemers; (e) processing the amplified head-to-tail concatemers to release double-stranded monomer precursor molecules; and (f) selectively removing the complement strand of the double-stranded monomer precursor molecules to generate a population of single-stranded nucleic acid probes each probe a predetermined nucleic acid sequence.
In one embodiment of the method, a population of target specific capture probes (e.g., a library of capture probes) is generated that may be used in solution based capture methods for enriching a population of DNA molecules for one or more target sequences of interest, such as for resequencing analysis. In accordance with this embodiment, each single-stranded capture probe oligonucleotide in the population of capture probes comprises (i) a target-specific binding region consisting of a nucleic acid sequence that is at least 95% identical to at least a portion of the sense or antisense strand of a target nucleic acid sequence of interest, and (ii) a region for binding to a capture reagent. The methods according to this embodiment of the invention can be used to create populations of single-stranded capture nucleic acid molecules (i.e., capture probes). A population of capture probes is also referred to as a “library” of capture probes. The capture probes generated using the methods described herein may be used for solution based capture to enrich for targets of interest.
A representative method of solution based capture is illustrated in
As shown in
Any library of DNA molecules comprising a subpopulation of nucleic acid target insert sequences of interest may be enriched using the solution based capture methods described herein. In some embodiments, the library of DNA molecules comprises a plurality of distinct insert sequences flanked by a first primer binding region and a second primer binding region within a larger population of nucleic acid insert sequences flanked by the first primer binding region and the second primer binding region may be enriched for target sequences using the capture probes 200 generated using the methods disclosed herein. For example, a library of DNA molecules comprising a subpopulation of nucleic acid target insert sequences of interest flanked by a first primer binding region and a second primer binding region within a larger population of nucleic acid insert sequences flanked by the first primer binding region and the second primer binding region may be enriched using the capture probes generated using the methods of the invention. In some embodiments, the library of DNA molecules further comprises at least one anchor probe binding site, such as a flow cell binding site for binding to a flow cell sequencing platform, such as an Illumina Genome Analyzer for sequence analysis.
The use of solution-based capture to enrich a library allows for the efficient creation of resequencing samples (sequence-ready libraries) that are largely composed of target sequences, as demonstrated in Example 2.
The Design of the Target Capture Probe 200
The general design of the target capture probe 200 is described as follows. As shown in
The length of a target capture probe 200 is typically in the range of from 20 nucleotides to about 200 nucleotides, such as from about 20 nucleotides to about 150 nucleotides, such as from about 30 nucleotides to about 100 nucleotides, or such as from about 40 nucleotides to about 80 nucleotides.
The target-specific binding region 202 of the target capture probe 200 is typically from about 10 to about 150 nucleotides in length (e.g., 35 nucleotides, 50 nucleotides, 100 nucleotides) and is chosen to specifically hybridize to a target sequence of interest. In one embodiment, the target capture probe is about 60 to 80 nucleotides in length, comprising a target-specific binding region of about 20 to 40 nucleotides in length, such as about 35 nucleotides in length.
The target specific binding region 202 comprises a sequence that is substantially complementary (i.e., at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical) to the target sequence of interest. Described in another way, for a target specific binding region 202 having a sequence with a length of from 10 to 100 nucleotides that is at least 95% complementary or at least 95% identical to a target sequence of interest, a region 202 that is from 20 nt to 35 nt in length may contain 1 mismatch, a region 202 that is from 40 nt to 50 nt in length may contain up to 2 mismatches, a region 202 that is from 60 nt to 70 nt may contain up to 3 mismatches, a region 202 that is from 80 nt to 90 nt may contain up to 4 mismatches, and a region 202 having a length of 100 nt may contain up to 5 mismatches with the target sequence.
In one embodiment, the method is used to generate a plurality of capture probes 200 each comprise a target-specific binding region 202 consisting of a sequence with a length of 35 nucleotides, that is at least 95% complementary, or at least 95% identical to a target sequence of interest (i.e., up to 1 mismatch with the target sequence).
The capture oligonucleotides may be designed to bind to a target region at selected positions spaced across the target region at various intervals. The capture oligo design and target selection process may also take into account genomic features of the target region such as genetic variation, G:C content, predicted oligo Tm, and the like. One of skill in the art can use art-recognized methods to determine the features of a target binding region that will hybridize to the target with minimal non-specific hybridization. For example, one of skill can determine experimentally the features such as length, base composition, and degree of complementarity that will enable a nucleic acid molecule (e.g., the target-specific binding region of a target capture probe) to specifically hybridize to another nucleic acid molecule (e.g., the nucleic acid target) under conditions of selected stringency, while minimizing non-specific hybridization to other substances or molecules. For example, for an exon target of interest, a target gene sequence is retrieved from a public database such as GenBank, and the sequence is searched for stretches of from 25 to 150 bp with a complementary sequence having a GC content in the range of 45% to 55%. The identified sequence may also be scanned to ensure the absence of potential secondary structure and may also be searched against a public database (e.g., a BLAST search) to ensure a lack of complementarity to other genes, as described in Example 3.
In some embodiments of the method, a set of capture probes (e.g., a library) is designed to specifically bind to target sequences across a genomic location, such as across a chromosomal region, and the capture probes are contacted with nucleic acid molecules from a total genomic library, or a whole-transcriptome library in order to analyze the whole transcriptome across the chosen genomic locus. In some embodiments of the method, a set of capture probes is designed to specifically bind to a plurality of target regions, such as the exons of a single gene, or multiple genes, such as at least 5 genes, at least 10 genes, at least 20 genes, at least 50 genes, at least 75 genes, at least 200 genes, at least 1000 genes, at least 10,000 genes, or more, as described in Examples 1-3. For example, as demonstrated in Example 3 herein, the methods according to this aspect of the invention were used to generate a set of capture probes comprising 1,148,286 distinct target-specific 35mer regions that were designed to capture all the exons from a total of 25,341 annotated genes from a sample containing nucleic acid sequences derived from a human.
In some embodiments of the method, a set of capture probes is designed to specifically bind to a genomic locus known to be associated with a clinical outcome or disease, or disease risk.
In some embodiments, the methods of the invention are used to capture and sequence a modified or mutated target, such as to determine the presence of a particular single nucleotide polymorphism (SNP), or deletion, addition, or other modification. In accordance with such embodiments, the set of target capture probes are typically designed such that there is a very dense array of capture probes that are closely spaced together such that a single target sequence, which may contain a mutation, will be bound by multiple capture probes that overlap the target sequence. For example, capture probes may be designed that cover every base of a target region, on one or both strands, (e.g., head to tail), or that are spaced at intervals of every 2, 3, 4, 5, 10, 15, 20, 40, 50, 90, 100, or more bases across a sequence region.
As another example, the selection of the target capture probes over a target region of interest is based on the size of the target region. For example, for a target region of less than 100 nucleotides in length, capture probes (either sense, antisense, or both) are typically designed to hybridize to target sequences spaced apart by from 0 to 100 nucleotides, such as every 45 nucleotides, or every 35 nucleotides. As another example, for a target region greater than 200 nucleotides, capture probes (either sense, antisense, or both) are typically designed to hybridize to target sequences spaced apart by from 0 to 200 nucleotides, such as at 45 to 65 nucleotide intervals, or at higher density coverage such as every 35 nucleotide intervals. In one embodiment, for a target region greater than 200 nucleotides (e.g., a 200,000-nucleotide target region), a set of sense and antisense capture probes are designed that are each about 35 nucleotides in length and are spaced about 45 nucleotides apart across the target region (alternating sense/antisense) in order to saturate the region (e.g., “tile” across the region of interest).
In one embodiment, a library of target specific probes are designed to bind to a desired target with high density coverage, such that at least one capture probe binds to at least every 35 nucleotide region of the target sequence. In one embodiment, a library of target specific probes are designed to bind to every nucleotide of the target, with alternating binding regions on the sense and antisense strands of the target sequence.
Referring now to
In operation, as shown in
As shown in
The 5′ flanking region 210 is typically from about 4 to about 30 nucleotides in length, such as from about 5 to about 15 nucleotides in length, or from about 5 to 10 nucleotides in length. The nucleotide sequence of the 5′ flanking region 210 is chosen to provide a first processing site, such as a first restriction enzyme recognition site, such as a type II restriction endonuclease (e.g., Bsm1), to generate the desired nucleotide overhang at the first end of the double-stranded precursor molecule for ligation in a head to tail configuration, and for precise cleavage of the 5′ flanking region 210 from the capture probe 200 region.
The 3′ flanking region 220 is typically from about 4 to about 30 nucleotides in length, such as from about 5 to about 25 nucleotides in length, or from about 10 to 20 nucleotides in length. The nucleotide sequence of the 3′ flanking region 220 is chosen to provide a second processing site, such as a second restriction enzyme recognition site (e.g., Bsm1), to generate the desired nucleotide overhang at the second end of the double-stranded molecule for ligation. In some embodiments, the 3′ flanking region 220 further comprises an additional processing site (e.g., a third restriction enzyme recognition site, such as Psi1 or HindIII) for precise cleavage of the 3′ flanking region 220 from the capture probe 200 region.
As illustrated in
It will be understood by one of skill in the art that Bsm1 is a non-limiting example of a type II restriction endonuclease that recognizes an asymmetric sequence and cleaves outside of that asymmetric sequence to yield the desired nucleotide overhangs at the first end and the second end of the double-stranded precursor molecule for ligation in a head to tail configuration. For example, dozens of type II restriction endonuclease enzymes are commercially available and known to those of skill in the art (see, e.g., New England Biolabs catalogue and REBASE web site) from which one of skill in the art could design a first processing site in the 5′ flanking region and a second processing site in the 3′ flanking region to provide ligation substrates that could only ligate in a head to tail configuration.
The double-stranded capture probe precursor 230 for use in various embodiments of the methods of the invention may be generated using a variety of methods. In one embodiment, the double-stranded capture probe precursor 230 is generated by synthesizing pairs of single-stranded complementary sense and antisense oligonucleotides comprising the full length sequence of the capture probe precursor 230 and annealing the strands together to form the double-stranded capture probe precursor. In another embodiment, a ligation ready, double-stranded capture probe precursor 230 having from one to four nucleotide overhangs on each end, is generated by annealing a pair of synthesized complementary strands together to produce the desired nucleotide overhang that will result in head-to-tail concatemers upon ligation.
In another embodiment, as shown in
Referring again to
At step 630, the processed capture probe precursors are ligated to generate head-to-tail concatemers 240, as illustrated in
At step 640, the head-to-tail concatemers 240 are amplified using any suitable amplification method, such as PCR amplification, in vitro transcription, Klenow, or isothermal amplification.
In one embodiment, the amplification of the head-to-tail concatemers 240 is carried out using isothermal amplification with either Bacillus subtilis phage phi29 polymerase (hereafter referred to as “phi29” polymerase) or Bacillus stearothermophilus (Bst) DNA polymerase large fragment, 5′→3′ exo− (hereafter referred to as “Bst DNA polymerase”). Isothermal amplification is based on random priming of denatured DNA, followed by strand-displacement synthesis at constant temperature, wherein multiple primers are extended over tens of kilobases, as described in Lage et al., Genome Res 13:294-307 (2003), incorporated herein by reference. The single-stranded DNA generated by strand displacement is targeted by new random priming events, and these new strands are elongated in the opposite direction, resulting in a hyperbranched network of amplified head-to-tail concatemers, as shown in
In one embodiment, as illustrated in
At step 650, the amplified head-to-tail concatemers are processed to release monomer double-stranded capture probe precursors. In one embodiment, as shown in
At step 660, the amplified monomer double-stranded precursor molecules are processed to remove the 5′ and/or 3′ flanking regions. In one embodiment, as shown in
At step 670, the monomer double-stranded precursor molecules are further processed to selectively remove the complementary strand of the capture probe to produce a population of single-stranded target specific capture probes 200.
In one embodiment, as shown in
Alternatively, the non-capture complementary strand may be removed by first adding exonuclease resistant adaptors to the capture probe strand and degrading away the non-capture complementary strand with any suitable double-strand DNA specific exonuclease, such as Exonuclease III. In another example, the capture strand may be selectively amplified by adding an amplification primer binding site in the 5′ flanking region 210 of the capture probe precursor 230, and selectively amplifying the plurality of amplified capture probe monomers 270 with an amplification primer that binds to the amplification primer binding site.
The synthesized library of single-stranded precursor molecules 100 is then cleaved off the substrate 102, annealed to a common reverse primer 150 and extended with Klenow, to generate a library of double-stranded capture probe precursor molecules 230 (best illustrated in
Referring again to
In some embodiments of the methods described herein, a library of capture probe precursors in the form of head-to-tail concatemers, with reference to
The population of single-stranded capture probes 200 can be used in solution based capture methods as described herein. As demonstrated in Example 2, and shown in
The level of representation of expected nucleic acid sequences in a library generated according to the methods of the invention typically has a variation of less than about 30% (such as a variation of less than about 20%). The level of representation of expected nucleic acid sequence in the final single stranded capture probe library 200 may be assessed using various methods. For example, as described in Example 2, a capture probe library 200 may be used for solution-based capture of a set of targets (e.g., the 13 exons of the AKT gene), and the standard deviation of the exon to exon capture efficiency, expressed as a percentage of the fold-enrichment can be determined, as shown in
Oligonucleotide Synthesis
DNA synthesis of the various oligonucleotides of the invention (e.g., single-stranded nucleic acid molecules having predetermined sequences, capture probe precursors and universal adaptor oligonucleotides) can be carried out by any art-recognized chemistry, including phosphodiester, phosphotriester, phosphate triester, or N-phosphonate and phosphoramidite chemistries (see, e.g., Froehler et al., Nucleic Acid Res. 14:5399-5407, 1986; McBride et al., Tetrahedron Lett. 24:246-248, 1983). Methods of oligonucleotide synthesis are well known in the art and generally involve coupling an activated phosphorous derivative on the 3′ hydroxyl group of a nucleotide with the 5′ hydroxyl group of the nucleic acid molecule (see, e.g., Gait, Oligonucleotide Synthesis: A Practical Approach, IRL Press, 1984).
A population of nucleic acid molecules can be synthesized on a substrate by any art-recognized means including, for example, photolithography (see, Lipshutz et al., Nat. Genet. 21(1 Suppl):20-24, 1999) and piezoelectric printing (see, Blanchard et al., Biosensors and Bioelectronics 11:687-690, 1996). In some embodiments, nucleic acid molecules are synthesized in a defined pattern on a solid substrate to form a high-density microarray. Techniques are known for producing arrays containing thousands of oligonucleotides comprising defined sequences at defined locations on a substrate (see, e.g., Pease et al., Proc. Nat'l. Acad. Sci. 91:5022-5026, 1994; Lockhart et al., Nature Biotechnol. 14:1675-80, 1996; and Lipshutz et al., Nat. Genet. 21 (1 Suppl):20-4, 1999).
In some embodiments, populations of nucleic acid molecules are synthesized on a substrate, to form a high density microarray, by means of an ink jet printing device for oligonucleotide synthesis, such as described by Blanchard in U.S. Pat. No. 6,028,189; Blanchard et al., Biosensors and Bioelectrics 11:687-690 (1996); Blanchard, Synthetic DNA Arrays in Genetic Engineering, Vol. 20, J. K. Setlow, Ed. Plenum Press, New York at pages 111-123; and U.S. Pat. No. 6,028,189 issued to Blanchard. The nucleic acid sequences in such microarrays are typically synthesized in arrays, for example, on a glass slide, by serially depositing individual nucleotide bases in “microdroplets” of a high surface tension solvent such as propylene carbonate. The microdroplets have small volumes (e.g., 100 picoliters (pL) or less, or 50 pL or less) and are separated from each other on the microarray (e.g., by hydrophobic domains) to form surface tension wells which define the areas containing the array elements (i.e., the different populations of nucleic acid molecules). Microarrays manufactured by this ink-jet method are typically of high density, typically having a density of at least about 2,000 different nucleic acid molecules per 1 cm2. The nucleic acid molecules may be covalently attached directly to the substrate, or to a linker attached to the substrate at either the 3′ or 5′ end of the polynucleotide. Exemplary chain lengths of the synthesized nucleic acid molecules suitable for use in the present methods are in the range of about 20 to about 100 nucleotides in length, such as 50 to 100, 60 to 100, 70 to 100, 80 to 100, or 90 to 100 nucleotides in length. In some embodiments, the nucleic acid molecules are in the range of 80 to 100 nucleotides in length.
Exemplary ink jet printing devices suitable for oligonucleotide synthesis in the practice of the present invention contain microfabricated ink-jet pumps, or nozzles, which are used to deliver specified volumes of synthesis reagents to an array of surface tension wells (see, Kyser et al., J. Appl. Photographic Eng. 7:73-79, 1981).
In some embodiments, a population of nucleic acid molecules is synthesized to form a high-density microarray. A DNA microarray, or chip, is an array of nucleic acid molecules, such as synthetic oligonucleotides, disposed in a defined pattern onto defined areas of a solid support (see, Schena, BioEssays 18:427, 1996). The arrays are preferably reproducible, allowing multiple copies of a given array to be produced and easily compared with each other. Microarrays are typically made from materials that are stable under nucleic acid molecule hybridization conditions. In some embodiments, the nucleic acid molecules on the array are single-stranded DNA sequences. Exemplary microarrays and methods for their manufacture and use are set forth in T. R. Hughes et al., Nature Biotechnology 19:342-347, April 2001, which publication is incorporated herein by reference.
In some embodiments, the methods of the invention utilizes oligonucleotides that are synthesized on a multiplex parallel DNA synthesis system based on an integrated microfluidic microarray platform for parallel production of oligonucleotides, wherein the DNA synthesis system utilizes photogenerated acid chemistry, parallel microfluidics and a programmable digital light controlled synthesizer, as described in U.S. Patent Pub. No. 2007/0059692, Gao et al., Biopolymers 73:579-596 (2004), and Zhou et al., Nucleic Acids Research 32(18):5409-5417 (2004), each of which is incorporated herein by reference.
In some embodiments, the methods of the invention utilize synthesized oligonucleotides that are cleaved off a substrate, such as a microarray. The synthesized nucleic acid molecules can be harvested from the substrate by any useful means. In some embodiments, the portion of the nucleic acid molecule that is directly attached to the substrate, or attached to a linker that is attached to the substrate, is attached to the substrate or linker by an ester bond which is susceptible to hydrolysis by exposure to a hydrolyzing agent, such as hydroxide ions, for example, an aqueous solution of sodium hydroxide or ammonium hydroxide. The entire substrate can be treated with a hydrolyzing agent, or alternatively, a hydrolyzing agent can be applied to a portion of the substrate. For example, a silane linker can be cleaved by exposure of the silica surface to ammonium hydroxide, yielding various silicate salts and releasing the nucleic acid molecules with the silane linker into solution. In some embodiments, ammonium hydroxide can be applied to the portion of a substrate that is covalently attached to the nucleic acid molecules, thereby releasing the nucleic acid molecules into the solution (see, Scott and McLean, Innovations and Perspectives in Solid Phase Synthesis, 3rd International Symposium, 1994, Mayflower Worldwide, pp. 115-124).
In another aspect, the present invention provides a method for enriching a library for target nucleic acid regions of interest. The methods according to this aspect of the invention comprise: (a) amplifying a plurality of head-to-tail concatemers formed from ligating a population of double-stranded nucleic acid precursor molecules, wherein each double-stranded precursor molecule in the starting population comprises a target capture probe region comprising (i) a target-specific binding region comprising a nucleic acid sequence that is at least 95% identical to at least a portion of the sense or antisense strand of a target nucleic acid sequence of interest and (ii) a region for binding to a capture reagent; wherein the target capture region is flanked on the 5′ end by a 5′ flanking region comprising a first processing site and is flanked on the 3′ end by a 3′ flanking region comprising a second processing site; (b) processing the amplified head-to-tail concatemers to release double-stranded monomer precursor molecules; (c) selectively removing the complement strand of the double-stranded monomer precursor molecules to generate a population of single-stranded capture probes, each capture probe comprising (i) a target-specific binding region comprising a nucleic acid sequence that is at least 95% identical to at least a portion of the sense or antisense strand of a target nucleic acid sequence of interest and (ii) a region for binding to a capture reagent; (d) contacting the population of single-stranded capture probes with a library comprising at least one target nucleic acid sequence of interest under conditions that allow binding between the capture probes and the at least one nucleic acid target region of interest, to form a mixture comprising a plurality of complexes between target regions of interest and capture probes; (e) contacting the mixture of step (d) with a capture reagent and separating the capture reagent bound complex from the mixture; and (f) eluting the target regions of interest from the capture reagent bound complex.
The steps (a) to (c) may be carried out as previously herein described. The steps (d) to (f) of enriching a library for target sequences with the population of single-stranded capture probes may be carried out as illustrated in
The annealing step is typically carried out by mixing a molar excess of capture probes (or capture probes plus universal adaptor oligos) with the library in a high salt solution comprising from 100 mM to 2 M NaCl (osmolarity=200 to 4000 molar). An exemplary high salt solution for annealing is 10 mM Tris pH 7.6, 0.1 mM EDTA, 1 M NaCl (osmolarity=2000 molar). The nucleic acid molecules in the mixture are then denatured (i.e., by heating to 94 degrees) and allowed to cool to room temperature. In one embodiment, the annealing step is carried out in a high salt solution comprising from 100 mM to 2 M NaCl with the addition of 0.1% triton X100 (or Tween or NP40) nonionic detergent.
An amount of capture reagent 400 is added to the annealed mixture sufficient to generate a plurality of complexes each containing a nucleic acid molecule, a capture probe (or a capture probe and a universal adaptor oligo), and a capture reagent. This step is carried out in a high salt solution comprising from 100 mM to 2 M NaCl (osmolarity=200 to 4000 molar). An exemplary high salt solution for anneal is 10 mM Tris pH 7.6, 0.1 mM EDTA, 1 M NaCl (osmolarity=2000 molar). The mixture is incubated at room temperature with mixing for about 15 minutes.
The complexes formed are then isolated or separated from solution with a sorting device 500 (e.g., a magnet) that pulls or sorts the capture reagent 400 out of solution.
The sorted complexes bound to the capture reagent 400 are washed with a low salt wash buffer (less than 10 mM NaCl, and more preferably no NaCl) to remove non-target nucleic acids. An exemplary low salt wash buffer is 10 mM Tris pH 7.6, 0.1 mM EDTA (osmolarity=10 millimolar). In some embodiments, the low salt wash optionally contains from 15% to 30% formamide, such as 25% formamide (osmolarity=6.3 molar). For each wash step, the capture reagent 400 bound to the complexes (e.g., magnetic beads) are resuspended in the low salt wash buffer and rocked for 5 minutes, then sorted again with the sorting device (magnet). The wash step may be repeated 2 to 4 times.
The nucleic acid molecules containing the target sequences are then eluted from the complexes bound to the capture reagent as follows. The washed complexes bound to the capture reagent 400 are resuspended in water, or in a low salt buffer (i.e., osmolarity less than 100 millimolar), heated to 94° C. for 30 seconds, the capture reagent (e.g., magnetic beads) is pulled out using a sorting device (e.g., magnet), and the supernatant (eluate) containing the target nucleic acid molecules is collected.
The eluate may optionally be amplified in a PCR reaction with a first PCR primer that binds to the first primer binding site 22 in the first linker and a second PCR primer that binds to the second primer binding site 32 in the second linker, producing an enriched library which can be optionally sequenced.
In another aspect, the present invention provides kits for generating a population of single-stranded nucleic acid molecules from a population of precursor double-stranded molecules. The kits according to this aspect of the invention are useful for carrying out various embodiments of the methods of the invention described herein. The kits in accordance with this aspect of the invention comprise (a) a plurality of random 7-mer oligonucleotide primers, (b) at least one of phi29 polymerase or Bst DNA polymerase large fragment 5′-3′ exo-; and (c) a lambda exonuclease enzyme. In some embodiments of the kit, the plurality of random 7-mer oligonucleotide primers each comprise an additional two nitroindole residues at the 5′ end and a phosphorothioate linkage at the 3′ end (SEQ ID NO:77). In some embodiments, the kit may further comprise at least one of the following: an alkaline phosphatase enzyme, at least one type II restriction enzyme, a DNA ligase, and a DNA polymerase enzyme (Klenow).
In an embodiment of the kit comprising phi29 polymerase, the kit may optionally further comprise one or more of the following reagents: (i) a phi29 concentrated stock reaction buffer comprising at least one of the following: Tris-HCL (e.g., at 50 mM for 10× buffer), (NH4)2SO4 (e.g., at 10 mM for 10× buffer), MgCl2 (e.g., 10 mM for 10× buffer) and dithiothreitol (e.g., 4 mM for 10× buffer); (ii) a concentrated stock of dNTPs (e.g. from 100 μM to 10 mM dNTPs); and (iii) a reducing agent, such as dithiothreitol. In further embodiments, the kit may optionally comprise at least one or more of the following; a common reverse primer oligo 150 designed to hybridize to the region 204 for binding to the synthesized oligos 100, Klenow enzyme, at least one Type II Restriction Enzyme, ligase, and alkaline phosphatase.
The following examples merely illustrate the best mode now contemplated for practicing the invention, but should not be construed to limit the invention.
All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. In some cases, the compositions and methods of this invention have been described in terms of embodiments, however these embodiments are in no way intended to limit the scope of the claims, and it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain components which are both chemically and physiologically related may be substituted for the components described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
This Example demonstrates a method for uniformly amplifying a library of target capture probes specific for the AKT gene designed for solution based capture of the 13 exons of the AKT gene.
Rationale:
Several recent studies describe the complete resequencing of human genomes (Wang et al., Nature 456:60 (2008); Bentley et al., Nature 456:53 (2008); Ley et al., Nature 456:66 (2008)). One in particular describes the full genome sequence of a tumor and normal adjacent tissue (Ley et al. (2008)). Remarkably, the authors distill their analysis of the complete human genome to ten mutated genes, highlighting the point that a small percentage of human genome sequence variations, including the protein coding regions and some surrounding flanking sequences, is interpretable. At the opposite end of the spectrum, candidate gene resequencing strives to test phenotype to genotype hypotheses by seeking sequence variation in specific genes that are thought to influence traits. The caveat is that the selection of candidate genes can be somewhat arbitrary. The ideal solution is one in which the interpretable regions, referred to as the “exome” of the genome, can be selectively resequenced. As used herein, the term “exome” refers to the collection of genomic segments that include protein coding regions, exons, promoters, known ncRNAs (non-coding RNAs) and UTRs, altogether comprising about 2% of the human genome. Interestingly, if the exome could be captured from a sample, then the sequencing data from a single flow cell from a highly parallel sequencing technology (e.g., a single Illumina GAII flow cell) would be adequate to address all of the diploid variation present in the exomic fraction of that sample.
Solution-based capture of the human exome would require ˜2 million oligonucleotides. Even with state-of-the-art, high-throughput oligo synthesis stations, the cost of each capture oligo is $7 and therefore the cost of synthesizing an exome solution based capture library would be approximately $14 million. An alternative—synthesized oligonucleotide libraries cleaved from microarrays, also initially appears to be an untenable solution due to the high cost involved to obtain sufficient yields of material. Each microarray typically produces about 50,000 individual oligonucleotide sequences that are high-quality, cleavable oligos in picomole quantities (e.g., commercially available from Agilent, Santa Clara, Calif.). Thus, the cost of synthesizing a library of exome capture oligonucleotide probes from microarrays (requiring ˜40 arrays at an estimated cost of $400,000) is substantially reduced relative to conventional oligonucleotide synthesis, however, the yield of material produced by microarray synthesis is sufficient for only four solution based capture experiments. Moreover, the use of high density probe coverage (i.e., more than 4 probes per exon, or at least one capture probe per 35 nt), as shown in
This Example demonstrates a method for uniformly amplifying a library of synthesized capture oligonucleotides for use in solution based capture methods.
Design of Capture Probe Oligonucleotide Precursors for Amplification and Processing into a Library of Capture Probes:
As shown in
Design of Capture Probe Precursor Oligonucleotides:
As shown in
Design of the Target-Specific Region 202 of the Capture Probe 200:
In this Example, a 64 oligonucleotide library was synthesized for high density solution based capture of the 13 exon AKT1 gene (NM—005163). As illustrated in
Design of the Region 204 for Binding to a Capture Reagent 300:
The AKT1 capture probes 200 in the library were designed such that each final processed single-stranded capture probe had a 34 nucleotide common region 204 for binding to the universal oligo adaptor capture reagent 300.
As shown in
Design of the 5′ Flanking Region 210 of the Capture Probe Precursor 230:
The double-stranded AKT1 capture probe precursors 230 were designed to include a 5′ flanking region 210 comprising a first restriction enzyme site for Bsm1, in order to create a two nucleotide “GG” overhang at the first end of the precursor molecules 230 after digestion with Bsm1 to facilitate ligation into head-to-tail concatemers.
The 5′ flanking region 210 of the capture probe precursors in this Example had the following sequence: 5′ CGCGAATGCC 3′ (SEQ ID NO:2) to provide a first Bsm1 site.
Design of the 3′ Flanking Region 220 of the Capture Probe Precursor 230:
The double-stranded AKT1 capture probe precursors 230 were designed to include a 3′ flanking region 220 comprising a second restriction enzyme site for Bsm1, in order to create a two nucleotide “CC” overhang at the second end of the precursor 230 molecule after digestion with Bsm1 to facilitate ligation into head-to-tail concatemers.
The 3′ flanking region was designed to also include a third restriction enzyme recognition sequence to precisely cleave off the 3′ flanking region after amplification, and also leave 5′ terminal phosphates on the complementary strands (e.g., Psi1 or HindIII).
Using the general design principles described above, several series of oligonucleotides were synthesized as follows:
“A” Series: a control set of 64 AKT capture probes that were directly synthesized (not amplified) having a total length of 69 nucleotides.
“B” Series: a set of 64 AKT capture probe precursor oligonucleotides for amplification, but which did not impose the head to tail ligation characteristics (later abandoned, as discussed below in the results section).
“C” Series: a set of 64 AKT capture probe precursor oligonucleotides for amplification, having a total length of 79 nucleotides (34 nt 204 region that binds to the reverse primer 150, with the same 5′ flanking region 210 and target specific region 202 as D series). Note: this series was more expensive to synthesize because they required 0.2 micromolar synthesis, due to the longer size.
“D” Series: a set of 64 AKT capture probe precursor oligonucleotides for amplification, having a total length of 60 nucleotides, including a 15 nt 204 region that binds to the reverse primer 150, with a 10 nucleotide 5′ flanking region 210 and a 35 nucleotide target-specific region 202. Note: this series was much less expensive because it was synthesized on a 50 nmole scale, due to shorter size, which was then extended with the reverse primer and Klenow fill-in reaction.
As described above, the C and D series oligos were designed such that after the fill-in reaction with the common reverse primer 150, a pair of asymmetric Bsm1 sites were created on the double-stranded precursor molecule 230, such that following digestion with Bsm1, a two nucleotide “CC” overhang is present on the top strand of each double-stranded oligonucleotide and a two nucleotide “GG” overhang is present on the bottom strand of each double-stranded oligonucleotide, thus creating a situation in which only head-to-tail ligation events are allowed. Moreover, ligation recreates the Bsm1 site for downstream processing after amplification. As described in detail below, the Bsm1 digested amplification products were then treated with Antarctic phosphatase to dephosphorylate the 5′ end of the capture strand while leaving an exposed 5′ phosphate on the complement strand to allow for degradation by treatment with lambda exonuclease, to generate the desired single-stranded capture probes.
For the “D” Series oligos each capture probe precursor 100 was synthesized as shown below in TABLE 1, with the 5′ regions 210 and the 3′ regions 220 underlined.
CGCGAATGCCGGTGCCCGAGGCTCCCGCGACGCTCACGCGCTCCTACG
CGTGGCGGATGT
CGCGAATGCCATGAGCGACGTGGCTATTGTGAAGGAGGGTTGGCTACG
CGTGGCGGATGT
CGCGAATGCCCCAGCCCTGGCAGCGGGTACTAACCTCGTTTGTGCACG
CGTGGCGGATGT
CGCGAATGCCGCCTGGGGAGGGAGAGATGGGGGTAGTAGCCCCAGACG
CGTGGCGGATGT
CGCGAATGCCCTACAGACGTGCGGGTGGTGAGAGCCACGCACACTACG
CGTGGCGGATGT
CGCGAATGCCGGGAGTACATCAAGACCTGGCGGCCACGCTACTTCACG
CGTGGCGGATGT
CGCGAATGCCTTGTAGCCAATGAAGGTGCCATCATTCTTGAGGAGACG
CGTGGCGGATGT
CGCGAATGCCGGAGCGGCCGCAGGATGTGGACCAACGTGAGGCTCACG
CGTGGCGGATGT
CGCGAATGCCGGGATACTTACGCGCCACAGAGAAGTTGTTGAGGGACG
CGTGGCGGATGT
CGCGAATGCCCTTGGCCTCTCGGGATTCAGATTTGGGGGGTTGGCACG
CGTGGCGGATGT
CGCGAATGCCCTGCGGGCAGGCAGAGCCTCTGTCTGCGTGCATCCACG
CGTGGCGGATGT
CGCGAATGCCGTGGTCCACTGCAGGCAGCGGATGATGAAGGTGTTACG
CGTGGCGGATGT
CGCGAATGCCTGTCATCGAACGCACCTTCCATGTGGAGACTCCTGACG
CGTGGCGGATGT
CGCGAATGCCCCTGGCCTGGCCGCCACAGCCCACGTACCGCTCCTACG
CGTGGCGGATGT
CGCGAATGCCCTGCAGGAGGTCAGGTGAGGCTGCAGGCCTGTACCACG
CGTGGCGGATGT
CGCGAATGCCGGAGGAGTGGACAACCGCCATCCAGACTGTGGCTGACG
CGTGGCGGATGT
CGCGAATGCCGTCCATCTCCTCCTCCTCCTGCTTCTTGAGGCCGTACG
CGTGGCGGATGT
CGCGAATGCCTTCCGGTCGGGCTCACCCAGTGACAACTCAGGGGCACG
CGTGGCGGATGT
CGCGAATGCCGCTTGGGCTTGGCCAGGGACACCTCCATCTCTTCAACG
CGTGGCGGATGT
CGCGAATGCCACCGCGTGGTGAGGCCTGTCCCCACTTCTGCCTGTACG
CGTGGCGGATGT
CGCGAATGCCCTATGGGCAGGCACCAGGGTCAGCAAGCGGCGCTGACG
CGTGGCGGATGT
CGCGAATGCCACCATGAACGAGTTTGAGTACCTGAAGCTGCTGGGACG
CGTGGCGGATGT
CGCGAATGCCCCTTCACCAGGATCACCTTGCCGAAAGTGCCCTTGACG
CGTGGCGGATGT
CGCGAATGCCAGAAGGCCACAGGCCGCTACTACGCCATGAAGATCACG
CGTGGCGGATGT
CGCGAATGCCGGCCCCACCTTGGCCACGATGACTTCCTTCTTGAGACG
CGTGGCGGATGT
CGCGAATGCCCTGTAAAGCAGGGCTGGGTGAGCTGCCACCCCGCAACG
CGTGGCGGATGT
CGCGAATGCCGACGAGGTGGCCCACACACTCACCGAGAACCGCGTACG
CGTGGCGGATGT
CGCGAATGCCTCACTGTGAGGAAGGGGTGCCTGGAGTTCTGCAGGACG
CGTGGCGGATGT
CGCGAATGCCGTGGGAGCCCAGATGGGGCTGAAGGGCTGGGGCCAACG
CGTGGCGGATGT
CGCGAATGCCCTGCAAGGAAGGGGAGCTGGAACTGCGGCCCCACAACG
CGTGGCGGATGT
CGCGAATGCCGCCCTGAAGTACTCTTTCCAGACCCACGACCGCCTACG
CGTGGCGGATGT
CGCGAATGCCCCTCGCCCCCGTTGGCGTACTCCATGACAAAGCAGACG
CGTGGCGGATGT
CGCGAATGCCTAGGGGCTGGGGCTGCGGGGGATGGACTTCGCGGCACG
CGTGGCGGATGT
CGCGAATGCCCTGCGGGAGGCGCAACCTGAGGCACAGCCGTGGCTACG
CGTGGCGGATGT
CGCGAATGCCCTGTTCTTCCACCTGTCCCGGGAGCGTGTGTTCTCACG
CGTGGCGGATGT
CGCGAATGCCCAATCTCAGCGCCATAGAAGCGGGCCCGGTCCTCGACG
CGTGGCGGATGT
CGCGAATGCCTGTCAGCCCTGGACTACCTGCACTCGGAGAAGAACACG
CGTGGCGGATGT
CGCGAATGCCGCCCGCCAGCGCACCTTGAGGTCCCGGTACACCACACG
CGTGGCGGATGT
CGCGAATGCCCTAGGGGAAAGGTGGCCTCAGGTCAGTGCCGCCAGACG
CGTGGCGGATGT
CGCGAATGCCCTGGAGAACCTCATGCTGGACAAGGACGGGCACATACG
CGTGGCGGATGT
CGCGAATGCCTCCCCTCCTTGCACAGCCCGAAGTCTGTGATCTTAACG
CGTGGCGGATGT
CGCGAATGCCTCAAGGACGGTGCCACCATGAAGACCTTTTGCGGCACG
CGTGGCGGATGT
CGCGAATGCCGGGGCGCACACCTCGGGGGCCAGGTACTCAGGTGTACG
CGTGGCGGATGT
CGCGAATGCCCTGCACGGGTGGCAGATGGGCAGGACTCGGCATCAACG
CGTGGCGGATGT
CGCGAATGCCGTGCTGGAGGACAATGACTACGGCCGTGCAGTGGAACG
CGTGGCGGATGT
CGCGAATGCCTCATCTCGTACATGACCACGCCCAGCCCCCACCAGACG
CGTGGCGGATGT
CGCGAATGCCTGTGCGGTCGCCTGCCCTTCTACAACCAGGACCATACG
CGTGGCGGATGT
CGCGAATGCCATCTCCTCCATGAGGATGAGCTCAAAAAGCTTCTCACG
CGTGGCGGATGT
CGCGAATGCCCCGCTTCCCGCGCACGCTTGGTCCCGAGGCCAAGTACG
CGTGGCGGATGT
CGCGAATGCCCTTGGGGTCCTTCTTGAGCAGCCCTGAAAGCAAGGACG
CGTGGCGGATGT
CGCGAATGCCCAGAGGTGAGGGCCGCCCATCCCAGCTACAGGCTAACG
CGTGGCGGATGT
CGCGAATGCCCTGCAGGCAGGAAACAAGGCCACAGTGTCGGTACCACG
CGTGGCGGATGT
CGCGAATGCCGCTTGGCGGGGGCTCCGAGGACGCCAAGGAGATCAACG
CGTGGCGGATGT
CGCGAATGCCCTGCCACACGATACCGGCAAAGAAGCGATGCTGCAACG
CGTGGCGGATGT
CGCGAATGCCCACGTGTACGAGAAGAAGGTGCGGCTGCTCCCCGCACG
CGTGGCGGATGT
CGCGAATGCCCTGCAGAGGTGGGCAGACGGGACAGTCATGAGCTTACG
CGTGGCGGATGT
CGCGAATGCCCTCAGCCCACCCTTCAAGCCCCAGGTCACGTCGGAACG
CGTGGCGGATGT
CGCGAATGCCCCGTGAACTCCTCATCAAAATACCTGGTGTCAGTCACG
CGTGGCGGATGT
CGCGAATGCCCCCAGATGATCACCATCACACCACCTGACCAAGGTACG
CGTGGCGGATGT
CGCGAATGCCCTGTGGGTGTAGACAGCTCAGACCCCGGTGCCCCAACG
CGTGGCGGATGT
CGCGAATGCCATGACAGCATGGAGTGTGTGGACAGCGAGCGCAGGACG
CGTGGCGGATGT
CGCGAATGCCCCGCTGGCCGAGTAGGAGAACTGGGGGAAGTGGGGACG
CGTGGCGGATGT
CGCGAATGCCCACGGCCTGAGGCGGCGGTGGACTGCGCTGGACGAACG
CGTGGCGGATGT
Oligo Synthesis: For this experiment, the initial population of oligonucleotides 100 were synthesized individually in solution by Operon (Huntsville, Ala.). In the future, the oligonucleotides will be synthesized on an array then cleaved. For example, synthesized and cleaved oligonucleotides are commercially available (e.g., available from LC Sciences, Houston, Tex., Agilent also manufactures “Sure-print” oligo arrays, cleaves the oligos and delivers pmol quantities of single-strand reagent).
Annealing and Fill-in to Generate Double-Stranded Capture Probe Precursors:
As shown in
Design of the Second Strand Reverse Oligonucleotide 150:
The following reverse primer oligonucleotides 150 were used in this Example:
Preparation of Oligo Pools and Annealing:
As shown in
Pooling of Oligonucleotides:
Each A, B, C, and D series oligos were resuspended to 100 μM, then each of the oligos in the A, B, C, and D series were pooled separately, to create an A series pool, a B series pool, a C series pool, and a D series pool at 100 μM, each pool containing a mixture of 64 different oligonucleotides. The DNA concentration of the oligo pool (100 μm of 70 to 80mer=2 μg/μl) was confirmed by agarose gel electrophoresis by diluting the pool 50-fold to 40 ng/μl, and loading 2.5 μl and 5 μl.
The common reverse primer 150 (#2: SEQ ID NO:68) for second strand synthesis was resuspended to 100 μM.
Annealing:
The following reagents were combined to give a 1 μM solution in each primer (40 ng/μl combined):
10 μl of 100 μM primer pool C or D (SEQ ID NO:3 to SEQ ID NO:66)
10 μl of 100 μM reverse primer #2 (SEQ ID NO:68)
100 μl New England Biolabs Buffer #4
880 μl H20
1000 μl total
A 100 μl aliquot of the above mixture was heated to 95° C., then cooled down as shown below to room temperature:
95° C., 2 minutes
80° C., 1 minute
75° C., 1 minute
70° C., 1 minute
65° C., 1 minute
60° C., 1 minute
55° C., 1 minute
Room temperature, hold.
A 2.5 μl and 5.0 μl aliquot of the annealed mixture was checked on an agarose gel.
Fill-in Reaction:
The annealed mixture was then treated with Klenow to fill in both strands, thus generating a population of blunt ended, double-stranded precursor molecules 230 as shown in
Second strand synthesis of each of the annealed oligo mixtures “C” and D″ were carried out as follows:
100 μl annealed primer mixture, described above
1 μl 10 mM dNTPs
2 μl Klenow (3′ to 5′ exo-) 5000 units/ml (M0212S), New England Biolabs, MA
Incubated at 37° C. for 30 minutes, 75° C. for 20 minutes. A 3 μl or 6 μl aliquot of each pool was checked on an agarose gel.
Exemplary double-stranded capture probe precursors 230 are provided below based on the “D” series oligo for exon 1 (−35) (SEQ ID NO:3), provided in Table 1:
The top strand of the filled-in double stranded product (SEQ ID NO:3 annealed to Reverse primer #1 SEQ ID NO:67) is:
GCGTGGCGGATGTGGACCCCTTCGAGCAATTATAAGAATGCCGACC
The bottom strand of the filled-in double-stranded product (SEQ ID NO:3 annealed to Reverse Primer #1 SEQ ID NO:67) is:
CG-3′
The top strand of the filled-in double-stranded product (SEQ ID NO:3 annealed to Reverse Primer #2 SEQ ID NO:68) is:
GCGTGGCGGATGTGGACCCCTTCGAGCAATTAAGCTTGAATGCCGACC
The bottom strand of the filled-in double stranded product shown is:
CG-3′
Digestion with Bsm1 to Generate Ligation Substrates:
As shown in
2 μl of Bsm1 (10,000 units/ml, R0134S, New England Biolabs) was added to each of the 100 μl heat inactivated fill-in reactions and incubated at 65° C. for 1 hour. 3 μl and 6 μl of each pool was checked on an agarose gel. The Bsm1 digests were then purified over a QIAQUICK column (Qiagen), eluted and quantified by nanodrop.
Resulting exemplary double-stranded oligonucleotide structures after Bsm1 digestion, as illustrated in
The top strand of the Bsm1 digested double-stranded oligo (SEQ ID NO:69/SEQ ID NO:70) is:
TGTGGACCCCTTCGAGCAATTATAAGAATGCC 3′
The bottom strand of the Bsm1 digested double-stranded oligo (SEQ ID NO:69/SEQ ID NO:70) is:
CGCGTGAGCGTCGCGGGAGCCTCGGGCACCGG3′
The top strand of the Bsm1 digested double-stranded oligo (SEQ ID NO:71/SEQ ID NO:72) is:
TGTGGACCCCTTCGAGCAATTAAGCTTGAATGCC 3′
The bottom strand of the Bsm1 digested double-stranded oligo (SEQ ID NO:71/SEQ ID NO:72) is:
AGCGCGTGAGCGTCGCGGGAGCCTCGGGCACCGG3′
Ligation of Bsm1 Digested Precursors to Form Head-to-Tail Concatemers:
As shown in
Ligation was carried out as follows:
The following reagents were combined for each pool “C” and “D”:
50 μl 2× quick ligase buffer (New England Biolabs)
11 μl of 48 ng/μl Bsm1 digested pool C or D
34 μl H2O
5 μl ligase (New England Biolabs)
100 μl total
Incubated at room temperature for ≧10 minutes. A no DNA control (no template) was also prepared.
As illustrated in
Amplification of Head-to-Tail Concatemers:
As shown in
The random amplification primer 250 used in this Example was a random 7-mer amplification primer with an additional two nitroindole residues at the 5′ end and a phosphorothioate linkage at the 3′ end: 5′[nitroindole]2-[N]6-(phosphothioate)-N (SEQ ID NO:77, wherein the “N” at positions 1-7 may be A, G, C or T), as described by Lage et al., Genome Res 13:294-307 (2003), incorporated herein by reference.
Isothermal Amplification:
Four reaction mixtures of “C” pool and “D” pool ligations were prepared as follows, along with 2 reaction mixtures of a “no template” control:
25 μl of 100 μM amplification primer [5-nitroindole]2-[N]6-(phosphothioate)-N (SEQ ID NO:77)
10 μl ligated template “C” or “D” pool (or no template control)
5 μl 10× phi29 buffer (New England Biolabs)
10 μl H20
50 μl total
The above reagents were mixed and incubated at 95° C. for 3 minutes, then cooled to room temperature, then 50 μl of the following enzyme premix was added:
Enzyme Premix:
25 μl of 100 μM amplification primer (SEQ ID NO:77)
10 μl H20
5 μl 10× phi29 buffer (NEB)
4 μl 10 mM dNTPs
2 μl 100 mM DTT
2 μl 10 mg/ml BSA (NEB)
2.5 μl phi29 polymerase (NEB)
50 μl total volume of enzyme premix
The 50 μl annealed mixture was combined with the 50 μl enzyme premix, then incubated at 30° C. for 12 hours. The polymerase was inactivated by incubation at 65° C. for 10 minutes, then cooled to 4° C. The mixture was briefly centrifuged to pellet the protein and the supernatant was transferred to a fresh tube.
The isothermal amplification reactions were then ethanol precipitated by combining the four “C” reactions and the four “D” reactions (separately), then adding 600 μl TEzero, split into two tubes of 500 μl each, then adding 120 μl 3M NaOAc at pH 5.2 to each tube, then 1200 μl ethanol. The reactions were centrifuged for 10 minutes at 12K RPM and the pellets were resuspended in 880 μl TEzero. The amount of DNA recovered was quantitated. The yield was determined to be 65 to 79 ng/μl for all four tubes, therefore, each 100 μl isothermal amplification reaction produced 28.5 to 30 μg of DNA, which was an unexpectedly high yield. It is noted that the isothermal amplification reaction carried out with 50 μM of the random amplification primer [5-nitroindole]2-[N]6-(phosphothioate)-N (SEQ ID NO:77) in combination with 400 nM dNTPs and DTT as described above provided reaction conditions that yielded a significantly higher amount of amplification product than was obtained from an amplification reaction with the same templates using the reagents from a commercially available kit (GE Healthcare Life Sciences).
It is noted that an initial attempt was made to simply ligate dsDNA blunt-end oligonucleotide probes together followed by amplification (Series “B” pool). However, it was determined that this initial approach was not suitable for uniformly amplifying a population of probes for solution-based capture because the oligonucleotides were ligated in random head to tail, head to head and tail to tail orientation. In the head to head and tail to tail orientations some common tail sequences hybridized together, thereby creating a snap-back stem in ssDNA. These ssDNA stem regions were poisonous to polymerases, resulting in under-representation of amplified products.
Digestion of Amplified Concatenated Strands into Monomer Double-Stranded Capture Probe Precursors:
As shown in
Digestion with Bsm1 after Isothermal Amplification:
55 μg from each amplification reaction was digested with 20 μl Bsm1 in a total volume of 1 ml in 1×NEB Buffer #4 buffer at 65° C. for two hours. After the two hour digestion, an aliquot of each digest was checked on an agarose gel. The majority of the “C” and “D” digested pool showed the expected 90 bp product, with a small amount of 130 bp product.
Phosphatase Treatment:
110 μl 10× Antarctic Phosphatase buffer (NEB) was added to the Bsm1 digested pool C and D. 50 μl Antarctic phosphatase was added (NEB), and incubated for one hour at 37° C. then 65° C. for 15 minutes. An aliquot of 10 μl was taken out of each sample, then the samples were ethanol precipitated.
After ethanol precipitation, each sample was split into four tubes of 250 μl each to which was added: 250 μl TEzero, 120 μl of 3M NaOAc pH5.2, 2 μl glycol and mixed. 1200 μl ethanol was added per tube, mixed, precipitated and centrifuged at 12K RPM, 10 minutes.
Digestion With Psi1 or HindIII to Liberate the Precise 3′ Ends of the Capture Probes:
As shown in
After the phosphatase treatment as described above, the pellets were then resuspended in 960 μl of 1×NEB #4 buffer and 40 μl PsiI, then digested for 2 hours at 37° C. The digests were then ethanol precipitated.
Selective Removal of the Complementary Strand of the Monomer Capture Probe Precursors to Produce a Population of Single-Stranded Capture Probes for Solution Based Capture:
As shown in
Lambda Exonuclease Digestion:
An enzyme titration was first run with lambda exonuclease starting at a concentration of 1 μl/10 μl and diluted down in 2-fold steps. Each reaction contained 10 μg substrate (digested with Bsm1, alkaline phosphatase and Psi1) in 100 μl and were digested at 37° C. for 10 minutes, 75° C. for 10 minutes, then cooled to 4° C. The reactions were run on an agarose gel and the conditions of 10 μg substrate in 100 μl with 5 μl exonuclease for 10 minutes at 37° C. was used for subsequent digestions of the pool C and pool D substrate.
Scaled Up Reaction:
For capture series “C”, 90 μl of 89 ng/μl dsDNA precursor was digested with 5 μl lambda exonuclease in 100 μl 1× lambda exonuclease buffer (NEB).
For capture series “D”, 100 μl of 208 ng/μl dsDNA precursor was digested with 5 μl lambda exonuclease in 200 μl 1× lambda exonuclease buffer (NEB).
The following is an exemplary structure of a final single-stranded capture probe 200 resulting from the capture probe precursor SEQ ID NO:69/70 or SEQ ID NO:71/72 after processing with lambda exonuclease:
TGTGGACCCCTTCGAGCAATTA3′ 3′
The capture probe SEQ ID NO:78 comprises a 5′ region 202 that hybridizes to the −35 exon 1 of the AKT gene, and a 3′ region 204 (underlined) that hybridizes to a universal biotinylated oligonucleotide 300.
The universal capture hybridizing region 204 of capture probe SEQ ID NO:78 is:
Discussion:
The isothermal amplification method described in this Example converted 2.5 ng (0.1 μmol) of starting material into 50 μg (2000 μmol) of raw, unprocessed double-stranded material, which is a 20,000-fold level of amplification. In comparison, the Agilent custom library array platform provides 250 ng (10 μmol) of cleaved oligonucleotide. Processing of the double-stranded DNA precursor capture probe 230 to single stranded capture probes 200 resulted in approximately 15 μg (1200 μmol) of single-stranded capture probe 200, which is a 60% yield from the starting amplified unprocessed double-stranded material 230. In addition to high yield of amplified products, the use of concatemers 240 as templates for amplification provides an equal distribution of amplified monomer products 270, resulting in an equal distribution of processed capture probes 200. The lambda exonuclease digested material (ssDNA) 200 was successfully used as a capture probe library for solution based capture of the AKT exons 1-13, as described in Example 2.
This Example describes solution-based capture using a pool of capture probes 200 generated as described in Example 1, each capture probe 200 comprising a target specific region 202 specific for binding to one of the 13 exons of AKT (NM—005163) and a common region 204 that hybridizes to a universal biotinylated adaptor oligo 300.
Rationale:
As shown in
While indirect capture is described in this Example, it will be understood by those of skill in the art that solution based capture may also be accomplished through the use of ssDNA probes 200 are directly labeled. For example, the probes 200 could be directly labeled by adding a biotin, deoxygenin, fluorescein, and the like (through the use of commercially available kits), followed by the use of antibody coated beads for purification.
Methods:
ssDNA capture probes were generated as described in Example 1. For capture series “C” and “D,” the concentration of capture probe after the lambda exonuclease digestion was approximately 40 ng/μl to 50 ng/μl.
The following universal 5′ biotinylated oligo (capture reagent 300) was used in this Example:
As described in Example 1, a library of 64 ssDNA chimeric capture oligos 200 were generated that each target one of the 13 exons of AKT1 that each have a first 5′ region 202 with the identical sequence to the oligos shown above in TABLE 1, and a second 3′ region 204 consisting of the following additional sequence that hybridizes to the universal biotinylated oligo 300:
Capture Mixture:
A master mix was prepared by combining 62.5 μl of 80 ng/μl of a genomic DNA library containing an average insert size of 100 bp flanked by a first and second primer binding site, 10 μl of 1 μM universal biotinylated oligo (SEQ ID NO:81), 125 μl 2× binding buffer (20 mM Tris pH 7.6, 0.2 mM EDTA, 2M NaCl).
The master mix was then combined (separately) with the following:
10 μl of 1 μM Maxwell 139 AKT1 set, a set of 28 directly synthesized capture oligonucleotides specific for AKT exons 1-13 (low density coverage).
10 μl of 1 μM “A” series high density AKT1 control set (64 oligo pool) that was directly synthesized (not amplified). Each capture probe was synthesized to contain only the target specific portion 202 and the universal oligo hybridizing portion 204, without the flanking sequences. For example, the probe for AKT exon 1 (−35) was identical to the final processed amplified probe for AKT exon 1 (−35) (SEQ ID NO:78).
10 μl of processed “C” Series Capture probes (in duplicate): a set of 64 AKT capture probes generated using the amplification method described in Example 1.
10 μl of processed “D” Series Capture probes: a set of 64 AKT capture probes generated using the amplification method described in Example 1.
20 μl of processed “D” Series Capture probes, as described for Sample #4 above.
Each reaction was brought to a total volume of 250 μl, mixed, and annealed as follows:
95° C. for 5 minutes
80° C. for 15 minutes
75° C. for 15 minutes
70° C. for 15 minutes
65° C. for 15 minutes
60° C. for 15 minutes
55° C. for 15 minutes
Room Temperature.
Capture Reagents:
Washed streptavidin-coated magnetic beads were prepared by combining 66 μl beads (MyOne streptavidin-coated beads, InVitrogen) 500 μl 2× binding buffer and 440 μl water.
Capture:
Each of the annealed 250 μl mixtures were combined with 10 μl of washed beads in a total volume of 1 ml (10 mM Tris, pH 7.6, 0.1 mM EDTA, 1 M NaCl, 0.1% Triton X100) and incubated with mixing for 15 minutes. The beads were then washed four times, 5 minutes each, with 1 ml of TEzero wash buffer (10 mM Tris pH 7.6, 0.1 mM EDTA) containing 25% formamide.
Elution:
The DNA bound to the beads was eluted with two 25 μl aliquots of water by incubation at 95° C. for 1 minute, pulling over the beads, and removing the eluate, for a total eluate volume of 50 μl
Amplification of Eluate:
PCR Reaction Mixture (Each Sample Performed in Duplicate)
10 μl template (eluate from enriched fragment library)
30 μl H2O
20 μl 5×PCR buffer (supplied by manufacturer with the EXPANDplus® kit, Roche)
10 μl 25 mM MgCl2
5 μl 10 mM dNTPs
5 μl DMSO
1 μl ExpandPLUS® polymerase (Roche)
100 μl total volume
PCR Cycling Conditions:
1 Cycle:
95° C. for 2 minutes
10 Cycles:
95° C. for 30 sec
60° C. for 30 sec
72° C. for 1 minute
10 Cycles:
95° C. for 30 sec
60° C. for 30 sec
72° C. for 1 minute plus 10 sec/cycle
1 Cycle:
72° C. for 5 minutes
4° C. hold
The PCR products were purified over a QIAQUICK column, quantified and diluted to 1 ng/μl for subsequent quantitative PCR (qPCR) (Taqman) analysis.
Quantitative PCR Analysis:
The PCR products generated as described above (1 ng/μl), no template control and genomic DNA control (10 ng/μl) were used as templates in Taqman assays directed against coding exons 1-13 of AKT1. Negative controls ANKHD, PIK3CA and TP53 were also included in this assay, which should not be captured as target sequences with the AKT specific probe pools.
Results:
The results of the qPCR analysis were analyzed in two ways. First, the fold-enrichment for each solution-based capture over genomic DNA was calculated, as shown in TABLE 2.
Second, the standard deviation of the exon-to-exon capture efficiency was calculated as a percent of the overall fold-enrichment, as shown below in TABLE 3. This latter number provides a measure of the uniformity of each capture reaction.
Discussion:
The “A” series oligos were directly synthesized 69 mers (non-amplified) containing the identical sequence as the probe sequences generated after processing amplified sequences. The “C” series oligos were initially synthesized as 79mers, annealed to a reverse primer, filled-in, digested with Bsm1, ligated into concatemers, amplified, digested back to monomers, and lambda-exo treated to generate ssDNA probes. The “D” series oligos were initially synthesized as 60mers (less overlap with reverse primer than the “C” series), annealed to a reverse primer, filled-in, digested with Bsm1, ligated into concatemers, amplified, digested back to monomers and lambda-exo treated to generate ssDNA probes. It is important to note that the “D” series oligos are the least expensive to generate due to the fact that they can be made on a 50 nmole synthesis scale due to their smaller size. As described above in Example 1, the amplification of the capture probe pool “C” or “D” was observed to be in the range of 10,000 to 20,000 fold amplification, thereby facilitating the cost-effective use of solution based capture for target enrichment in a variety of applications.
It is noted that the absolute magnitude of the fold-enrichment shown in TABLE 2 may be exaggerated because the critical stoichiometry between the capture oligonucleotides and the common, biotinylated adaptor capture reagent oligonucleotide was not optimized in this experiment. However, the key metric is that the standard deviation of exon-to-exon capture efficiency, expressed as a percentage of the fold-enrichment, as shown in TABLE 3 and
Agarose gel analysis of the capture probes generated by the amplification methods described herein showed that they formed bimolecular complexes during solution based capture that were indistinguishable from the biomolecular complexes formed with directly synthesized capture probes. Importantly, as demonstrated in TABLES 2 and 3 and
Therefore, this Example demonstrates that the capture probes generated from amplified head-tail concatemers may be successfully used for solution based capture, and provide an advantage in targeted resequencing by reducing the cost of resequencing while increasing the feasibility of profiling applications that are dependent on complex oligonucleotide libraries.
This Example describes a method for designing and uniformly amplifying a library of target capture probes designed to capture the entire collection of exons that include protein coding regions from a human genomic DNA library.
Rationale:
As described in Examples 1 and 2, a method for uniformly amplifying a library of synthesized capture oligonucleotides for use in solution based capture methods can be applied to capture all the exons of a gene, such as AKT. This Example demonstrates that the methods described herein can be applied on a very large scale, in order to generate a library of capture probes that capture the entire collection of genomic segments that include all protein coding regions from a human genomic library.
Methods:
Design of Capture Probe Oligonucleotide Precursors for Amplification and Processing into a Library of Capture Probes:
As shown in
Design of the Target-Specific Region 202 of the Capture Probe 200:
In this Example, a capture probe library 200 comprising 1,148,286 distinct target-specific regions 202 was generated for high density solution based capture of the entire collection of genomic segments that include protein coding regions (exons) for 25,341 annotated human genes.
The overall design principles for designing a capture probe library 200 comprising target-specific regions 202 for capture of all the exons of 25,341 human genes were as follows. Each target-specific region 202 was 35 nucleotides in length. The target-specific region was designed such that each target exon had at least 4 or more probes (high density), and the capture probes alternated in strand orientation and were oriented in a head to tail arrangement, with oligo probes alternating with respect to hybridizing to the coding or non-coding strand of the target exon.
In this Example, the term “candidate oligonucleotide” refers to a 35mer nucleotide sequence that was analyzed for potential use as target-specific region in a capture probe, to determine whether the candidate oligonucleotide sequence met the desired criteria, as described below. The 35mer nucleotide sequences that met all of the design criteria outlined herein were chosen as the set of target-specific regions 202 and were synthesized on a microarray along with flanking sequences to generate an oligonucleotide library having 1,148,286 distinct target-specific regions.
Step 1: Obtain Input Sequence:
In order to design the target-specific sequences of the library of capture probes, the genes and transcripts of interest were first identified. In this Example, the entire list of human high quality mRNA transcripts provided in the publicly accessible NCBI database “RefSeq NM transcripts” was selected as input sequence, which was a total of 25,341 human annotated mRNA transcripts. The protein coding exons in the 25,341 input human mRNA transcripts were then identified using the publicly accessible “UCSC Genome Browser” database. The UCSC Genome Browser is developed and maintained by the Genome Bioinformatics Group, a cross-departmental team within the Center for Biomolecular Science and Engineering at the University of California Santa Cruz. Once the protein coding exons were identified, the genomic sequences of interest plus 100 nucleotides of adjacent intronic sequences on either side of the exons were extracted. Overlapping regions were then identified and removed from the list of sequences by using genomic coordinates. For overlapping regions that were identified, the sequence that was retained was based on the 5′ most and 3′ most genomic coordinates between all the pairs of exons in the overlapping region.
Step 2: Upfront Sequence Classification:
The list of sequences generated as described in Step 1 was then searched using the software program “repeatmasker” to identify, but not mask, the repeat and low complexity elements.
Step 3: Determination of Uniqueness Score of Candidate Oligonucleotide Probe Sequences:
The uniqueness of all 35mer sequences in the sequenced human genome relative to the human genome was determined as follows. First, the “UCSC Genome Browser” database was used to extract every 35mer sequence from the sequenced human genome. Second, the software algorithm “Burrows-Wheeler Alignment” (hereinafter referred to as “BWA”) was used to align (i.e., blast) these 35mer sequences against the entire sequenced human genome. BWA is a fast light-weighted tool that aligns short sequences to a sequence database, such as the human reference genome. For each 35mer candidate oligo sequence, BWA returned an alignment score that measures the confidence in the identified location, as described in Li, H., et al., Genome Res. 18(11):1851-8 (2008), incorporated herein by reference. The alignment score provided by BWA is a −log 10 p-value, ranging from 37 (unique in the genome) to 0 (multiple perfect matches). Intermediate values reflect alignments that are similar but not 100% (e.g., with one nucleotide mismatch).
Step 4: Final Selection of the Target-Specific Regions 202 for Synthesis to Generate a Library of Capture Probes 200 for Total Exon Capture of the Human Genome:
The final selection of the 1,148,286 distinct target-specific regions for synthesis in order to generate a library of capture probes 200 that hybridizes to all the exons of 25,341 annotated genes was carried out as follows.
For each target exon at least 140 nucleotides or longer, analysis for candidate oligos began at the 5′ edge of the exon. For target exons 140 nucleotides or shorter, the analysis began in the 5′ intron such that the candidate oligos were equally spaced at the exon center.
From the starting position, each candidate oligo location was then “jittered” by shifting the region of analysis by +/−4 nucleotides as follows. The nucleotide sequence of the candidate oligo closest to the desired location was first examined, and the examination of candidate oligos was carried out by shifting the region of analysis in the order: +1, −1, +2, −2, +3, −3, +4, −4. The +/−4 nucleotide range was chosen because a wider range (more “jitter”) may allow oligo dimers to form with adjacent oligos, which would be undesirable.
The best candidate oligos from each “jittered” position were then selected based on the following criteria. First, the BWA alignment score (uniqueness) of the candidate 35mer capture oligo sequences was maximized relative to the entire genome (wherein 37=unique and 0=multiple perfect matches). Second, among the candidate 35mer capture oligo sequences with the same BWA alignment score, the number of repeat/low-complexity nucleotides contained in the 35mer sequence was minimized.
The nucleic acid sequences of the 1,148,286 distinct target-specific 35mer regions were output as a text file to an oligonucleotide synthesis platform and synthesized.
In this Example, the target-specific regions were flanked by a 3′ flanking region (SEQ ID NO:1) for annealing to a common reverse primer for second strand synthesis, and by a 5′ flanking region (SEQ ID NO:2) which provides at least one processing site for ligation and amplification, resulting in a structure corresponding to the single-stranded capture probe precursor 100 as illustrated in
TABLE 4 below provides the sequence information and scores from the selection criteria described above for a representative, randomly chosen subset of 2170 capture probe precursors from the total library of 1,148,286 capture probe precursors that were synthesized.
In particular, TABLE 4 provides the following information: Column 1: gene name; Column 2: Genbank transcript reference number; Column 3: target exon number; Column 4: the chromosome of the target exon; Column 5: the gene strand; Column 6: the distance of the capture probe from the 5′ edge of the target exon; Column 7: the strand of the capture probe; Column 8: the number of repeat nucleotides; Column 9: the BWA alignment score (wherein 37=unique and 0=multiple perfect matches); Column 10: the number of hits from the BWA alignment (1=unique); Column 11: the number of hits from BWA mismatch (0=no mismatch; 1=mismatch); Column 12: the sequence of the single-stranded precursors, each including the common 5′ flanking region (SEQ ID NO:2) and common 3′ flanking region (SEQ ID NO:1); and Column 13: the SEQ ID NO: from SEQ ID NO:84 to SEQ ID NO:2253.
As shown in TABLE 4, Column 9, a score of “1” for the “number of hits from the BWA analysis corresponds to a uniqueness score of a perfect 37, in which capture oligo sequence only recognizes the intended target.
While the preferred embodiment of the invention has been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.
This application claims the filing date benefit of U.S. Provisional Application No. 61/164,859, filed on Mar. 30, 2009. The contents of each foregoing patent applications are incorporated by reference in their entirety. Throughout this application various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
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| 61164859 | Mar 2009 | US |
