Patent Application
20210171939
This invention relates generally to the cell capture and cell processing field, and more specifically to new and useful systems, methods, and compositions for sample processing barcoded beads for target material reactions.
With an increased interest in cell-specific drug testing, diagnosis, and other assays, systems and methods that allow for individual cell isolation, identification, and retrieval are becoming highly desirable. Single cell capture systems and methods have been shown to be particularly advantageous for these applications. However, associated processes and protocols for single cell capture and subsequent analysis must often be performed in a particular manner and with a high precision in order to properly maintain the cells. Furthermore, efficient retrieval of target material from high density platforms is subject to many challenges. Additionally, compositions of materials can be improved significantly for applications involving capture and retrieval of target material in a manner that allows for single-cell analysis. As such, these processes can be time consuming for the user, can require extensive and iterative manual library preparation and selection processes, may not amenable to automation, and may thus result in damage to the cells (e.g., in terms of undesired loss of viability), high background noise rates, elevated false positive rates, or otherwise unreliable experimental results.
Thus, there is a need in the cell capture and cell processing field to create a new and useful system and method for sample processing and target material retrieval and minimize steps required in the library preparation of the target biomaterials, where some embodiments utilize molecular barcoding (e.g., through the use of barcoded oligonucleotides in the workflow typically delivered to a reaction environment involving functional particles). There is also a need for creating methods for streamlined manufacturing of described embodiments of barcoded beads in large quantities.
The following description of the preferred embodiments of the invention is not intended to limit the invention to these preferred embodiments, but rather to enable any person skilled in the art to make and use this invention.
The invention(s) described can confer several benefits over conventional systems, methods, and compositions.
The invention(s) confer(s) the benefit of providing non-naturally occurring compositions for facilitating capture, extraction, and/or retrieval of target biological material from a sample, while providing barcoding for each biomarker molecule retrieved from a partition of a sample which may be discrete single cells in the sample. Such compositions can include materials that have been modified from their natural states (e.g., in terms of providing structural differences from natural compositions). Furthermore, the invention(s) relate to combinations of materials, where the combinations of materials are non-naturally occurring (e.g., there is no naturally occurring counterpart to the compositions described and claimed).
The invention(s) also include novel compositions of base material and chemistry of components, to produce simplifications in library preparation processes.
The invention(s) also include novel compositions with cleavable sites that allow for separation of target material, with the ability to monitor cleavage and/or quantify components processed from a biological sample.
The invention(s) also confer(s) the benefit of providing mechanisms for efficient retrieval of target material (e.g., beads, cells, released nucleic acid material, etc.) from high-aspect wells of a high-density capture platform. Retrieval is typically difficult and non-efficient in this scenario due to close packing of wells of the capture platform. Retrieval mechanisms described also subject target material to acceptable amounts of shear and other potential stresses that would otherwise obstruct downstream processing steps.
The invention(s) also confer(s) the benefit of providing methods for manufacturing beads for capturing target molecules and/or molecules coupled to a substrate (e.g., chamber wall), where the molecules include a set of unique barcodes that can be detected for sample processing.
The invention(s) also confer(s) the benefit of reducing burden on system operators in relation to target material retrieval processes from wells, where standard processes can be inefficient/labor intensive.
The invention(s) also confer(s) the benefit of increasing the efficiency at which target material is retrieved (and non-target material is not retrieved). Selective retrieval efficiency can thus reduce downstream costs in relation to processing reagent and other material costs (due to reduced volumes needed), processing burden, and improved signal to noise ratios. For instance, the invention(s) can enable a system operator to purchase smaller volumes of reagents, reduce the number of splits required for successful amplification of target molecules and obviate the need for doing SPRI-based clean-up and size selection of target oligonucleotide products from other oligonucleotide tags that do not contain products but get carried over from one process step to the next. Such improved recovery of target products and reduction of carryover of non-target products can also reduce the complexity of data analysis and also provide more useable data pertaining to the desired biomarker analysis as well. This can function to save costs, reduce reagent waste, or have any other suitable outcome.
The invention(s) also confer(s) the benefit of providing greater sequencing depth with respect to desired target, due to greater numbers of target reads provided by the compositions, methods, and systems described.
The invention(s) also confer the benefit of enabling at least partial automation of the protocols involved in single cell capture, target material retrieval, and subsequent processing. For instance, a human operator user can be removed from part or all of the method. Furthermore, the system(s) and/or method(s) can enable better accuracy in performance of a protocol over conventional systems and methods. Some of these inventions are also much more amenable to full automation with a liquid handling robot.
Additionally or alternatively, the invention(s) can confer any other suitable benefit.
As shown in
The composition 100 can be configured to operate with systems configured to perform single-cell analyses, in manual, semi-automatic, and/or automatic operation modes. Embodiments, variations, and examples of such systems are described in one or more of: U.S. application Ser. No. 13/557,510 titled “Cell Capture System and Method of Use” and filed on 25 Jul. 2012, U.S. application Ser. No. 14/289,155 titled “System and Method for Isolating and Analyzing Cells” and filed on 28 May 2014, U.S. application Ser. No. 15/422,222 titled “System and Method for Isolating and Analyzing Cells” and filed on 24 Feb. 2017, U.S. application Ser. No. 15/815,532 titled “System and Method for Retrieving and Analyzing Particles” and filed on 16 Nov. 2017, and U.S. application Ser. No. 16/115,059 titled “System and Method for Isolating and Analyzing Cells” and filed on 28 Aug. 2018 which are each incorporated in their entireties by this reference.
The composition 100 can be configured for processes and reactions associated with one or more of: a reverse transcription reaction (RT-reaction), immunochemistry, DNA reactions, mRNA FISH reactions, proximity ligation reactions, bridge amplification reactions, catalytic enzymatic reactions, hybridization reactions, restriction digestion reactions, amplification reactions (e.g., mRNA and/or DNA PCR), and other suitable reactions. Such reactions can be performed on-chip and/or off-chip, where embodiments, variations, and examples of microfluidic chips for single-cell analyses are described in U.S. application Ser. No. 13/557,510 titled “Cell Capture System and Method of Use” and filed on 25 Jul. 2012, U.S. application Ser. No. 14/289,155 titled “System and Method for Isolating and Analyzing Cells” and filed on 28 May 2014, U.S. application Ser. No. 15/422,222 titled “System and Method for Isolating and Analyzing Cells” and filed on 24 Feb. 2017, and U.S. application Ser. No. 15/815,532 titled “System and Method for Retrieving and Analyzing Particles” and filed on 16 Nov. 2017, which are each incorporated in their entireties by this reference.
The body 110 functions to provide a substrate to which the one or more molecules 120 can be coupled to, in order to provide functionalization for the composition with respect to implementation of respective assays and reactions.
In relation to morphology, the body 110 can have the form of a microsphere. Alternatively, the body 110 can have the form of a non-spherical (e.g., ellipsoidal, prismatic, polyhedral, amorphous, etc.) body, where a cross section taken through the body 110 is non-circular. However, the body 110 can alternatively have another suitable form. In relation to dimensions, the body 110 can have a diameter (or characteristic width) from 5-50 microns, with a tolerance of ±0.05 to 5 microns. Additionally, the uniformity of the body 110 across a population of particles can enable a desired retrieval efficiency behavior upon completion of various steps of an intended single cell process. In a specific example, the body 110 has a diameter of 20 microns±1 micron; however, variations of the example body 110 can have other morphology.
In embodiments, the body 110 has a characteristic dimension configured such that only a single body 110 of the composition 100 can enter a well of the chip described above, along with a single target cell, in order to co-localize and co-capture the single cell-particle pair within an individual well. However, the body 110 of the composition 100 can have another suitable characteristic dimension configured for other microfluidic or non-microfluidic assay applications.
In relation to density, the body 110 is configured to have a density greater than the density of process liquids intended for use with the composition 100 (e.g., in relation to specific reactions or assays), such that the composition 100 settles within the process liquid(s) by gravity during operation. In an embodiment, the density of the body no is greater than 1.02 g/cm3, however, the body 110 can have other suitable densities in variations. For instance, the body 110 can be configured to be of the same density as an intended process liquid in some embodiments (e.g., in order to facilitate steps where the body 110 is desired to be carried with flow of the process liquid). In still other embodiments, the body 110 can be configured to be buoyant relative to a process liquid, such that the body 110 is buoyant and can be used for separation of target or non-target material of a sample.
In relation to density and morphology, the body 110 can be a continuous body (e.g., at micron scale, at nanometer scale, at sub-nanometer scale). Alternatively, a variation of which is shown in
In an example, a composite microsphere made of a number of small microspheres (e.g., having 0.5 micrometer diameters) reacted to the surface of a larger microsphere (e.g., having a 19 micron diameter), such that the composite microsphere had a total diameter of 20 micron but the surface area of the surface of the composite particle was significantly enhanced by the presence of the smaller microspheres or presence of certain reactive groups ordered in a specific pre-designed array.
In embodiments, the base materials and surface properties can be different to offer significant flexibilities of performance. For example, the bigger microsphere may be a hard material while the small microspheres could be of hydrogel. In another example, the larger microspheres can be non-magnetic but the smaller microspheres can be magnetic. In another example, the larger microsphere is magnetic and the smaller microspheres are magnetic or paramagnetic. In another example, the larger microsphere can be made of transparent material while the smaller microspheres may be of optically (e.g., brightfield or fluorescent) coded. In another example, the larger microsphere can be made dissolvable while the smaller microsphere are non-dissolvable. Another embodiment of a composite microsphere could include a set of base hard microspheres coated with a thin (e.g., 1-3 micron layer) of hydrogel or other material(s) providing increased surface area of reactions. Such an innovative microsphere would also provide an added advantage of allowing biomarkers of certain size to permeate into the microsphere to part-take in a specific reaction. Yet another example of composite microspheres could include solid particles (e.g., 20 micron diameter) with micro-tunnels (e.g., 0.1-2 micron diameter) that span from the surface of the composite microsphere to the center of the microspheres. In some cases, these micro-tunnels could go across the diameter of the entire particle. In still other embodiments the micro-tunnels are pores which increase the total surface area of the composite materials. In yet another embodiment, the large microsphere may have a thin coating on the surface that has a different functional composition compared to the composition inside. The top surface may be cross-linked but the inside material may be soft or dissolvable.
In variations, each of the smaller bodies 115 can be the same in properties and composition; however, in other variations, one or more of the smaller bodies 115 can be configured to have different properties, compositions, and distributions within the cluster (e.g., from the core to the surface), in order to provide different functionality for different portions of an assay or reaction. For instance, a first region (e.g., surface) of the cluster can have a first set of properties, composition, and/or surface chemistry to perform a first part of an assay or reaction, be dissolved or otherwise removed, and then a second region (e.g., core) of the cluster can have a second set of properties, composition, and/or surface chemistry to perform a second part of an assay or reaction.
In a specific example, a set of approximately 750 smaller bodies 115 each composed of polystyrene with divinylbenzene crosslinking (PS-DVB) having a diameter of 1 micron (with suitable tolerance) are clustered in a dissolvable hydrogel to provide a gross diameter of 20 microns, with overall surface area ˜7.5 times that of a single contiguous 20 micron particles. In another example, the body 110 can be composed of a hydrogel where the smaller bodies are made up of poly-acrylamide matrix and the clustering material comprises a disulfide crosslinking agent (e.g., BAC). However, variations of the example can be configured in another suitable manner.
In relation to thermal properties, the body 110 is configured to operate between a lower temperature limit (e.g., associated with low temperature reactions and processes, associated with storage, etc.) and an upper temperature limit (e.g., associated with high temperature reactions and processes). In specific examples, the lower temperature limit is from −20 C through 4 C (e.g., for cold storage), and the upper temperature limit is from 90 C through 120 C (e.g., for denaturation reactions). However, the body 110 can be configured for other operating temperatures.
In relation to physical properties, the body 110 is configured to maintain structure in solution (e.g., in buffer during storage, in solution during performance of an assay). As such, the body 110 is configured to be non-swelling and non-leaching. However, in alternative embodiments, the body 110 can be configured to swell a desired amount (e.g., in relation to achieving a desired size or morphology for processing or use in an application), configured to leach certain compounds (e.g., process reagents) for performance of an assay, and/or to dissolve in a desired manner during performance of an assay or other process. In yet another embodiment, the particle may have well-defined tailored swellability such that its use in specific buffer and/or physical conditions allows the particles to easily enter a microwell but may be trapped in the microwell under specific buffer conditions. Further in relation to physical properties, the body 110 can be configured with a desired degree of hydrophilicity (e.g., on a spectrum from hydrophilic to hydrophobic) in relation to performance of an assay or other process. In relation to surface properties associated with fluid contact, the body 110 can be configured to have a desired wettability (e.g., in terms of contact angle, etc.). Variations of the body 110 can thus have a suitable type of crosslinking (e.g., chemical crosslinking, physical crosslinking, etc.) and percentage of crosslinking (e.g., from 1-10% crosslinking for acrylamide, 30-99% crosslinking for other materials, another suitable range of crosslinking), to provide a desired level of stability in conditions of use.
In relation to other surface properties, the body 110 can be configured with a desired porosity (e.g., 200-2000 A, etc.). The body 110 can additionally or alternatively be configured with a desired loading density (LD), in order to enable achievement of a suitable linker density (e.g., by providing points of attachment on the body 110 to provide more robust detectible signals during use), where additions to the body 110 are described in more detail in Section 2.2 below. Furthermore, the body 110 can include surface groups (e.g., hydroxyl groups, amine groups, carboxyl groups, sulfide groups, silanol groups, etc.) for coupling of linker molecules described in Section 2.2 below. In examples, desired loading density (LD) can be as low as 1 umol/g or as high as few hundred umol/g of functional group density.
In relation to magnetic properties, the body 110 can be configured to respond to magnetic fields (e.g., in relation to assays involving separation and/or retrieval of target or non-target material). Certain regions (e.g., a core region) of the body 110 can be magnetic (e.g., magnetic, paramagnetic, etc.), and certain regions (e.g., a shell region) of the body 110 can be non-magnetic in variations of the body 110. In relation to surface properties, the body 110 can be configured with or without charge, in order to facilitate binding to target material, or to facilitate fabrication involving molecules with functionality.
In relation to optical properties, the body 110 can be configured to be non-fluorescent (e.g., so as to not interfere with optical-based detection assays). However, in variations, the body 110 can be configured to be optically detectable (e.g., via a non-fluorescent modality, via a fluorescent modality, via an infrared detection modality, via a thermal detection modality, etc.), for instance, for tracking purposes.
In relation to mechanical properties, the body 110 can be configured to have a desired hardness (e.g., measured on the Mohs scale, measured on another hardness scale), in order to retain a desired level of hardness during applications of use. Additionally or alternatively, the body 110 can be configured with desired mechanical properties associated with one or more of: rigidity, elastic behavior (e.g., in terms of moduli, in terms of plastic and elastic deformation, etc.), viscoelastic behavior, fatigue resistance, fracture resistance, shear strength, compressive strength, tensile strength, rheological behavior (e.g., under conditions of wear), and other mechanical properties.
In relation to composition, the body 110 can be composed of one or more of: polystyrene, polystyrene-divinylbenzene, polymethylmethacrylate (PMMA), silica, silica-gel, non-porous glass, porous glass, coated glass, agarose, acrylamide, polyacrylamide, iron, steel, or ceramic materials and/or a combination of one or more suitable materials. As noted above and below, different regions of the body 110 can be composed of different materials (e.g., a core region can be composed of a first material and a shell region can be composed of a second material). In some embodiments there may be multiple regions either as multiple shell regions, or in other configurations such as amorphous or ordered spatial arrangements.
Specific examples of the body 110 are composed of polyacrylamide (e.g., as described in more detail below, silica (e.g., silica gel), polystyrene, or PMMA, 15-25 microns in diameter (e.g., where smaller diameters allow for minor swelling in a manner that is still appropriate for use within microfluidic structures), with a surface porosity from 80-1500 A, with between 20% and 80% crosslinking (e.g., Polystyrene with 60% crosslinking by divinylbenzene or Polystyrene with 80% crosslinking by divinylbenzene) for polymeric beads, with surface groups (e.g. amine groups, hydroxyl groups, silanol groups) for coupling of linker chemistry (e.g., C18 tag linker), and polyethylene glycol (PEG) functionalization for reaction efficiency. Variations of the specific examples can have magnetic (e.g., magnetic, paramagnetic) cores or shells to allow for magnetic functionality (e.g., for separation and retrieval).
As shown in
In embodiments, the one or more molecules 120 can include a single molecule, a set of identical molecules, or a set of different molecules (e.g., a first and a second molecule, a plurality of different molecules) distributed across a body 110. For instance, in reactions involving mRNA capture and cDNA synthesis, the one or more molecules 120 can include oligonucleotide molecules having a first sequence for mRNA binding, and having a second sequence associated with generation of complementary cDNA strands. Similarly, in reactions involving binding of protein tags, the one or more molecules can include molecules having a first sequence for detecting antibody binding through detecting tagging of antibodies with an oligonucleotide tag, and molecules having a second sequence for synthesis. In another embodiment, different sets of molecules for providing forward as well as reverse primers may be present in the one or more molecules 120 to allow for bridge amplifications to amplify certain nucleic acid fragments from single cells that are initially bound to the microspheres. Relative proportions of various forward or reverse primers may be adjusted such that only cDNA of certain sizes are maximized during bridge amplification (e.g., for example products less than 600 base pairs or more than 300 base pairs). However, the sequences of the one or more molecules 120 can be adapted for other reactions and processes, variations of which are described below in relation to different structural features of the one or more molecules 120. Binding groups may also be present in 120 in certain proportion for enzymes to be tethered to the microsphere during enzymatic reactions such that these enzymes can process and create reaction products for mRNA to reach only a certain size or prevent products to be more than certain base sizes. Alternatively, the structural features may exclude certain enzymes (e.g., nucleases or restriction enzymes) or other functional moieties from close proximity to the body 110 in order to adjust the size of the retained molecules to a desirable size (eg., anything longer than 300 bp is digested to smaller size).
As shown in
In embodiments, the number of linkers in the set of linkers is configured to be greater than the number of target molecules per single cell being targeted for binding reactions, In one example, the number of target molecules per cell is on the order of 0.5 to 1 million molecules or molecule fragments; thus, in the example, the set of linkers can include 107-1010 linkers for positioning 107-1010 full-length oligonucleotides per body 110, wherein an excess of full-length oligonucleotides result in more mRNAs (or other molecules) captured during a reaction. However, the set of linkers can include other numbers of linkers in other embodiments.
In embodiments, the linker 130 comprises a branched linker configured to provide suitable density of oligonucleotide molecules at the surface of the body 110, and to provide suitable spacing between adjacent oligonucleotide molecules. In variations, the branched linker is a dendrimer (e.g., symmetric dendrimer, asymmetric dendrimer, doubler, trebler, labelled, non-labelled, etc.), that provides branching with nodes of attachment. In one variation, the dendrimer can be a y-shaped dendrimer that includes a source node (e.g., for attachment at a region of the body 110 or proximal to the body 110), and two terminal nodes (e.g., for attachment to functional oligonucleotide molecules of the one or more molecules 120 or for attachment to subsequent dendrimers distal to the body 110). In a specific example, the branched linker is a symmetric doubling phosphoramidite dendrimer; however, variations of the specific example can use another core chemistry (e.g., carbosilane, thiolated, etc.) and structure. As such, in other variations, the dendrimer can have any other suitable number of attachment points, chemistry, and/or structure, to provide spacing and sites of coupling for oligonucleotide molecules to the body 110.
Furthermore, the branched linker can be configured for selectable attachment (e.g., with functional groups specific to specific chemistries) and/or selectable cleavage (e.g., for release of oligonucleotide segments, such as molecular scissors, during processing).
As shown in
As shown in
In other variations, the PCR segment(s) 140 can additionally or alternatively include a PCR handle segment 143 that is detectable and configured for quality control of the composition. However, variations of the one or more molecules 120 can additionally or alternatively omit the PCR handle segment 143.
In embodiments, the PCR segment(s) 140 are coupled directly to a terminal portion (or other portion) of one of the set of linkers 130. However, in other variations, the PCR segment(s) can be coupled relative to other portions of an oligonucleotide molecule in another manner.
In embodiments, the PCR segment(s)140 can have from 5-30 bases and can include custom or non-custom primers; however, in alternative variations the PCR segment(s) 140 can have other suitable numbers of bases.
As shown in
As shown in
The barcode region 150 can include one or more barcode segments, where manufacture and assembly of the barcode segments are described in more detail in Section 4 below. In some variations the barcode segment may include portions used for assembly (e.g., a handle such as a ligation handle or PCR extension handle) which can alternately be used as portions of barcode or independently from the barcode segments. In variations, each barcode segment can be from 2-20 nucleotides long; however, in alternative variations, each barcode segment can have another suitable length. Preferably, each barcode segment has a Hamming distance (e.g., number of substitutions required to make two strings of nucleic acids identical) greater than 2; however, in alternative variations, the barcode segments can have another suitable Hamming distance. Furthermore, each barcode segment can be configured to not end in GG (or other sequences that are less suitable for specific sequencing platforms); however, the barcode segments can be configured in another suitable manner. The barcode region 150 can be constructed from one or more segments to create 1-100 million unique barcodes of suitable length; however, variations can produce other suitable numbers of unique barcodes. In a specific example, the barcode segments are selected from a set of 875 (or more) 7-mers having a Hamming distance of 2 without termination in GG bases, where the sequences are non-naturally occurring. In the specific example, the barcode region is composed of multiple segments that, when assembled together, create 50 million unique barcodes. However, variations of the specific example can be configured in another suitable manner.
As shown in
As shown in
In one variation, as shown in
However, in other variations, the preparation-facilitating segment(s) 170 can additionally or alternatively include other sequences configured to reduce steps (e.g., manual steps) associated with operations (e.g., for specific platforms, for specific processes, etc.).
As shown in
In variations, as shown in
In additional variations as shown in
In embodiments, two different oligonucleotide tags present in the same particle as in
In other variations, as shown in
The composition can additionally or alternatively include other active segments in the one or more molecules 120, for performing other processes involving binding/other interactions.
For instance, as shown in
During use, as shown in
In more detail as shown in
While thermolabile mechanisms are described, the active region 180′ can additionally or alternatively include other cleavable mechanisms whereby products can be detected to confirm cleavage. For instance, the active region 180′ can additionally or alternatively include photocleavable regions, chemically cleavable regions, enzymatically cleavable regions, or regions cleavable by another suitable mechanism.
Furthermore, as described above, the reverse orientation of the fluorophore 180a′ and the quencher 180b′ can be implemented, in order to monitor cleavage and/or capture with emitted fluorescent signals.
In related variations, the active region 180′ can alternatively include a fluorophore, where the fluorophore acts as both a fluorophore and quencher. In particular, when the density of fluorophores on a bead is high enough for self quenching, the removal of some fluorophores from the bead will result in an increase in the total fluorescence even when no specific quencher molecule is included. The cleavage can thus be monitored by an increase in fluorescence (e.g., fluorescence from a bead or fluorescence from a well containing beads and/or released fluorophores in the supernatant) even if the number of beads and number of fluorophores being monitored remains unchanged.
Still alternatively, in another variation of the active region 180′, the quencher 180b may not be a dark quencher but rather another fluorophore (e.g., FRET partner) that affects signals detected from the reaction during operation. For example, the active region 180 can incorporate a first fluorophore (e.g., Fluorescein) on the portion configured to remain on the body 110 post-cleavage, and a second fluorophore (e.g., TAMRA) configured to be released by cleavage, which would result in quenching of the fluorescein signal from the first fluorophore when in close proximity, but an increase in the signal when the oligonucleotide with the second fluorophore is released. Furthermore, the signal from the second fluorophore could be monitored in both cleaved and uncleaved configurations.
In one alternative configuration shown in
However, other configurations or combinations of configurations described can be envisioned.
As shown in
In relation to mRNA binding-cDNA synthesis reactions, the molecular scissor(s) can be configured to be used for cleavage of product pre or post-denaturation to remove mRNA. As such, the molecular scissor region(s) 190 can be used to remove both mRNA-cDNA products, target mRNA, and/or synthesized cDNA products (without mRNA).
In one example embodiment, double stranded specific molecular scissors can be implemented, such that strands are released only after polymerase extension or reverse transcription or similar processes have completed the second strand. In this manner, unreacted products can be washed away, and then completed products can be selectively released and recovered without background contamination from the one or more molecules 120 or other portions of the composition 100. In an alternative variation to the composition shown in
Furthermore, in alternative embodiments, the one or more molecules 120 and/or other portions of the composition 100 can include regions designed for controlled cleavage of oligonucleotide sequences and/or other products using other mechanisms (e.g., photocleaving, thermal cleaving, chemical cleavage, etc.).
As shown in
In more detail, as shown in
As shown in
As shown in
Alternatively,
In a first variation, as shown in
Methods and configurations shown in
As shown in
As shown in
Variations of the example shown in
In an alternative variation shown in
During experimentation according to an example, untreated beads with ostensibly ss oligonucleotides showed the highest number of molecules released by BamHI cleavage (e.g., approximately twice the number compared to treatment where double stranded products were created by hybridization of a reverse primer and extension by polymerase), and beads with ssDNA and denatured with sodium hydroxide shortly before restriction digestion showed lower cleavage indicating that the cleavage is dependent upon the double stranded state with time requirements for reannealing.
In these variations, the molecule(s) form correct double stranded motifs by transient hybridization between different oligo strands (i.e., they do not form hairpin or other secondary structures within a single strand). Furthermore, no sequences that would complete restriction site are in the rest of a respective molecule strand indicating that intermolecular interactions are required. Furthermore, use of a BamHI restriction site is not only palindromic, but also GC rich to facilitate cleavage; however, other restriction sites can be used although the efficiency of cleavage may vary. In more detail, ssDNA can form loop structures with only a handful of bases, and often can assume a “random coil” configuration, but the linker length and flexibility of the linker region 230′ play a role in getting oligonucleotide pairs to match up to enable targeted cleavage. Furthermore, in these embodiments, it is not required that both strands be attached prior to every cleavage. For instance, Bam HI both strands will be cleaved with the same resulting products due to the manner in which the restriction endonuclease cut, but a missing base will not completely inhibit cleavage; thus, one cleaved oligonucleotide could hybridize with an uncleaved oligonucleotide and induce a second cut (e.g., nick) in the previously uncleaved strand, but without the need for the addition of exogenous complementary strands. As such, density of oligonucleotides coupled to the body plays a role in rate of reactions, but is not strictly required to enable cleavage.
Another specific example of a cleavable linker is shown in
The segments of the molecule can, however, additionally or alternatively include other suitable segments as described, and/or be coupled to the body 210 in another suitable manner. As non-limiting examples, the restriction site 290′ of
In one example, an embodiment of the composition 200 can be implemented in a method 300 for single-cell ATAC sequencing, where, as shown in
Variations of the method 300 can further include library cleanup and next generation sequencing loading steps.
Variations of the method 300, can however, be implemented in another suitable manner (e.g., using another capture and processing platform, etc.).
As shown in
The method 400 functions to efficiently create a composition that allows for processing, separation, and retrieval of target material from a sample, according to one or more benefits described in Section 1 above. The method 400 can produce compositions with complex oligonucleotide structures in a phased-attachment manner, that reduces the compounding error associated with base-by-base oligonucleotide attachment methods (e.g., phosphoramidite based oligonucleotide synthesis). The method 400 can also produce compositions that provide simplification of library preparation processes, by inclusion of molecular adaptors specific to sequencing platforms (e.g., Illumina™ adaptors, etc.). The method 400 can thus be used for manufacturing of functionalized particles in a scalable manner, and in a manner that provides quality control and improvements in the amount of recoverable product.
In embodiments, the method 400 can produce embodiments, variations, and examples of the compositions 100 and 200 described above. However, portions of the method 300 can be adapted to produce other related compositions.
Block S410 recites: providing a body as a base substrate, which functions to provide a base substrate for attachment of functional molecules specific to various processes. As noted above, the base can be provided as a contiguous body or can alternatively be provided as a cluster of smaller bodies. In either continuous or clustered form, Block S410 can include coupling of functional groups (e.g., amines, hydroxyl groups, silanol groups, etc.) to the body in order to facilitate subsequent attachment of linker molecules to surfaces of the body.
In an alternative variation, as noted above, Block S410 can include aggregating a set of smaller bodies to form the body. In a first variation, as shown in
In another alternative variation, as shown in
Other variations of Block S410 can, however, involve additional or alternative steps for formation of a set of clustered smaller bodies having suitable surface chemistry (and/or core material features, such as magnetism), in order to provide a substrate for functionalization with oligonucleotides.
In a first variation, Block S410 can include generating base substrates in the form of beads, where the beads are composed of a polymer that dissolves in controlled environments. In a specific example, the beads can be composed of a polyacrylamide material processed from an acrylamide solution (e.g., 40% v/v acrylamide, another percentage of acrylamide), Bis(acryloyl) cystamine (e.g., 0.8% w/v BAC, another percentage of BAC, deionized water, and a buffer (e.g., a buffer composed of Tris-HCL, NaCl, KCl, EDTA, Triton X-100, and water, another suitable buffer, etc.), where the polyacrylamide beads are configured to polymerize with Ammonium persulfate (e.g., 10% APS, another percentage of APS) and Tetramethylethylenediamine (TEMED) under low oxygen conditions (e.g., under Argon gas) and later to dissolve in the presence of a reducing agent such as dithiothreitol (DT).
In this variation, as shown in
In a variation of the example associated with
Block S420 recites: coupling a set of linkers to the body, which functions to control spacing and density of a set of oligonucleotide molecules coupled to the body to produce functionalization of the composition. In embodiments, the linker can be an embodiment, variation, or example of the linker 130 described above; however, the linker can be another suitable linker.
In variations involving asymmetric linkers (e.g., linkers having branches of different lengths or linkers of similar length but with different functional or protecting groups), Block S420 can include building a first oligonucleotide segment off of a first branch of the asymmetric linker while protecting a second branch with a second protecting group, and separately building a second oligonucleotide segment off of the second branch of the asymmetric linker while protecting the first branch with a first protecting group (and deprotecting the second branch) S425. Variations of Block S425 can, however, be configured to operate without using a linker, or by coupling an oligonucleotide that has already been synthesized, to an attachment site of the composition.
Block S430 recites: coupling one or more molecules to the set of linkers with a phased/sequential attachment operation, which functions to reduce compounding error and lot-to-lot variability associated with typical chemical synthesis of oligonucleotide chains. In more detail, Block S430 functions to provide a method that involves fewer addition events to produce lower compounding error, in order to create higher accuracy oligonucleotide molecules, more control over design of the molecules, and higher efficiency of synthesis, in relation to the amount of usable full-length product (e.g., over 97% usable product). In some embodiments it further serves to confine the incomplete products to discrete units that are larger than a single base which provides advantages that may keep the partial products from participating in downstream workflows, and facilitates data analysis that can distinguish manufacturing errors from artefacts of downstream processes which can improve subsequent data analysis.
As shown in
In relation to barcode segments or other segments described above, as shown in
In the specific example, 3 segments of barcode sequences can be generated with unique overhangs (e.g., having associated identifiers), where the overhangs can be used to facilitate correct assembly of the oligonucleotide molecule in a desired order. For instance, as shown in
In still more detail regarding the specific example, a precursor of the composition can be constructed with a body (e.g., bead) coupled to a linker (e.g., C18 linker) coupled to an oligonucleodie comprising a primer binding site (e.g., TSO primer) followed by a set of bases (e.g., 8 thymine bases). Then, a first barcode segment with overhangs on each side of the first barcode segment can be pre-hybridized and then coupled to the precursor of the composition with an appropriate ligase enzyme. Subsequent barcode segments with overhangs can then be coupled to the running build of the barcode region, until a desired barcode region length is achieved. For each step of assembly of barcode segments, complementary segments comprising a detection portion (e.g., fluorophore segment) can be tagged onto the current segment being added, where detection of the detection portion (e.g., by an optical detection process) can be used for quality control at each step of phased attachment. However, quality control at each phase of the phased attachment method can be performed in another suitable manner, or omitted.
Still alternative variations of Block S430 can include performing a synthesis operation configured for single-base addition of nucleotides to form an oligonucleotide product. In a specific example of the alternative variation, chemical synthesis involves addition of nucleotide bases, base-by-base, to a linker (e.g., C18 linker) to produce a full length product. Furthermore, variations of the method 400 can include a hybrid approach, whereby a portion of an oligonucleotide molecule (e.g., linker and primer segments) are formed by base-by-base synthesis, and remaining portions of the oligonucleotide molecule are formed by a phased attachment approach involving assembly of shorter sub-segments of oligonucleotides.
The method 400 can additionally or alternatively include other suitable steps. For instance, variations of the method 400 can include steps associated with manufacturing, scale-up, and quality control in order to improve efficiency of generating usable product, including one or more of: performing a reaction with a ligase (e.g., NEB-M0202M) in a controlled environment (e.g., with a desired concentration per number of particles being generated) in order to couple generated oligonucleotide segments; providing a desired concentration of oligonucleotide material per number of particles being generated; providing a desired reaction volume (e.g., within a container that allows sufficient headroom for wash steps); providing a stabilization reagent (e.g., polyethylene glycol) during manufacturing in order to improve reaction efficiency; implementing a shaking procedure (or other procedure to thoroughly disperse or create uniform product with desired reaction conditions); implementing an incubation procedure (e.g., 16±5° C. or 16±1° C.) during manufacturing of the composition; and performing a suitable number of wash steps. Additionally, variations of the method 400 can exclude certain elements from the manufacturing process such as manufacturing with DTT free ligase and removing DTT from the other reagents in the process, or excluding other potential release agents for the smaller bodies (temperature, chemical, etc.) from the manufacturing process. However, the method 400 can additionally or alternatively include other suitable steps of processes for mass production of units of the composition 100, 200.
In one example, the method 400′ can be adapted to create multiple barcode sets on each body, in a manner where a single bead has different combinations of barcode sequences, using a limited (e.g., a few) sets of barcodes combined in known and unique combinations. All of the combinations of barcode sequences on a single bead can be unique to the bead, or can be otherwise configured. As such, the method 400′ can implement a limited set of barcodes combined together in known combinations so that a single manufacturing build results in multiple barcodes (CBC's) per bead in a controlled and predictable manner, such that all the different barcodes can map back to the same bead.
In more detail, each barcode unit can include a barcode unit subsequence having a set of bases (e.g., less than 10 bases, more than 10 bases) and a handle or handles (e.g., one of a set of different ligation handles or one of a set of ligation handles on either end or other handle(s) such as polymerase extension handle(s)), where the barcode unit subsequences can be configured as sets defined primarily by the handle. In variations, the handles can each have between 3 and 15 bases, or another suitable number of bases. Each of the barcode unit subsequences in an assembled set is thus configured with the same handle(s) (e.g., one of a set of different ligation handles), with different sets having other handles of the set of different handles. The number of ligation handles can thus be determined based upon the number of barcode sequences desired per bead and total barcode diversity desired.
In examples, the method 400′ can implement a number of barcode unit subsequences (e.g., 96 barcode units, 384 barcode units, another number of barcode units), along with a set of ligation handles (e.g., 4 ligation handles, less than 4 ligation handles, more than four ligation handles) to achieve a desired level of diversity for the sample(s) being processed and desired number of different barcodes per bead. Each of the sets could have unique barcodes, but alternatively, the same set (e.g., of 96 barcode subsequences, of 384 barcode subsequences, etc.) could be used for all the sets. In one example, 96 barcode unit subsequences with a 7-mer barcode can be implemented with a 4 base ligation handle, where the barcode unit subsequences are selected from four different sets of 96 barcode unit subsequences; however, other numbers of sets of barcode unit subsequences could be used including a single set differentiated in context only by the handle sequences.
Expanding the example, to provide four uniquely different barcode sequences on one bead, the method 400′ can implement a first set having barcode subsequences of XXXXXX with the ligation handle ATCG, where XXXXXXX is a 7-mer barcode sequence (e.g., one of a set of 96 barcode sequences, one of 384 barcode sequences, one of another number of barcode sequences); a second set having barcode subsequences of XXXXXXX with the ligation handle TCGA, where XXXXXXX is the 7-mer barcode sequence; a third set having barcode subsequences of XXXXXXX with the ligation handle CGAT, where XXXXXXX is a 7-mer barcode sequence; and a fourth set 404′ having barcode subsequences of XXXXXXX with the ligation handle GATC, where XXXXXXX is a 7-mer barcode sequence. As such, the ligation handles ATCG, TCGA, CGAT, and GATC are specific to the set, but the subsequences XXXXXXX may not be specific to the set. In this example, the specific 4 base ligation handles are different for the first (e.g., ATCG, TCGA, CGAT, and GATC), second (e.g., TCAG, AATC, ATTA, TCC), third, and 4th ligation reactions associated with an individual bead and are also be different for each set of barcode unit subsequences. As such, this configuration provides 16 different handles across four sets of barcode unit subsequences with 4 ligation events (e.g., the number of handles is a product of the number of sets of barcode unit subsequences and the number of desired ligation events).
During implementation of the method 400′, all of a first set of barcode versions can be provided in a first well, all of a second set of barcode versions can be provided in a second well, and so on, in order to generate uniquely barcoded beads with different barcodes coupled to each bead (e.g., well 1 contains barcode 1 ATCG, barcode 1 TCGA. Barcode 1 CGAT, and barcode 1 GATC; well 2 has barcode 2 ATCG, barcode 2 TCGA. Barcode 2 CGAT, and barcode 2 GATC, etc.). In alternative variations, different barcode versions from each set can be provided in each well as long as each well has one uniquely identifiable barcode from each barcode set (e.g., well 1 has barcode 1 ATCG, barcode 25 TCGA. Barcode 49 CGAT, and barcode 76 GATC; well 2 has barcode 2 ATCG, barcode 33 TCGA. Barcode 82 CGAT, and barcode 25 GATC, or alternatively if each barcode set originates from a different set of 96, for example, well 1 has barcode 1 ATCG, Barcode 97 TCGA, barcode 193 CGAT, and barcode 290 GATC, etc.)
As shown in
As shown in
As shown in
In more detail, if the same capture sequence is applied to all oligonucleotide strands of a particular bead, even as they comprise different composite barcodes, the pool of sequences from any cell will all map to one of a limited whitelist set of barcode subsequences associated with that particular bead allowing better identification of sequencing errors or chimeric sequences. The ligation handles used further correspond to a particular set for all positions of an aggregate barcode sequence aggregated from individual barcode unit subsequences. As such, any crossing of sets could be detected and those sequences flagged. In clinical applications, the ability to have even relatively rare (e.g., greater than 1) captured sequences (e.g., transcripts) confirmed to originate from the same cell because there are multiple different barcodes that ALL map to the same bead (and thus same cell) would greatly improve the certainty of any calls associated with the barcodes, and thus any potential diagnoses. A particular transcript or a set of transcripts associated with aggregate barcode sequence but different UMI's is probable to be different transcripts from a single target cell, but could result from chimeric sequences. As such, mapping to 4 different aggregate barcodes, all of which are associated with a single bead, provides much greater confidence that they originated from a single cell.
An additional benefit of using individual sets of barcode unit subsequences according to the example method 400′ is that the “invariant” ligation handles will now, collectively in association with each single bead, have diversity and thus avoid sequencing flags allowing more cost effective use of the downstream processes.
While three sets of barcode unit subsequences are described, the method 400′ can include addition of any other suitable number of barcode unit subsequences. In relation to the example method 400, the result after 3 (or however many) rounds of ligation with pooling and splitting between rounds is beads with the same barcode diversity we would have with single barcode sequences, but 4 different barcode sequences on each bead. It would be possible to put 4 different capture sequences on these beads using the different ends, and because the barcode unit subsequence associations are known, any barcode sets should match not only at a single barcode position, but across the set of 3 barcode unit subsequences making up an aggregate barcode sequence.
In a variation of the method 400′, as shown in
In still another variation of the method 400′, As shown in
As described above, methods 400′ and 400″ are shown to append oligonucleotide sequences to a bead; however, the methods 400′ and 400″ can additionally or alternatively be adapted to incorporate cleavage sites (e.g., molecular scissors, restriction sites, etc.) as described in various variations above. Furthermore, in some applications, the oligonucleotides may be attached to bead by the 5′ end and have free 3′-OH group. In other applications, the oligonucleotides may be attached to the bead by the 3′ end. In other applications, different barcode sets could include oligonucleotides assembled to potentially have identical sequences after ligation, but are configured in a manner where one barcode set is added by extending the oligonucleotide along a 5′ to 3′ direction, and the other oligonucleotide is extended along a 3′ to 5′ direction.
With respect to steps of the methods 400′ and 400″ described above, keeping the beads in suspension during the ligation is beneficial to the overall ligation and likely to the uniformity of the ligation among beads. The precise speed will vary with the size and shape of the container and the number of beads in the reaction. Associated mixtures were shaken at 1500 RPM in a shaking apparatus in an example; however, other shaking parameters can be implemented. With respect to timing of each ligation step., ligation times of less than 1 hour may reduce the overall ligation efficiency, or require additional enzyme to achieve the same efficiency. In examples, ligation time periods of between 4 hours and 24 hours per ligation were implemented, with incubation at 16 C; however other ligation times and incubation temperatures can be implemented.
Inherent in the split and pool synthesis approach for bead manufacturing is that beads with incomplete oligonucleotides will be combined together. As such, there is the potential for un-ligated barcodes from one well to become ligated onto oligos on beads that were originally in different wells. This is particularly true when the number of “stubs” (i.e., incomplete oligos attached to bead) is not completely saturated with barcodes. The result would be beads with more than one barcode on the same bead, and this would result in incorrect assignment of sequence data during analysis. This type of contamination would be very undesirable. If the beads (and ligation reaction components) from multiple wells are collected into larger tubes, collection of the beads, followed by pelleting to retain the beads and remove supernatant, followed by washing of the beads, significantly reduces cross-contamination to mitigate the above described effects (e.g., if performed rapidly). Alternatively, for the automation system or when any beads are left in mixed solution at intermediate states, ligation should be inhibitied (e.g, with a stop solution, with heat killing of enzymes, with dephosphorylating the barcode oligonucleotides, with adding blocking oligos, with depleting the ATP from the ligation solution, in another suitable manner). An example stop solution can include EDTA combined with approximately 2× the molar equivalents of Mg++ present.
The ideal number of oligonucleotides per bead further vary based on bead composition and final application of use. For instance, improved performance and reduced cost can be achieved for ligations with sub-maximal amounts of barcode oligonucleotides. An example process implemented 850 nanomoles of partially double stranded oligonucleotides in a ligation reaction with approximately 3.5 million beads, or about 0.25 picomols per bead. By reducing the amount of partially double stranded oligonucleotides to 172 nanomoles per 3.5 million beads, or about 50 femtomoles per bead, the cost of manufacture was significantly reduced with improved performance. This example achieved more optimal distribution of oligonucleotides around each bead, resulting in less steric hindrance as adjacent oligonucleotides where steric hindrance would be an issue were ligated at lower rates resulting in a more distributed set of full length oligonucleotides. The amount of ligase also scales with number of beads and with the number of ligation events per bead. In the example, 33,333 cohesive end units per 3.5 million beads were implemented, or about 0.0095 cohesive end units per bead.
Other ligation reaction components that can improve ligation include PEG 6000 to a final concentration of 10% w/v, Mg++ to a concentration of 10 mM (or by replacing up to ˜50% of the Magnesium with another divalent cation or with a much larger amount of monovalent cations where monovalent=120*square root of [divalent]). Other ligation reaction components can additionally or alternatively be implemented to produce suitable reaction environments.
Furthermore, while ligation is described in the example methods 400′, 400″, other methods of assembly or extension could be implemented (e.g., templated polymerase extension or chemical attachment, such as click attachment, etc.).
In relation to barcode unit subsequence lists described in relation to the methods above, various example lists can include between 96 (or less) and 932 (or more) barcode unit subsequences. In particular, sets can be configured for greater Hamming distance, Levenshtein distance, or other distance, in order to provide characteristics for easy correctability by post-sequencing analyses. Sets can additionally or alternatively be configured for producing beads with lower total barcode diversity.
However, other suitable configurations and/or numbers of barcode units per list can be implemented.
In examples, methods for manufacturing may start with multiple wells (e.g., 96 wells), each well containing over 1 million microspheres and one unique oligonucleotide segment attached (e.g., by ligation) to each bead under optimal conditions of time, temperature and shaking and compositions (e.g., enzyme concentration, oligonucleotide concentration, reaction enhancers, molecules to provide crowding). After ligation of the unique oligonucleotide tag to all the particles present in each tube (e.g., 96 tubes), the beads could be washed such that no carryover of products happen after washing when all the beads from 96 tubes would be pooled together (e.g., 1 million beads per tube×96 tubes=96 million beads pooled).
After washing an additional time, the beads are re-distributed into 96 different tubes containing a unique barcoded oligonucleotide segment and then additional reagents added (e.g., ligase, ATP, PEG, reaction enhancers) to continue the second phase of attachment. This process of barcode segment reaction, washing, pooling, redistributing is continued until all the different oligonucleotide segments area added to complete the entire process. The liquid handling process for split-pooling-washing and reaction of beads may be automated in 96 well plates or may be automated in other plate sizes such as 384 well plates or 1536 well plates. The dispensing of reagents in each well may be done by a liquid pipettor or may be done by other methods such as ink-jet-type nozzles, or acoustically ejected from an inverted well plate. The pooling of beads can be done by a pipettor or done by using a specially designed received lid plate that can be placed on the 96 well plate and then the plate-lid assembly inverted and shaken to collect all the beads in the receiver lid plate. Liquid handling operations are designed such that contamination of steps during the entire operations are minimized to prevent any errors to propagate through the entire process. This invention described herein will allow the workflow for manufacturing these barcoded beads to be significantly streamlined. The total number of beads that can be manufactured can be as low as 10 million to as high a 10 billion, with a bead diversity of more than 100,000 (or 1 million or >10 million) different unique combinations.
In some embodiments, the unique oligonucleotides present in each well may include different size fragments in different wells. For clarity, a specific example might be that 32 of the wells might each contain of a partially double stranded construct including of 6 bases providing overlap with the previous segment to facilitate ligation, 7 unique bases that define a barcode segment, and 4 bases to provide overlap with the following segment. An additional 32 wells contain of a partially double stranded construct including the same 6 bases providing overlap with the previous segment, 8 unique bases that define a barcode segment, and 4 bases to provide overlap with the following segment. A third set of 32 wells each contain a partially double stranded construct including the same 6 bases providing overlap with the previous segment, 9 unique bases that define a barcode segment, and 4 bases to provide overlap with the following segment. When used in the manufacturing method described above this would result in full length oligonucleotides that differ in length due to the inclusion of the different length fragments. When sequences are subsequently generated that read through the barcode regions, the barcodes manufactured in this way would have multiple distinct benefits for the sequence generation and analysis that are not present with a typical manufacturing process. In particular, when a plurality of sequences are generated from a plurality of beads, those generated by the above process can have the beneficial attribute that the overlap sections for some or all of the sequences should be identical. They can thus serve as alignment markers and provide other benefits to the analysis such as identifying chimeric molecules, sequencing or manufacturing errors, and other benefits.
The sequencers typically used for these analyses will produce errors and terminate the run, thus failing to collect the desired experimental data, if too large a portion of the sequences all contain the same base at a particular position. As such, inclusion of identical sequences, such as the identical overlap regions described, can be problematic when all of the sequences are the same length. By varying the length of the barcode units preceding the constant regions in the manner described herein, the resulting sequences become offset. While the overlap region or regions may be fundamentally invariant across the plurality of sequences, they are effectively out of phase such that the benefits of identical or near identical markers can be achieved without causing errors in the sequencing process itself. This can be implemented in the described manufacturing process with different numbers of wells or tubes and different configurations of sequence length variation that those used here for illustration as long as they are suitable to provide the dual benefit of working with the constraints of the sequencing instrumentation limitations and providing improved analysis post sequencing.
The FIGURES illustrate the architecture, functionality and operation of possible implementations of systems, methods and computer program products according to preferred embodiments, example configurations, and variations thereof. In this regard, each block in the flowchart or block diagrams may represent a module, segment, or portion of code, which comprises one or more executable instructions for implementing the specified logical function(s). It should also be noted that, in some alternative implementations, the functions noted in the block can occur out of the order noted in the FIGURES. For example, two blocks shown in succession may, in fact, be executed substantially concurrently, or the blocks may sometimes be executed in the reverse order, depending upon the functionality involved. It will also be noted that each block of the block diagrams and/or flowchart illustration, and combinations of blocks in the block diagrams and/or flowchart illustration, can be implemented by special purpose hardware-based systems that perform the specified functions or acts, or combinations of special purpose hardware and computer instructions.
As a person skilled in the art will recognize from the previous detailed description and from the figures and claims, modifications and changes can be made to the preferred embodiments of the invention without departing from the scope of this invention defined in the following claims.
This application claims the benefit of U.S. Provisional Application No. 62/945,006 filed on 6 Dec. 2019, which is incorporated in its entirety herein by this reference.
| Number | Date | Country | |
|---|---|---|---|
| 62945006 | Dec 2019 | US |
