BACKGROUND OF THE INVENTION

Accurate analysis of nucleic acids having a limited mass is required for many applications. When nucleic acids have an insufficient mass to be sequenced directly, a process of amplification is required. High fidelity amplification is essential so as to ensure an accurate representation of the template, which, in turn, can affect the accuracy of any downstream measurement. This process is required for applications where there are only small numbers of cells (including only one cell) available for analysis.

For example, preimplantation genetic diagnosis (PGD) can be used to determine whether a specific embryo has a genetic abnormality or known aberration that could lead to either failed implantation, subsequent miscarriage or the birth of a child with physical and/or developmental disability. The diagnosis can be used to make decisions on which embryo(s) should be transferred to the uterus for implantation and will with high likelihood result in a normal pregnancy.

PGD can be performed on genomic DNA from cells that are biopsied from embryos. Because the quantity of genomic DNA extracted from this biopsy can be insufficient for current genomic analysis techniques, including DNA sequencing, the DNA is typically amplified. In performing this amplification, a requirement is to accurately represent the state of the embryonic genome in the amplified product. However, amplification methods can result in an over-representation of certain regions of the genome and an under-representation of others, a phenomenon known as “amplification bias”. This bias can render the amplification products a distorted version of the original genomic DNA and thus limit the accuracy of PGD.

The present invention addresses this and other needs by providing methods, devices and systems to perform high fidelity amplification of nucleic acids with the goal of accurately analyzing nucleic acids from samples with limited template mass. These methods and devices can be used for numerous applications, including but not limited to PGD.

SUMMARY OF THE INVENTION

In one aspect of the invention, a flexible and scalable microfluidic device is provided, for example, for the is genomic analysis of single-cells is described in more detail. The device comprises a plurality of variable volume chambers that provides a robust and scalable tool for the direct preparation of single-cell libraries.

In one embodiment, a variable volume chamber is provided. The chamber comprises a flow layer defining a reaction chamber and flow channels, wherein at least one wall of the reaction chamber or a portion thereof is an elastomeric membrane, the membrane having: (i) a neutral position; and (ii) a plurality of expanded positions; wherein the expanded positions determine the volume of the reaction chamber, whereby the volume of the reaction chamber is V at the neutral position and the volume of the reaction chamber is greater than V in the plurality of expanded positions, wherein V is less than 1 μl, and wherein the volume at a maximally expanded position is equal to or greater than 2×V; and a blank layer to seal the flow layer.

In another embodiment, a variable volume microfluidic chamber comprising a flow layer and a control layer is provided. The flow layer defines a reaction chamber and flow channels, wherein at least one wall of the reaction chamber or a portion thereof is an elastomeric membrane. The membrane includes a neutral position; a plurality of expanded positions; and a plurality of reduced positions; wherein the expanded and reduced positions determine the volume of the reaction chamber, whereby the volume of the reaction chamber is V at the neutral position, the volume of the reaction chamber is greater than V in the plurality of expanded positions and the volume is less than V in the plurality of reduced positions, such that the volume at a maximally reduced position is essentially zero and wherein V is less than 1 μl. The chamber further comprises a control layer adjacent the flow layer defining control lines; a seal layer adjacent the control, to seal the flow; and a pressure chamber that surrounds the variable volume chamber or chambers, whereby the pressure chamber is operable to produce a positive pressure on the exterior surface of the elastomeric membrane and wherein the displacement chamber is operable to constrain the expansion of the reaction chamber.

In another aspect of the invention, a system for analyzing one or more particles is disclosed, for example a single cell or nucleus, or a plurality thereof. The system includes a transfer device configured to transfer one or more specifically chosen particles to a uniquely specified location on a surface, wherein the transfer device is configured to deposit the one or more particles in a stationary droplet on the surface. The one or more particles may be deposited by contact dispensing. The stationary droplet may comprise a solution of less than about 400 nL. The solution may be 400 nL or less.

In another aspect, methods are provided to analyze polynucleotides from a small number of cells. A small number of cells (e.g., less than 10 cells) can have a limited amount of polynucleotides. Methods provided herein can enhance the accuracy and sensitivity of the analysis of such a limited amount of polynucleotides. The method can enhance the analysis accuracy by performing the analysis in a small volume solution (e.g., less than 400 nL). For example, a sequencing library constructed in a solution of less than 400 nL can yield a greater coverage breadth for a given sequencing depth than that constructed in a solution of larger volume.

One embodiment of the methods described herein is directed to a method for analyzing heterogeneity within a tumor sample. The method comprises isolating a plurality of cells and/or nuclei from the tumor sample. Isolating can also comprise a combination of cell(s) and nuclei. For example, one cell and one nucleus can be isolated by the methods described herein. Alternatively, the plurality of cells and/or nuclei can be obtained, i.e., the plurality of cells and/or nuclei can already have been isolated. Isolating can be carried out e.g., with one of the transfer devices described herein, e.g., a microcapillary or a micropipette or a microdispenser Individual cell(s) and/or nuclei are deposited into individual containers. The individual chambers in some embodiments are individual microwells. In another embodiment, the individual chambers are individual microfluidic chambers. In yet another embodiment, the individual chambers are individual open microwells, droplets on a surface, or droplets in an emulsion. A cell and/or a nucleus in an individual chamber is also referred to herein as a tumor subpopulation. Preparation of sequencing libraries is carried out on nucleic acid from the plurality of individual cells and/or nuclei without prior amplification. In one embodiment, the library preparation method comprises a transposase reaction. In another embodiment, the library preparation comprises DNA fragmentation (e.g. enzymatically or mechanically), end-repair, A-tailing and adapter ligation. In yet another embodiment, libraries can be prepared by targeted PCR. Libraries may also be enriched using capture or pull-down techniques. Methods for generating such libraries are described herein and can be used in the present method for analyzing heterogeneity within a tumor sample. Index sequences are added to the nucleic acid in each individual container such that a unique index sequence is associated with a unique container. Index sequences are added during the transposase reaction, during a ligation step, or during a PCR reaction (or other amplification reaction) or a combination thereof. Individual libraries are then pooled and sequenced to provide sequence information of individual cells and/or nuclei. From this sequence information, the copy number profiles or other genomic structural abnormalities (e.g. translocations, deletions, amplifications, inversions, breakpoints, insertions) of individual cells and/or nuclei is determined. Based on the genomic alterations of each individual cell and/or nuclei, the clonal composition of the tumor is determined.

In another aspect, a method of amplifying polynucleotides from one or more cells is disclosed. The method involves depositing one or more specifically chosen cells in a solution having a volume of less than about 400 nL, wherein the one or more cells contains a polynucleotide; and amplifying the polynucleotides in a solution having a volume less than about 400 nL. The amplification may be carried out by numerous methods including by multiple displacement amplification (MDA) or polymerase chain reaction (PCR). The method described herein may further involve fragmenting the polynucleotide to generate a plurality of polynucleotide fragments prior to amplification. The method may further involve fragmenting the polynucleotide to generate a plurality of polynucleotide fragments wherein the fragmenting occurs in a single solution with the amplification step.

In another aspect, a method of generating a sequencing library with polynucleotides is disclosed. The method involves fragmenting the polynucleotides to generate a plurality of polynucleotide fragments; attaching a first adaptor to a first end and a second adaptor to a second end of each of the polynucleotide fragments; and amplifying the polynucleotide fragments with the first and second adaptors from the aforementioned second step using a primer set, wherein the primer set introduces a third adaptor on the first end and a fourth adaptor on the second end of each of the amplified polynucleotide fragments, wherein the first or second steps are performed in a solution having a volume less than about 400 nL. The polynucleotide fragments from the first (fragmentation) step may not be amplified before the second step. The polynucleotides may be obtained from a single cell. The polynucleotides may be obtained from a specifically chosen single cell or specifically chosen small number of cells placed in a volume less than about 400 nL.

In another embodiment of the methods described herein, a method of analyzing a polynucleotide is disclosed. The method involves depositing one or more cells in a solution having a volume less than about 400 nL to a uniquely specified location on a surface, wherein the one or more cells contains a polynucleotide; amplifying the polynucleotide in the solution; fragmenting the amplified polynucleotide to generate a plurality of polynucleotide fragments; separating the plurality of polynucleotide fragments; amplifying the polynucleotide fragments from the preceding step; and pooling the amplified polynucleotide fragments from the preceding step. The one or more cells may be deposited by contact dispensing. The separating may involve dividing the plurality of polynucleotide fragments into multiple reaction solutions, wherein the multiple reaction solutions are deposited on the surface. Further, the separating may involve depositing the plurality of polynucleotide fragments into an emulsion.

In yet another embodiment of the methods described herein, a method is disclosed comprising dividing the pooled polynucleotide fragments from a preceding and above-mentioned step into multiple pools; amplifying the polynucleotide fragments in the multiple pools; fragmenting the amplified polynucleotide fragments in the multiple pools; generating sequencing libraries in the multiple pools; sequencing the sequencing libraries to obtain sequences of the polynucleotide fragments in the multiple pools; assembling the sequences from a preceding step to obtain an assembled sequence; and comparing the assembled sequence to a reference sequence to determine whether the assembled sequence is inherited from the reference sequence.

In even another embodiment of the methods described herein, a method of determining a haplotype of a polynucleotide is disclosed. The method involves depositing one or more cells in a solution having a volume less than about 400 nL to a uniquely specified location on a surface by contact dispensing, wherein the one or more cells contains a polynucleotide; fragmenting the polynucleotide to generate a sequencing library; sequencing the fragments of the polynucleotide to obtain a plurality of sequence reads; assembling the sequence reads from a preceding step; and comparing the assembled sequence to a reference sequence to determine whether the assembled sequence is inherited from the reference sequence.

In one embodiment of the methods described herein, a method of selecting an embryo to screen for an inheritable disease is disclosed. The method involves depositing one or more cells from the embryo in a solution having a volume less than about 400 nL, wherein the one or more cells comprise a polynucleotide; using a reaction in a volume less than about 400 nL to obtain sequence information from the polynucleotide, thereby obtaining a sequence read of the polynucleotide; and determining whether the polynucleotide carries a genetic variation associated with an inheritable disease using the sequence reads from a preceding step. This method may be further modified by analyzing parental genetic sequences in addition to embryo screening. Sequencing libraries are also generated in embodiments of the methods described herein. Generating a sequencing library may include fragmenting the polynucleotide and tagging the fragmented polynucleotide. Generating a sequencing library may include use of a transposome, wherein the transposome may include a transposase and a custom tagging sequence. The polynucleotide may be fragmented and tagged with the custom tagging sequence by the transposase. The method may further include use of a primer set to introduce adaptors to the sequencing library. The breadth of coverage of the sequencing library may be at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%, wherein the breadth of coverage represents the fraction of the polynucleotide represented by at least one molecule in the sequencing library. The analyzing may include amplifying the polynucleotide. The fidelity of the amplification may be such that for at least about 3.3 Gbp of aligned sequence data, the breadth of coverage is greater than about 35%. The amplifying may be performed by in vitro clone expansion, multiple displacement amplification (MDA), or polymerase chain reaction (PCR). The method may further involve pre-amplifying the polynucleotide before the amplifying. The pre-amplifying may be performed by in vitro clone expansion, MDA, or PCR. The method may further involve determining whether the polynucleotide carries a genetic variation associated with a disease. The genetic variation may include a copy number variation. The polynucleotide fragments may be sequenced to a depth that allow to detect copy number variation of at least 10 kb, at least 20 kb, or at least 50 kb nucleotides in length. The disease may be a genetic disorder. The method may further involve treating the disease, including a heritable disease, by correcting the genetic variation. The genetic variation may be corrected using a nuclease. The nuclease may include a CRISPR-associated protein. The method may further involve fragmenting the polynucleotide, thereby generating a plurality of polynucleotide fragments. The fragmenting may be performed before an amplification step, and the fragmenting and the amplifying may be performed in a single reaction solution. The fragmenting may be performed before an MDA step. The polynucleotide fragments may be distributed into a plurality of reaction solutions. The method may further involve amplifying the polynucleotide fragments in the plurality of reaction solutions. The polynucleotide fragments may be greater than 1000 bp in length. The method may further involve sequencing at least one of the polynucleotide fragments, wherein the fragmentation sites of the sequenced polynucleotide fragments may allow determination of a haplotype of the polynucleotide fragment. The method may further involve determining a haplotype of the polynucleotide. The haplotype determination may include determining haplotype of a region of length in the polynucleotide as described herein. The method may further involve sub-fragmenting the amplified polynucleotide fragments in the plurality of reaction solutions. The method may further involve sequencing the sub-fragmented polynucleotides in the plurality of reaction solutions, thereby obtaining sequence reads of the sub-fragmented polynucleotides. The method may further involve assembling the sequence reads from more than one of the reaction solutions. The assembling may be performed using overlapping heterozygous single nucleotide variant sites. The one or more cells may be circulating cells. The one or more circulating cells may be from a pregnant subject. The one or more cells may be from an embryo, including a mammalian embryo. The method may further include determining whether the embryo is selected for implantation. The polynucleotide may include DNA. The DNA may be genomic DNA. The polynucleotide may be RNA. An above-mentioned method may further involve determining an epigenetic modification in the polynucleotide. The epigenetic modification may include methylation or histone modification. The method may further involve determining expression of a gene in the one or more cells. The expression may be determined using reverse-transcription PCR or whole transcriptome sequencing.

In another embodiment, a method of generating a sequencing library from a polynucleotide from a sample comprising one or more cells is disclosed. The method involves fragmenting the polynucleotide to generate fragmented polynucleotides; and attaching an adaptor to the fragmented polynucleotides to generate a sequencing library of fragmented polynucleotides comprising an adaptor, wherein the above-mentioned steps are performed in a volume less than 400 nL, and wherein the polynucleotide or fragmented polynucleotides of the first step is not amplified before the second step. The steps may be performed in a volume less than 100 nL. The method may further involve sequencing the fragmented polynucleotides comprising an adaptor. The method may further include determining a genetic variation in the fragmented polynucleotides comprising an adaptor. The genetic variation may include a copy number variation. The method may further involve determining a haplotype of the polynucleotide. The haplotype determination may include determining haplotype of a region of length in the polynucleotide as described herein. The method may further involve determining an epigenetic modification in the polynucleotide. The epigenetic modification may include methylation or histone modification. The method may further involve determining expression of a gene in the one or more cells. The expression may be determined using reverse-transcription PCR or whole transcriptome sequencing. The one or more cells may or may not be isolated by fluorescence-activated cell sorting. The fragmenting and attaching may be performed sequentially or simultaneously. The fragmenting and attaching may be performed using a transposase.

In another aspect, a method of analyzing at least one polynucleotide from a sample comprising amplifying the polynucleotides, wherein the fidelity of the amplification is as described herein. In yet another aspect, a method for selecting an embryo with a genetic characteristic is disclosed. The method involves transferring a cluster of cells from the embryo to a uniquely specified location on a surface; and analyzing at least one polynucleotide from the cluster of cells, thereby determining whether the embryo has the genetic characteristic, wherein the cluster of cells is transferred in a solution of no more than 400 nL to the uniquely specified location. The volume of the solution may be less than 100 nL. The cluster of cells may include no more than 30 cells. The cluster of cells may be split into individual cells prior to the analyzing. The analyzing may include analyzing at least one polynucleotide from each of the split cells. The analyzing may involve generating a sequencing library using the polynucleotide. The sequencing library may be generated using a transposase. The analyzing may involve amplifying the polynucleotide. The amplifying may be performed by in vitro clone expansion, MDA, or PCR. The method may further involve pre-amplifying the polynucleotide before the amplifying. The pre-amplifying may be performed by in vitro clone expansion, MDA, or PCR. The analyzing may include determining whether the polynucleotide carries a genetic variation associated with a disease. The disease may be a genetic disorder. The genetic variation may include a copy number variation. The method may further involve treating the disease by correcting the genetic variation. The correcting may be performed using a CRISPR-associated protein. The method may further involve fragmenting the polynucleotide, thereby generating a plurality of polynucleotide fragments. The fragmenting may be performed after an amplification step and the fragmenting and the amplifying may be performed in a single reaction solution. The fragments of the fragmented polynucleotide may be distributed into a plurality of reaction solutions. The method may further involve amplifying the fragmented polynucleotide in the plurality of reaction solutions. The polynucleotide fragments may be greater than 1000 bp in length. The method may further involve sequencing at least one of the polynucleotide fragments. The fragmentation sites of the sequenced polynucleotide fragments may allow determination of a haplotype of the polynucleotide fragment. The polynucleotide fragments may be sequenced to a breadth that allows detection of copy number variation of at least 10 kb, at least 20 kb, or at least 50 kb nucleotides in length. The method may further involve determining a haplotype of the polynucleotide. The haplotype determination may include determining haplotype of a region of length in the polynucleotide. The method may further involve sub-fragmenting the amplified polynucleotide in the plurality of reaction solutions. The method may further involve sequencing the sub-fragmented polynucleotide in the plurality of reaction solutions, thereby obtaining sequence reads of the sub-fragmented polynucleotide. The method may further involve assembling the sequence reads from more than one reaction solution using overlapping heterozygous single nucleotide variant sites. The polynucleotide may be DNA or RNA. The method may further involve determining an epigenetic modification in the polynucleotide. The epigenetic modification may include methylation or histone modification. The method may further involve determining expression of a gene in the one or more cells. The expression may be determined by reverse-transcription PCR or whole transcriptome sequencing. The analyzing may be performed using a microarray.

In yet another embodiment, a method for determining a haplotype is disclosed. The method involves depositing a plurality of polynucleotides into a plurality of first solutions, wherein the first solutions have a volume of less than about 400 nL; contacting each of the plurality of first solutions with one or more transposomes to tag the plurality of polynucleotides, wherein each of the one or more transposomes comprises a transposase and one or more adaptor sequences, thereby generating a plurality of tagged polynucleotides; creating one or more pools of the plurality of tagged polynucleotides; depositing the one or more pools of tagged polynucleotides into a plurality of second solutions, wherein the second solutions have a volume of less than about 400 nL; fragmenting the plurality of tagged polynucleotides in the plurality of second solutions to generate a plurality of fragmented polynucleotides within each of the second solutions, wherein each of the plurality of tagged polynucleotides has the one or more adaptor sequences appended at a first and a second end; sequencing the plurality of fragmented polynucleotides with the one or more adaptor sequences appended at the first and second ends to obtain a plurality of sequence reads; and comparing the sequence reads to a reference sequence to determine the haplotype. Either of the depositing steps, or both of the depositing steps, may include non-contact dispensing. Either of the depositing steps, or both of the depositing steps may include depositing the solutions to a plurality of uniquely specified locations on a surface. The fragmenting may include contacting the plurality of first solutions with a denaturant. The denaturant may be sodium dodecyl sulfate (SDS). The one or more adaptor sequences may be different for each of the plurality of first solutions. The plurality of polynucleotides may be deposited in the plurality of first solutions at a limiting dilution. The plurality of polynucleotides may be deposited in the plurality of first solutions at a dilution such that the probability of two polynucleotides with overlapping sequences is less than 10%, 5%, 2%, 1%, or 0.1%, as determined by the diversity of sequences of the polynucleotides being analyzed. Each of the plurality of polynucleotides in each of the plurality of first solutions may align to a different region of the reference sequence. The diversity and the dilution of polynucleotides may be selected such that the large majority of the plurality of polynucleotides in the majority of each of the plurality of first solutions may align to a different region of the reference sequence. Each of the plurality of first solutions may comprise, on average, one polynucleotide. Each of the plurality of first solutions may comprise, on average, more than one polynucleotide. Determining the haplotype may include constructing a haplotype block. The method may further include, prior to the sequencing, amplifying the plurality of fragmented polynucleotides. The amplifying may include annealing primers containing one or more index sequences to the one or more adaptor sequences appended to the first and second ends of the fragmented polynucleotides. The one or more index sequences may be different for each of the plurality of solutions and may be used to associate the sequence reads with the identity of the first and second solutions from which they were produced. The transposase may be Tn5 transposase. The plurality of polynucleotides may include genomic DNA. The method may further include, prior to the first depositing step, obtaining the plurality of polynucleotides by lysing one or more cells.

In another aspect, a method is provided for determining a haplotype. The method includes fragmenting a plurality of polynucleotides to generate a plurality of fragmented polynucleotides; depositing the plurality of fragmented polynucleotides into a plurality of solutions, wherein the solutions have a volume of less than about 400 nL; contacting each of the plurality of solutions with one or more transposomes to tag the plurality of fragmented polynucleotides, wherein each of the one or more transposomes includes a transposase and one or more adaptor sequences, thereby generating a plurality of tagged polynucleotides with the one or more adaptor sequences appended at a first and a second end; sequencing the plurality of tagged polynucleotides to obtain a plurality of sequence reads; and comparing the sequence reads to a reference sequence to determine the haplotype. The depositing may include non-contact dispensing. The depositing may include depositing the solutions to a plurality of uniquely specified locations on a surface. The method may further include, prior to the fragmenting, obtaining the plurality of polynucleotides by lysing one or more cells. The plurality of fragmented polynucleotides may be deposited in the plurality of solutions at a limiting dilution. Each of the plurality of fragmented polynucleotides in each of the plurality of solutions may align to a different region of the reference sequence. Each of the plurality of solutions may include, on average, one polynucleotide. Determining the haplotype may further include constructing a haplotype block. The method may further include, prior to the sequencing, amplifying the plurality of tagged polynucleotides. The amplifying may include annealing primers containing one or more index sequences to the one or more adaptor sequences appended to the first and second ends of the tagged polynucleotides. The one or more index sequences may be different for each of the plurality of solutions. The transposase may be Tn5 transposase. The plurality of polynucleotides may be genomic DNA. The one or more adapter sequences used in the creation of the tagged polynucleotides during the transposome reaction, and/or the one or more index sequences appended to the first and second ends of the tagged polynucleotides during the amplification, may include one or more unique polynucleotide sequences that may be used to associate the sequenced polynucleotide fragments with the one or more solutions into which the original polynucleotide fragments were deposited.

