EMBRYOS

BACKGROUND OF THE INVENTION

[0001] The embodiments disclosed herein are generally directed towards methods for identifying de novo mutations in human embryos.

[0002] In vitro fertilization (IVF) has become increasingly popular as a type of assisted reproductive technology, typically for women of advanced maternal age or couples with difficulties conceiving. Advanced maternal age is associated with decreased fertility rates and increased child diseases (such as Down syndrome and other diseases associated with genetic dysfunctions such variants in copy number or mutations ), and as women in developed nations continue to forego conception until later ages, they become more at risk of producing affected babies or none at all.

[0003] With such risks, many patients undergoing assisted reproduction choose to have their embryos analyzed with embryo screening methods such as preimplantation genetic screening (PGS) and preimplantation genetic diagnosis (PGD). PGD, for example, is a procedure that samples embryos to render a genetic diagnosis of their viability and normalcy. Currently, PGD is primarily used to detect specific heritable diseases or chromosome abnormalities known to be present in parents. As such, current PGD approaches screen for both parents to detect the diseases that can be passed to the embryos, so PGD can screen them. However, de novo mutations can occur in the sperm or eggs (i.e. not present in one or both of the parents), thus current embryo screening methods are unable to target preimplantation or prenatal diagnoses. Recent studies have found that de novo variations spread across many different genes are likely to be the cause of many autism spectrum disorders, as well as many other rare congenital disorders.

[0004] Therefore, current methods are lacking in the PGD space to identify embryonic de novo mutations, and by extension, non-heritable genetic diseases.

[0005] Consequently, there is a need in the industry to provide accurate and comprehensive preimplantation, as well as prenatal, diagnoses of embryos that can specifically capture embryonic de novo mutations. SUMMARY OF THE INVENTION

[0006] In one aspect, a method for identifying de novo mutations in a tissue sample is provided. The method comprises obtaining a plurality of cells from a tissue sample and separating the cells into a plurality of partitions, wherein each partition includes a single cell, amplifying the DNA in the partitions. The method further comprises sequencing the DNA in the partitions, identifying genomic variants in the partitions, and labeling a genomic variant as a de novo mutation when it is present in at least a threshold percentage of partitions. Alternatively, the threshold percentage of partitions is 50%. Alternatively, at least four cells are obtained from the tissue sample. Alternatively, the sequencing is whole exome sequencing. The sequencing can also be whole genome sequencing. Further in the alternative, the genomic variants are copy number variants.

[0007] The method can further comprise enzymatically disaggregating the plurality of cells into individual cells prior to separating the cells. Even further, the method can comprise adding one or more enzymes in sufficient amount to immerse the plurality of cells, dispersing individual cells disaggregated by the one or more enzymes, and selecting individual cells for transporting to a respective one of the plurality of partitions. Furthermore, the method can also comprise inhibiting the one or more enzymes prior to transporting the individual cells. The one or more enzymes can comprise a proteolytic enzyme. The one or more enzymes can comprise a collagenolytic enzyme. The one or more enzymes can comprise a proteolytic enzyme and a collagenolytic enzyme.

[0008] The method alternatively can further comprise comparing the sequenced DNA to parental DNA to remove inherited genomic variants from being labeled as de novo mutations. Even further, the method can comprise applying a filter to the identified genomic variants to remove genomic variants selected from the group consisting of: amplification artifacts, inherited variants, sequencing error variants, mapping error variants, polymerase error variants, and combinations thereof.

[0009] In another aspect, a method for disaggregating a plurality of cells obtained from a tissue sample is provided. The method comprises providing a plurality of cells in a vessel, immersing the plurality of cells in one or more enzymes, incubating the immersed plurality of cells for a time period necessary for cells to disaggregate, dispersing individual cells disaggregated by the one or more enzymes, and selecting individual cells for transporting to a respective one of a plurality of partitions for analysis. Alternatively, the method further comprises inhibiting the one or more enzymes after incubating the immersed plurality of cells. [0010] Alternatively, the time period for incubating the immersed plurality of cells is at least 5 minutes, or the time period is at least 10 minutes, or is at least 50 minutes, or is at least 100 minutes. Alternatively, the time period is between 100 and 120 minutes. Alternatively, the time period is up to 150 minutes.

[0011] In the alternative, the one or more enzymes comprises a proteolytic enzyme. Alternatively, the one or more enzymes comprises a collagenolytic enzyme. Alternatively, the one or more enzymes comprises a proteolytic enzyme and a collagenolytic enzyme.

[0012] In yet another aspect, a method for identifying de novo mutations in human embryos indicative of an autism spectrum disorder is provided. The method comprises obtaining a plurality of cells from a human embryo and separating the plurality of cells into a plurality of partitions, wherein each partition includes a single cell. The method further comprises amplifying the DNA in the partitions, sequencing the DNA in the partitions, identifying genomic variants in the partitions, labeling a genomic variant as a de novo mutation when it is present in at least a threshold percentage of partitions, and determining that the human embryo is at high risk for developing an autism spectrum disorder based on a threshold number of genes having de novo mutations occurring at specific chromosomal locations. Alternatively, the genomic variants are copy number variants. Alternatively, the threshold percentage of partitions is 50%. Alternatively, at least four cells are obtained from the embryo. Further in the alternative, the sequencing is whole exome sequencing. Alternatively, the sequencing is whole genome sequencing.

[0013] Alternatively, the method further comprises enzymatically disaggregating the plurality of cells into individual cells before separating the cells into the plurality of partitions. Further, the method can comprises adding one or more enzymes in sufficient amount to immerse the plurality of cells, dispersing the individual cells disaggregated by the one or more enzymes, and selecting individual cells for transporting to a respective one of the plurality of partitions. Even further, the method can comprise inhibiting the one or more enzymes prior to transporting the individual cells.

[0014] In the alternative, the one or more enzymes comprises a proteolytic enzyme. Alternatively, the one or more enzymes comprises a collagenolytic enzyme. Alternatively, the one or more enzymes comprises a proteolytic enzyme and a collagenolytic enzyme. [0015] Alternatively, the method can further comprise comparing the sequenced DNA to parental DNA to remove inherited genomic variants from being labeled as de novo mutations.

[0016] In the alternative, the threshold number of genes is greater than one. The threshold number of genes can also be greater than three. The threshold number of genes can also be greater than ten. The threshold number of genes can also be greater than twenty.

[0017] Further in the alternative, the method further comprises applying a filter to the identified genomic variants to remove genomic variants selected from the group consisting of: amplification artifacts, inherited variants, sequencing error variants, mapping error variants, polymerase error variants, and combinations thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG. 1 is a flow chart illustrating a general workflow or system for identifying de novo mutations, in accordance with various embodiments.

[0019] FIG. 2 is a flow chart illustrating a method for identifying de novo mutations, in accordance with various embodiments.

[0020] FIG. 3 is a flow chart illustrating a method for enzymatically disaggregating cells of an embryo, in accordance with various embodiments.

[0021] FIG. 4 is a flow chart illustrating a method for identifying de novo mutations that are indicative of an autism spectrum disorder, in accordance with various embodiments.

[0022] FIG. 5 is a block diagram that illustrates a computer system, in accordance with various embodiments.

[0023] It is to be understood that the figures are not necessarily drawn to scale, nor are the objects in the figures necessarily drawn to scale in relationship to one another. The figures are depictions that are intended to bring clarity and understanding to various embodiments of apparatuses, systems, and methods disclosed herein. Wherever possible, the same reference numbers will be used throughout the drawings to refer to the same or like parts. Moreover, it should be appreciated that the drawings are not intended to limit the scope of the present teachings in any way. DETAILED DESCRIPTION OF THE INVENTION

[0024] The following description of various embodiments is exemplary and explanatory only and is not to be construed as limiting or restrictive in any way. Other embodiments, features, objects, and advantages of the present teachings will be apparent from the description and accompanying drawings, and from the claims.

[0025] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, nomenclatures utilized in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are those well-known and commonly used in the art. Standard techniques are used, for example, for nucleic acid purification and preparation, chemical analysis, recombinant nucleic acid, and oligonucleotide synthesis. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The techniques and procedures described herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the instant specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (Third ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 2000). The nomenclatures utilized in connection with, and the laboratory procedures and techniques described herein are those well-known and commonly used in the art.

[0026] The phrase "next generation sequencing" (NGS) refers to sequencing technologies having increased throughput as compared to traditional Sanger- and capillary electrophoresis-based approaches, for example with the ability to generate hundreds of thousands of relatively small sequence reads at a time. Some examples of next generation sequencing techniques include, but are not limited to, sequencing by synthesis, sequencing by ligation, and sequencing by hybridization. More specifically, the MISEQ, HISEQ and NEXTSEQ Systems of Illumina and the Personal Genome Machine (PGM) and SOLiD Sequencing System of Life Technologies Corp, provide massively parallel sequencing of whole or targeted genomes. The SOLiD System and associated workflows, protocols, chemistries, etc. are described in more detail in PCT Publication No. WO 2006/084132, entitled "Reagents, Methods, and Libraries for Bead-Based Sequencing," international filing date Feb. 1, 2006, U.S. patent application Ser. No. 12/873,190, entitled "Low-Volume Sequencing System and Method of Use," filed on Aug. 31, 2010, and U.S. patent application Ser. No. 12/873, 132, entitled "Fast-Indexing Filter Wheel and Method of Use," filed on Aug. 31, 2010, the entirety of each of these applications being incorporated herein by reference thereto.

[0027] The phrase "sequencing run" refers to any step or portion of a sequencing experiment performed to determine some information relating to at least one biomolecule (e.g., nucleic acid molecule).

[0028] DNA (deoxyribonucleic acid) is a chain of nucleotides consisting of 4 types of nucleotides; A (adenine), T (thymine), C (cytosine), and G (guanine), and that RNA (ribonucleic acid) is comprised of 4 types of nucleotides; A, U (uracil), G, and C. Certain pairs of nucleotides specifically bind to one another in a complementary fashion (called complementary base pairing). That is, adenine (A) pairs with thymine (T) (in the case of RNA, however, adenine (A) pairs with uracil (U)), and cytosine (C) pairs with guanine (G). When a first nucleic acid strand binds to a second nucleic acid strand made up of nucleotides that are complementary to those in the first strand, the two strands bind to form a double strand. As used herein, "nucleic acid sequencing data," "nucleic acid sequencing information," "nucleic acid sequence," "genomic sequence," "genetic sequence," or "fragment sequence," or "nucleic acid sequencing read" denotes any information or data that is indicative of the order of the nucleotide bases (e.g., adenine, guanine, cytosine, and thymine/uracil) in a molecule (e.g., whole genome, whole transcriptome, exome, oligonucleotide, polynucleotide, fragment, etc.) of DNA or RNA. It should be understood that the present teachings contemplate sequence information obtained using all available varieties of techniques, platforms or technologies, including, but not limited to: capillary electrophoresis, microarrays, ligation-based systems, polymerase-based systems, hybridization-based systems, direct or indirect nucleotide identification systems, pyrosequencing, ion- or pH-based detection systems, electronic signature- based systems, etc.

[0029] A "polynucleotide", "nucleic acid", or "oligonucleotide" refers to a linear polymer of nucleosides (including deoxyribonucleosides, ribonucleosides, or analogs thereof) joined by intemucleosidic linkages. Typically, a polynucleotide comprises at least three nucleosides. Usually oligonucleotides range in size from a few monomeric units, e.g. 3-4, to several hundreds of monomelic units. Whenever a polynucleotide such as an oligonucleotide is represented by a sequence of letters, such as "ATGCCTG," it will be understood that the nucleotides are in 5?->3? order from left to right and that "A" denotes deoxyadenosine, "C" denotes deoxycytidine, "G" denotes deoxyguanosine, and "T" denotes thymidine, unless otherwise noted. The letters A, C, G, and T may be used to refer to the bases themselves, to nucleosides, or to nucleotides comprising the bases, as is standard in the art.

[0030] The phrase "fragment library" refers to a collection of nucleic acid fragments, wherein one or more fragments are used as a sequencing template. A fragment library can be generated, for example, by cutting or shearing a larger nucleic acid into smaller fragments. Fragment libraries can be generated from naturally occurring nucleic acids, such as mammalian or bacterial nucleic acids. Libraries comprising similarly sized synthetic nucleic acid sequences can also be generated to create a synthetic fragment library.

[0031] In various embodiments, a sequence alignment method can align a fragment sequence to a reference sequence or another fragment sequence. The fragment sequence can be obtained from a fragment library, a paired-end library, a mate-pair library, a concatenated fragment library, or another type of library that may be reflected or represented by nucleic acid sequence information including for example, RNA, DNA, and protein based sequence information. Generally, the length of the fragment sequence can be substantially less than the length of the reference sequence. The fragment sequence and the reference sequence can each include a sequence of symbols. The alignment of the fragment sequence and the reference sequence can include a limited number of mismatches between the symbols of the fragment sequence and the symbols of the reference sequence. Generally, the fragment sequence can be aligned to a portion of the reference sequence to minimize the number of mismatches between the fragment sequence and the reference sequence.

[0032] In particular embodiments, the symbols of the fragment sequence and the reference sequence can represent the composition of biomolecules. For example, the symbols can correspond to identity of nucleotides in a nucleic acid, such as RNA or DNA, or the identity of amino acids in a protein. In some embodiments, the symbols can have a direct correlation to these subcomponents of the biomolecules. For example, each symbol can represent a single base of a polynucleotide. In other embodiments, each symbol can represent two or more adjacent subcomponents of the biomolecules, such as two adjacent bases of a polynucleotide. Additionally, the symbols can represent overlapping sets of adjacent subcomponents or distinct sets of adjacent subcomponents. For example, when each symbol represents two adjacent bases of a polynucleotide, two adjacent symbols representing overlapping sets can correspond to three bases of polynucleotide sequence, whereas two adjacent symbols representing distinct sets can represent a sequence of four bases. Further, the symbols can correspond directly to the subcomponents, such as nucleotides, or they can correspond to a color call or other indirect measure of the subcomponents. For example, the symbols can correspond to an incorporation or non-incorporation for a particular nucleotide flow.

[0033] In various embodiments, a computer program product can include instructions to select a contiguous portion of a fragment sequence; instructions to map the contiguous portion of the fragment sequence to a reference sequence using an approximate string matching method that produces at least one match of the contiguous portion to the reference sequence.

[0034] All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing devices, compositions, formulations and methodologies which are described in the publication and which might be used in connection with the present disclosure.

[0035] As used herein, the terms "comprise", "comprises", "comprising", "contain", "contains", "containing", "have", "having" "include", "includes", and "including" and their variants are not intended to be limiting, are inclusive or open-ended and do not exclude additional, unrecited additives, components, integers, elements or method steps. For example, a process, method, system, composition, kit, or apparatus that comprises a list of features is not necessarily limited only to those features but may include other features not expressly listed or inherent to such process, method, system, composition, kit, or apparatus.

[0036] As used herein, the term "cell" is used interchangeably with the term "biological cell." Non-limiting examples of biological cells include eukaryotic cells, plant cells, animal cells, such as mammalian cells, reptilian cells, avian cells, fish cells, or the like, prokaryotic cells, bacterial cells, fungal cells, protozoan cells, or the like, cells dissociated from a tissue, such as muscle, cartilage, fat, skin, liver, lung, neural tissue, and the like, immunological cells, such as T cells, B cells, natural killer cells, macrophages, and the like, embryos (e.g., zygotes), oocytes, ova, sperm cells, hybridomas, cultured cells, cells from a cell line, cancer cells, infected cells, transfected and/or transformed cells, reporter cells, and the like. A mammalian cell can be, for example, from a human, a mouse, a rat, a horse, a goat, a sheep, a cow, a primate, or the like.

[0037] The practice of the present subject matter may employ, unless otherwise indicated, conventional techniques and descriptions of organic chemistry, molecular biology (including recombinant techniques), cell biology, and biochemistry, which are within the skill of the art.

[0038] FIG. 1 is a flow chart illustrating a general workflow or system 100 for identifying de novo mutations, in accordance with various embodiments. Note that, as this is a general workflow, much of the detailed discussion related to the steps of FIG. 1 will be provided in greater detail with regard to embodiments described in FIGS. 2-4. Those detailed descriptions are still applicable to the embodiments of FIG. 1.

[0039] Though the workflow or system is applicable in various ways, FIG. 1 illustrates the workflow or system using an embryo. At step 110, fertilization of an egg with sperm occurs. This fertilization can occur naturally or through assisted reproduction techniques such as IVF. At step 120, an embryo is allowed to incubate for a sufficient period until it is capable of being tested for de novo mutations. For example, embryos that reach day three (cleavage stage) are capable of being tested. At step 130, the embryo is biopsied by having a plurality of cells removed for testing.

[0040] At step 140, a known process is provided where DNA from the obtained cells is isolated, diluted and aliquoted into several tubes. This commonly known method requires lysing embryo cells, denaturing the DNA contained therein, diluting the denatured DNA sample, and splitting aliquots of the diluted DNA-containing solution into separate wells or tubes to provide an estimated equivalent of approximately one cell per well or tube. At best, such methods can provide only an estimated cell equivalent in each well or tube. This creates, at the outset, deficiencies in the methodology, particularly when cell to cell comparisons are highly valued in making accurate diagnoses, with autism spectrum disorder (ASD) diagnoses being one such example.

[0041] Alternative to step 140, in accordance with the present embodiments, step 150 provides that the obtained plurality of cells are separated into individual cells and transferred to a plurality of partitions where one cell is provided per partition. The cells can also be initially disaggregated for example, by one or more enzymes, into individual cells before separating the cells. Further discussion related to the disaggregation process will be provided in detail later. Of particular note is that rather than splitting aliquots of the diluted DNA-containing solution into separate wells or tubes for testing (step 140), which provides only an estimated cell count per well/tube, the separated individual cells of step 150 are transferred to a plurality of partitions for further testing such that exactly one cell is provided per partition.

[0042] It has advantageously been found that testing individually separated tissue cells (e.g., human embryo cells), particularly in a PGD context, ensures that exactly one cell is provided per partition, providing a more accurate read of each cell's contents than aliquots of the diluted DNA- containing solution of lysed tissue cells (e.g., human embryo cells). Besides increased accuracy, the process of separating, or disaggregating and separating, a plurality of cells requires less time and processing, making the overall process of de novo mutation detection more efficient for the user.

[0043] At step 160, the diluted DNA-containing solution of step 140, or the DNA in the partitioned individual cells of step 150, are amplified. In step 170, the amplification products of step 160 are sequenced. Finally, in step 180, the sequenced DNA is analyzed to identify genomic variants in the tubes/wells (step 140) or partitions (step 150), and specific genomic variants are labeled as de novo mutations when present in at least a threshold percentage of tubes/wells (step 140) or partitions (step 150). This threshold analysis further enforces the advantage of using separated cells versus diluted DNA-containing solution aliquots, as the purpose of this threshold analysis is to understand the prevalence of de novo mutations across multiple cells. Whereas the aliquots of step 140 aim for a cell per well/tube equivalent, the cell separation technique of 150 accurately accomplishes the cell per partition target. The results of the threshold analysis are therefore more accurate using the cell separation technique of the present embodiments.

[0044] FIG. 2 is a flow chart illustrating a method 200 for identifying de novo mutations in accordance with various embodiments. Method 200 is illustrative only and embodiments can use variations of method 200. Method 200 can include steps for identification of de novo mutations specifically in tissue samples such as, for example, a human embryo.

[0045] In step 210 of method 200 of FIG. 1, a plurality of cells is obtained from a tissue sample. The cells can be obtained via known biopsy methods such as, for example, excisional, incisional/core, needle aspiration, liquid, and various embryo biopsies. The tissue sample can be from, for example, a human embryo. With in vitro fertilization (IVF), after successful fertilization of an egg (e.g., human egg) with sperm during the co-incubation period, resulting embryos are generally allowed to grow for a period in a medium until embryo transfer to the uterus. This medium can be an artificial medium. The period can be, for example, until the cleavage stage (day two to four after the co-incubation period) or the blastocyst stage (day five or six after the co-incubation period). Embryos that at least reach the day three cell stage (cleavage stage) can be tested for chromosomal or specific genetic defects by PGD.

[0046] The number of cells obtained from the tissue sample can be, for example, at least four. More particularly, the number of cells obtained can be, for example, four to ten. It has been advantageously found that, in identifying de novo mutations in tissue samples such as, for example, human embryos, capturing at least four cells from each sample produces more accurate results than a lower cell count per sample.

[0047] In step 220, the cells from the tissue sample are separated from each other into a plurality of partitions, wherein each partition includes a single cell. The partitions can be in any form, known in the art, which allows for physical separation of one cell from another into separate sample regions. Partitions can include, but are not limited to, wells, tubes, spots, depressions, through-holes, reaction chambers, and reaction sites. The partitions can be provided, for example and not limited to, in a regular or irregular pattern formed on the surface of a sample holder substrate, within a sample holder substrate, within test sites or volumes of a microfluidic system, or within or on small beads or spheres. Some examples of a sample holder may include, but are not limited to, any size multi- partition plate, card or array including, but not limited to, a collection of tubes, a microtiter plate, a microcard, a through-hole array, or a substantially planar holder, such as, for example, a glass or plastic slide.

[0048] Alternatively, at step 220, the plurality of cells can be enzymatically disaggregated (referred to also as, for example, dissected or dissociated) into individual cells before separating the cells into a plurality of partitions, wherein each partition includes a single cell. Enzymatic disaggregation is discussed in more detail below in relation to FIG. 3. In general, enzymatic disaggregation includes adding one or more enzymes in sufficient amount to immerse the plurality of cells, dispersing individual cells disaggregated by the one or more enzymes, and selecting individual cells for transporting to a respective one of the plurality of partitions. Enzymatic disaggregation can further include inhibiting the one or more enzymes prior to transporting the individual cells. Discussion related to the one or more enzymes will be provided in detail below.

[0049] Methods known in the art do not separate cells of a tissue sample such as, for example, cells of a human embryo, for PGD analysis, particularly for ASD diagnosis. For example, as discussed above, known methods for PGD analysis require lysing embryo cells, denaturing the DNA contained therein, diluting the denatured DNA sample, and splitting aliquots of the diluted DNA- containing solution into separate wells or tubes to provide an estimated equivalent of approximately one cell per well or tube. Because of the above known process, such methods at best can provide only an estimated cell equivalent in each well or tube. This creates, at the outset, deficiencies in the methodology, particularly when cell to cell comparisons are highly valued in making accurate diagnoses, with ASD diagnoses being one such example. [0050] Therefore, it has advantageously been found that testing individually separated tissue cells (e.g., human embryo cells) ensures that exactly one cell is provided per partition, providing a more accurate read of each cell's contents than diluted solution of lysed tissue cells (e.g., human embryo cells). Besides increased accuracy, the process of separating, or disaggregating and separating, a plurality of cells requires less time and processing, making the overall process of de novo mutation detection more efficient.

[0051] In step 230, DNA in each single cell is amplified. Amplification can occur while each single cell is in a respective partition and can include, for example, exponential (or non-linear), linear and quasilinear amplification methods. Alternatively, a linear or quasilinear amplification method can be used.

[0052] Examples of exponential (or non-linear) amplification include, but are not limited to, multiple displacement amplification (MDA) or modified MDA, which is a non-PCR based DNA amplification technique. Generally, this reaction is initiated with multiple hexamer primers annealing to the template, with DNA synthesis carried out via a high fidelity enzyme such as, for example, phi 29 DNA polymerase. Compared with conventional PCR amplification techniques, MDA generates larger sized products with a lower error frequency. This method has been actively used in whole genome amplification (WGA) and is a promising method for application to single cell genome sequencing and sequencing -based genetic studies.

[0053] Exponential (or non-linear) amplification, allows for a much faster rate of DNA amplification versus linear or quasi-linear amplification. Traditional exponential amplification techniques can have the tendency to magnify discrepancies in amplification as well. With certain exponential amplification techniques, while regions that amplify particularly well will exponentially multiply, any errors introduced during amplification may also be magnified. In the end, this can lead to quicker amplification at the cost of decreasing accuracy in the overall amplification process and increasing the time necessary to deal with increased numbers of, for example, amplification artifacts.

[0054] Linear or quasilinear amplification, as compared to traditional exponential amplification, allows for amplification of only the original genomic DNA, which can reduce amplification bias by reducing the magnification of errors introduced during amplification.

[0055] One example of linear or quasilinear amplification is the Multiple Annealing and Looping Based Amplification Cycles (MALBAC) method of quasilinear whole genome amplification. The MALBAC amplification method uses special primers that allow amplicons to have complementary ends and therefore to loop, preventing DNA from being copied exponentially. This results in amplification of only the original genomic DNA. This controlled amplification consequently reduces amplification bias and, by extension, can lower production of artifacts and lower incidences of false positive and false negative mutations.

[0056] Another example of linear or quasilinear amplification is the Linear Amplification via Transposon Insertion (LIANTI) method. This single cell method can use a unique transposon that can include a 19-base pair double-stranded transposase binding site and a single -stranded T7 promoter loop. The insertion of this transposon enables the desired linear amplification, and similar corresponding advantages as discussed above with MALBEC amplification.

[0057] Both MALBAC and LIANTI methods are examples of single-cell whole genome amplification. These exemplary methods can be particularly well suited for single cell analysis in fields such as forensics, pre-natal screening for genetic diseases, and PGD, where it can be especially valuable to observe the frequency with which mutations accumulate in single cells, as well as detect chromosomal abnormalities and genomic variants (e.g., insertions, deletions, single nucleotide variants, and copy number variations) within and between cells.

[0058] In step 240, the amplified DNA in each cell is then sequenced. Sequencing can occur while each single cell is in a respective partition. Sequencing techniques can include, for example, whole exome sequencing (WES) or whole genome sequencing (WGS) via traditional and next- generation sequencing methods. As compared to WGS, WES is only able to identify those variants found in the coding region of genes which affect protein function. As a result, approximately 99% of the human genome is not covered using exome sequencing. Accordingly, WES is not able to identify the structural and non-coding variants associated with a disease, which can be found using WGS. However, for a similar cost, WES allows sequencing of portions of the genome over at least 20 times as many samples compared to WGS. Therefore, while WGS is slowly being adopted in clinical applications, WES can provide cost and time advantages in translating identified rare variants into the clinic, interpreting results, and providing clinical diagnoses.

[0059] In step 250, the sequenced DNA is analyzed to identify genomic variants in the partitions. The genomic variants can include any structural variations including, but not limited to, insertions/deletions (InDels), single nucleotide variants (SNVs), copy number variations (CNVs), single nucleotide polymorphisms (SNPs), duplications, inversions and translocations. [0060] In step 260, a specific genomic variant is labeled as a de novo mutation when present in at least a threshold percentage of partitions. The threshold percentage can be, for example, 50%. Alternatively, the threshold percentage can be 40% if, for example, at least five cells are analyzed. Alternatively, the threshold percentage can be 30% if, for example, at least ten cells are analyzed. Alternatively, the threshold percentage of partitions is 50% of DNA of the aggregate of cells. As such, a concordance in the plurality of partitions over the given threshold percentage will be considered sufficient to label a genomic variant as a de novo mutation.

[0061] Alternatively, a filter, or multiple filters, can be applied to the identified genomic variants to remove particular genomic variants (i.e., false-positive genomic variants) that can affect detection of de novo mutations. These removed genomic variants are subsequently not considered for labeling as de novo mutations. By applying a filter, or multiple filters, an original genomic variant count can be reduced significantly to a number better suited for de novo mutation analysis. For example, by applying appropriate filters, an original variant count of 100,000 can be reduced to between approximately 1000 to 2000 variants. In some cases, the count can be reduced, for example, to approximately 100 variants. As the count is reduced, the chances increase that a high percentage of remaining variants are true de novo mutations.

[0062] False-positive genomic variants can occur for many different reasons such as, for example, errors, artifacts, and inherited variants. Therefore, a group of false-positive genomic variants can include, for example, amplification artifacts, inherited variants, sequencing error variants, mapping error variants, polymerase error variants via amplification method used, and combinations thereof. There are numerous corresponding filters available, the use of which depend on, for example, the experimental design for de novo mutation detection and type of variants being detected (e.g., SNV vs. CNV). As such, examples of filters include, but are not limited to, batch artifact, haplotype analysis, exclusive well presence, coverage depth, quality score, structural variation proximity (near InDels or SNV clusters), and repetitive sequence filters.

[0063] As discussed previously, use of linear or quasi-linear amplification can help reduce amplification bias, and therefore assist in reducing the proliferation of false-positive genomic variants. Inherited variants, however, still could remain. As inherited, these variants inherently do not qualify as de novo mutations. To remove inherited variants, for example, sequenced embryonic DNA would be compared to parental DNA to identify common variants between the two DNA sets, and remove those common variants from consideration. [0064] Alternatively, when the sequenced DNA is analyzed to specifically identify CNVs (as opposed to, for example, SNVs or InDels); potential filtering needs can be lessened. As compared to smaller sized structural variations, (e.g., InDels and SNVs), CNVs are less likely to be mistaken for amplification artifacts, and therefore will not require as much filtering, or at least require a simpler filter protocol. The associated reduced complexity, cost and time for analysis makes targeting CNVs advantageous, assuming sufficient data for diagnosis can be gleaned directly from CNV detection and analysis. Targeted CNV analysis is particularly advantageous for PGD in general and, in particular, for autism spectrum disorders (ASDs). Many recent large scale sequencing studies have found that de novo CNV mutations are likely to be the cause of a large fraction of ASD cases, as well as other rare congenital disorders.

[0065] In summary, particularly for de novo mutation identification, it has been found to be advantageous to employ methods that: utilize test samples of at least four tissue cells, separate or disaggregate (discussed in more detail below) tissue sample cells into individual partitions, perform linear/quasilinear amplification on DNA of tissue sample cells, perform WES sequencing on amplified DNA, or analyze sequenced DNA specifically for CNVs. Any one of these methods, as provided in various embodiments herein, can result in more accurate, less costly, less complex, and quicker execution of de novo mutation detection. Furthermore, it has been found to be advantageous to employ, as provided in various embodiments herein, at least two of these methods together to achieve de novo mutation identification methods superior in accuracy, cost, timing and simplicity versus those methods known in the art.

[0066] As discussed briefly above, at step 220, the plurality of cells can be enzymatically disaggregated (referred to also as, for example, dissected or dissociated) into individual cells prior to separating the cells into a plurality of partitions, wherein each partition includes a single cell. Methods for enzymatically disaggregating cells can include adding one or more enzymes in sufficient amount to immerse plurality of cells. The immersion would promote enzymatic reaction with the cells to disaggregate the cells into individual cells. Those individual cells could then be dispersed and selected for transporting to a respective one of the plurality of partitions. The method can further include inhibiting the one or more enzymes prior to transporting the individual cells to a respective one of the plurality of partitions.

[0067] FIG. 3 is a flow chart illustrating a method 300 for enzymatically disaggregating a plurality of cells obtained from a tissue sample, in accordance with various embodiments. As stated above, the cells can be obtained via known biopsy methods such as, for example, excisional, incisional/core, needle aspiration, liquid, and various embryo biopsies. The tissue sample can be from, for example, a human embryo.

[0068] With in vitro fertilization (IVF), for example, after successful fertilization of an egg (e.g., human egg) with sperm during the co-incubation period, resulting embryos are generally allowed to grow for a period in a culture medium until embryo transfer to the uterus. A culture oil can also be included to in order to maintain stable temperature, osmolality and pH.

[0069] The culture medium used can be an artificial medium with the culture generally taking place on a culture plate. With artificial culture medium, different methodologies are possible. For example, the same culture medium can be used throughout the co-incubation period. In another example, a sequential system can be used, where the embryo is sequentially placed in different media. For example, when culturing to the blastocyst stage, one medium may be used for culture to day three (cleavage stage), and a second medium is used for culture thereafter. Artificial embryo culture media can include, for example, glucose, pyruvate, and energy-providing components. Artificial culture media can additionally include amino acids, nucleotides, vitamins, and cholesterol to improve the performance of embryonic growth and development.

[0070] Embryos that at least reach the day three cell stage (cleavage stage), or alternatively those that reach blastocyst stage, can be tested for chromosomal or specific genetic defects by PGD. A biopsy can then be taken from the embryo via, for example, aspiration by micropipette to remove the plurality of cells. Prior to the subsequent cell disaggregation, culture oil can be removed from the culture medium, as the culture oil can affect disaggregation. Also, the medium can be removed from the plate, as medium containing serum can inactivate (or inhibit) enzyme used for subsequent disaggregation. The removed plurality of cells can then, at step 310 of FIG. 3, be provided in a vessel for the disaggregation process. The remaining embryo can then placed in fresh media to maintain embryo viability.

[0071] In step 320, the cells are immersed in one or more enzymes. In step 330, the immersed plurality of cells are incubated for a time period necessary for the cells to disaggregate. Over the time period, the added one or more enzymes break down the cell adhesion structure on the outside of the cells that attaches them to the vessel, such as the bottom of a flask. For certain enzymes, the enzyme (or enzyme solution) should be defrosted prior to adding to the cells. For certain enzymes, to maximize effectiveness of the enzyme, defrosting should not occur via heating, nor should the defrosted enzyme be preheated prior to addition to the cells. In certain enzyme systems, treatment of cells can begin as early as 4 degrees Celsius. Regardless, since many enzymes perform most optimally at ambient room temperature, these types of enzymes should be allowed to come to ambient room temperature without heat treatment. Furthermore, to maintain a controlled temperature during the enzymatic disaggregation procedure, the procedure should be conducted on, for example, a heated stage or air incubator. The temperature can be maintained, for example, at approximately 37°C.

[0072] The break down of cell adhesion structure by the enzyme occurs over a period of time. That time period can be, for example, at least 5 minutes, at least 10 minutes, at least 50 minutes, or at least 100 minutes. The time period can be, for example, 100 to 120 minutes. The time period can be, for example, up to 150 minutes. The time period can be, for example, 5 to 10 minutes. The time period can be, for example, 30 to 45 minutes. The disaggregation time period can take place at room temperature. As cell adhesion structure breaks down, the structural appearance of the cells will change. Initially, the cells may appear spidery or shrunken while remaining attached to the bottom of the vessel (e.g., flask). Over time, the enzymatic reaction will cause the cells to have a more rounded, spherical appearance.

[0073] Once observed as sufficiently disaggregated, or after incubation is complete, the enzyme can be inhibited. Inhibition can be accomplished by removing the enzyme to lessen further unnecessary enzymatic activity. However, residual enzyme may still be present one the cells and can cause residual enzymatic activity with the disaggregated cells. This can be dangerous to the health of cells if left unchecked.

[0074] Alternatively, the cells can be mechanically disaggregated rather than, or in addition to, enzymatic disaggregation. There are many techniques for mechanical disaggregation cells. For example, cells can be disaggregated by scraping (or spillage), where a cutting action, or abrasion of a cut surface, releases cells. Cells can also be disaggregated by sieving, where tissue is forced though a sieve using, for example, a syringe piston. Another example of a technique is syringing, which involves drawing tissue into a syringe through a wide bore needle or cannula, and expressing said contents. Yet another technique is repeated pipetting, where tissue fragments are repeatedly pipetted up and down through a wide bore pipette. A pipette such as, for example, a very fine borosilicate pipette, could be used, where the pipette has a diameter of, for example, approximately 50μπι. In some instances, mechanical disaggregation can be used in addition to enzymatic disaggregation to aid in separating cells into separate entities because the enzyme, while destroying the bridges between cells, may not succeed in actually separating the cells sufficiently into separate entities for subsequent dispersing, as will be discussed below.

[0075] In step 340, disaggregated individual cells are dispersed in preparation for selection and transfer to individual partitions. Dispersing can be performed in various ways. For example, the cells can be re-suspended in fresh medium. The re-suspended cells can then be further diluted into fresh vessels for transfer. If residual enzyme remains with the cells, this residual activity can be neutralized, for example, by re-suspending the cells in fresh medium that contains serum. The serum in the fresh medium neutralizes remaining enzyme. If, on the other hand, a serum-free fresh medium is used at dispersing step 340, residual enzyme may still be present after cells are dispersed, thereby requiring a separate inhibition step. In this case, neutralization can occur by adding, for example, serum-containing medium or an inhibitor specific to the enzyme used.

[0076] It should be noted that the need for an inhibition (or inactivation or neutralization) step via an enzyme inhibitor or separate serum-containing medium, depends on the one or more enzymes used. Some enzymes, such as Accutase®, are gentle on cells and auto-inhibit at a certain temperature, thereby eliminating the need for an inhibition step. With such enzymes, residual enzyme is less of an issue and disaggregated cells can simply be dispersed appropriately, often in a vessel with fresh media. On the other hand, other enzymes, such as trypsin, usually require an accompanying trypsin neutralizing solution to inhibit residual enzyme present after disaggregation.

[0077] Alternatively, the cells can be washed before and/or after disaggregation. Regardless of enzyme, washing cells before disaggregation with a solution such as, for example, the buffer solution phosphate-buffered saline (PBS), can assist with removing serum to ensure proper enzymatic activity. Washing cells after disaggregation can assist with removing remaining enzyme so as not to interfere with downstream applications. Moreover, an additive such as, for example, acetylated albumin can be included in the washing solution to assist in preventing cells from sticking against surfaces of a plate or pipette.

[0078] Regarding use of enzymes herein, many different enzymes, or enzyme mixes, can promote cell disaggregation. Enzyme examples include, but are not limited to, proteolytic and collagenolytic enzymes. The one or more enzymes can comprise a proteolytic enzyme. The one or more enzymes can comprise a collagenolytic enzyme. The one or more enzymes can comprise a proteolytic enzyme and a collagenolytic enzyme. Proteolytic enzymes (or proteases) refer to the various enzymes that function to digest protein. These enzymes can include, for example, the pancreatic proteases (such as, for example, chymotrypsin and trypsin), bromelain (stem and fruit bromelain), elastase, hyaluronidase, dispase, papain (papaya proteinase), fungal proteases, and serratia peptidase. Collagenolytic enzymes (or collagenases) refer to enzymes that function to break the peptide bonds in collagen. These enzyme systems assist in destroying extracellular structures in the pathogenesis of bacteria such as Clostridium. They normally target the connective tissue in muscle cells and other body organs.

[0079] Note that media remaining with cells prior to disaggregation can have a negative effect on enzyme performance (see above at step 310). However, fresh media, particularly serum-containing media, becomes advantageous for inactivating residual enzyme after disaggregation is complete.

[0080] In step 350, individual cells are selected for transporting to a respective one of a plurality of partitions in preparation for amplification and subsequent analysis for the presence of de novo mutations.

[0081] FIG. 4 is a flow chart illustrating a method 400 for identifying de novo mutations in human embryos indicative of an autism spectrum disorder, in accordance with various embodiments. Method 400 is illustrative only and embodiments can use variations of method 400.

[0082] Method 400 includes obtaining a plurality of cells (e.g., at least four cells) from a human embryo at step 410; separating the plurality of cells into a plurality of partitions at step 420, wherein each partition includes a single cell; amplifying the DNA in the partitions at step 430, sequencing the DNA in the partitions at step 440, identifying genomic variants in the partitions at step 450, and labeling a genomic variant as a de novo mutation when it is present in a threshold percentage of partitions at step 460. Specific details related to these steps were discussed previously.

[0083] Alternatively, at step 420, the cells can be enzymatically disaggregated (referred to also as, for example, dissected or dissociated) into individual cells before separating the cells into a plurality of partitions, wherein each partition includes a single cell. As discussed previously, enzymatic disaggregation can include adding one or more enzymes in sufficient amount to immerse the plurality of cells, dispersing individual cells disaggregated by the enzyme, and selecting individual cells for transporting to a respective one of the plurality of partitions. Enzymatic disaggregation can further include inhibiting the one or more enzymes prior to transporting the individual cells. Discussion related to the types of one or more enzymes was provided previously. [0084] Alternatively, at step 450, sequenced DNA can be analyzed to identify copy number variations, and labeling a specific copy number variation as a de novo copy number variation at step 460 when the specific copy number variation is present in at least a threshold percentage of partitions. The threshold percentage can be, for example, 50%. As discussed above, when sequenced DNA is analyzed to specifically identify CNVs (as opposed to, for example, SNVs or InDels), CNVs are less likely to be mistaken for amplification artifacts, and therefore will not require as much filtering, or at least require a simpler filter protocol. The associated reduced complexity, cost and time for analysis makes targeting CNVs advantageous, assuming sufficient data for diagnosis can be gleaned directly from CNV detection and analysis. Targeted CNV analysis is particularly advantageous for PGD in general and, in particular, autism spectrum disorders (ASDs). Many recent large scale sequencing studies have found that de novo CNV mutations are likely to be the cause of a large fraction of ASD cases, as well as other rare congenital disorders.

[0085] At step 470, labeled de novo mutations (or de novo copy number variations) are analyzed to determine that the human embryo is at high risk for developing an autism spectrum disorder based on a threshold number of genes having de novo mutations occurring at specific chromosomal locations. For example, copy number variations that have a threshold number of genes greater than one may be analyzed. Alternatively, copy number variations that have a threshold number of genes greater than three may be analyzed. Alternatively, copy number variations that have a threshold number of genes greater than ten may be analyzed. Alternatively, copy number variations that have a threshold number of genes greater than twenty may be analyzed. Research suggests a positively correlation between the number of genes having de novo mutations and the number of ASD risk genes contained therein. As such, raising the threshold gene number requirements for CNV during detection can limit the number of CNVs subsequently analyzed while likely still accurately embryos having high risk for developing an autism spectrum disorder. This threshold, combined with the advantages already identified for the embodiments herein, may provide even further cost, time, accuracy, and simplicity advantages to what presently exists.

[0086] As discussed above, particularly for de novo mutation identification, it has been found to be advantageous to employ methods that: utilize test samples of at least four embryonic cells, separate or disaggregate or enzymatically disaggregate embryonic cells into individual partitions, perform linear/quasilinear amplification on DNA of embryonic cells, perform WES sequencing on amplified DNA, or analyze sequenced DNA specifically for CNVs. Any one of these methods, as provided in various embodiments herein, can result in more accurate, less costly, less complex, and quicker execution of de novo mutation detection. Furthermore, it has been found to be advantageous to employ, as provided in various embodiments herein, at least two of these methods together to achieve de novo mutation identification methods superior in accuracy, cost, timing and simplicity versus those methods known in the art. In addition, when specifically detecting de novo CNV mutations, particularly for identification of embryos having high risk of an ASD, targeting CNVs of a specific threshold gene number can assist in providing the same advantages discussed above with minimal negative impact to the accuracy of results.

[0087] While discussed in the context of PGD, the various embodiments discussed herein could be applied for testing many different types of tissue where sufficient tissue cells are available for biopsy. For example, the embodiments described herein can be applicable in non-invasive prenatal testing (NIPT). In NIPT, 1-5% of DNA of the fetus is provided in maternal blood. Moreover, there are single fetal cells in circulation, and a variety of methods to capture them, which can be utilized for testing. Because of this availability of fetal DNA, the embodiments described herein could still be applicable. Further, the embodiments described herein can be applicable in cancer screening using circulating tumor cells or cells from micro biopsies, again, capturing these cells through different existing methods and applying the methods described herein to differentiate artifact results from true de novo mutations.

[0088] It should be understood that the preceding embodiments can be provided, whole or in part, as a system of components integrated to perform the methods described. For example, the workflow of FIG. 1 can be provided as a system of components or stations for identifying de novo mutations in a tissue sample. For example, the fertilization, embryo incubation and embryo biopsy steps (1 10- 130) can be provided in a cell capture station. Cell separation step 150 can be provided, for example, as part of the cell capture station or as part of a separate cell separation station.

[0089] Finally, the DNA amplification, DNA sequencing and de novo mutation detection and labeling steps (160- 180) can be provided, for example, in a DNA analysis station, with the de novo mutation detection and labeling step 180 being performed on an engine, module, or component that can be implemented as computer hardware, firmware, software, or any combination thereof. Likewise, the identifying step 250 and labeling step 260 of method 200 of FIG. 2 can be performed on an engine, module, or component that can be implemented as computer hardware, firmware, software, or any combination thereof. Moreover, the identifying step 450, labeling step 460, and determining step 470 of method 400 of FIG. 4 can be performed on an engine, module, or component that can be implemented as computer hardware, firmware, software, or any combination thereof. FIG. 5 below describes the computer system in more detail.

[0090] FIG. 5 is a block diagram that illustrates a computer system 500, upon which embodiments, or portions of the embodiments, of the present teachings may be implemented. In various embodiments of the present teachings, computer system 500 can include a bus 502 or other communication mechanism for communicating information, and a processor 504 coupled with bus 502 for processing information. In various embodiments, computer system 500 can also include a memory 506, which can be a random access memory (RAM) or other dynamic storage device, coupled to bus 502 for determining instructions to be executed by processor 504. Memory 506 also can be used for storing temporary variables or other intermediate information during execution of instructions to be executed by processor 504. In various embodiments, computer system 500 can further include a read only memory (ROM) 508 or other static storage device coupled to bus 502 for storing static information and instructions for processor 504. A storage device 510, such as a magnetic disk or optical disk, can be provided and coupled to bus 502 for storing information and instructions.

[0091] In various embodiments, computer system 500 can be coupled via bus 502 to a display 512, such as a cathode ray tube (CRT) or liquid crystal display (LCD), for displaying information to a computer user. An input device 514, including alphanumeric and other keys, can be coupled to bus 502 for communicating information and command selections to processor 504. Another type of user input device is a cursor control 516, such as a mouse, a trackball or cursor direction keys for communicating direction information and command selections to processor 504 and for controlling cursor movement on display 512. This input device 514 typically has two degrees of freedom in two axes, a first axis (i.e., x) and a second axis (i.e., y), that allows the device to specify positions in a plane. However, it should be understood that input devices 514 allowing for 3 -dimensional (x, y and z) cursor movement are also contemplated herein.

[0092] Consistent with certain implementations of the present teachings, results can be provided by computer system 500 in response to processor 504 executing one or more sequences of one or more instructions contained in memory 506. Such instructions can be read into memory 506 from another computer-readable medium or computer-readable storage medium, such as storage device 510. Execution of the sequences of instructions contained in memory 506 can cause processor 504 to perform the processes described herein. Alternatively hard-wired circuitry can be used in place of or in combination with software instructions to implement the present teachings. Thus implementations of the present teachings are not limited to any specific combination of hardware circuitry and software.

[0093] The term "computer-readable medium" (e.g., data store, data storage, etc.) or "computer- readable storage medium" as used herein refers to any media that participates in providing instructions to processor 504 for execution. Such a medium can take many forms, including but not limited to, non-volatile media, volatile media, and transmission media. Examples of non-volatile media can include, but are not limited to, optical, solid state, magnetic disks, such as storage device 510. Examples of volatile media can include, but are not limited to, dynamic memory, such as memory 506. Examples of transmission media can include, but are not limited to, coaxial cables, copper wire, and fiber optics, including the wires that comprise bus 502.

[0094] Common forms of computer-readable media include, for example, a floppy disk, a flexible disk, hard disk, magnetic tape, or any other magnetic medium, a CD-ROM, any other optical medium, punch cards, paper tape, any other physical medium with patterns of holes, a RAM, PROM, and EPROM, a FLASH-EPROM, any other memory chip or cartridge, or any other tangible medium from which a computer can read.

[0095] In addition to computer readable medium, instructions or data can be provided as signals on transmission media included in a communications apparatus or system to provide sequences of one or more instructions to processor 504 of computer system 500 for execution. For example, a communication apparatus may include a transceiver having signals indicative of instructions and data. The instructions and data are configured to cause one or more processors to implement the functions outlined in the disclosure herein. Representative examples of data communications transmission connections can include, but are not limited to, telephone modem connections, wide area networks (WAN), local area networks (LAN), infrared data connections, NFC connections, etc.

[0096] It should be appreciated that the methodologies described herein flow charts, diagrams and accompanying disclosure can be implemented using computer system 500 as a standalone device or on a distributed network of shared computer processing resources such as a cloud computing network.

[0097] While the present teachings are described in conjunction with various embodiments, it is not intended that the present teachings be limited to such embodiments. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art. [0098] Further, in describing various embodiments, the specification may have presented a method and/or process as a particular sequence of steps. However, to the extent that the method or process does not rely on the particular order of steps set forth herein, the method or process should not be limited to the particular sequence of steps described. As one of ordinary skill in the art would appreciate, other sequences of steps may be possible. Therefore, the particular order of the steps set forth in the specification should not be construed as limitations on the claims. In addition, the claims directed to the method and/or process should not be limited to the performance of their steps in the order written, and one skilled in the art can readily appreciate that the sequences may be varied and still remain within the spirit and scope of the various embodiments.

[0099] Embodiment 1. A method for identifying de novo mutations in a tissue sample, comprising: obtaining a plurality of cells from a tissue sample, separating the cells into a plurality of partitions, wherein each partition includes a single cell, amplifying the DNA in the partitions, sequencing the DNA in the partitions, identifying genomic variants in the partitions, and labeling a genomic variant as a de novo mutation when it is present in at least a threshold percentage of partitions.

[00100] Embodiment 2. The method of Embodiment 1, further comprising: enzymatically disaggregating the plurality of cells into individual cells prior to separating the cells.

[00101] Embodiment 3. The method of Embodiment 2, further comprising: adding one or more enzymes in sufficient amount to immerse the plurality of cells, dispersing individual cells disaggregated by the one or more enzymes, and selecting individual cells for transporting to a respective one of the plurality of partitions.

[00102] Embodiment 4. The method of Embodiment 3, further comprising: inhibiting the one or more enzymes prior to transporting the individual cells.

[00103] Embodiment 5. The method of Embodiments 3 or 4, wherein the one or more enzymes comprises a proteolytic enzyme.

[00104] Embodiment 6. The method of any one of Embodiments 3 to 5, wherein the one or more enzymes comprises a collagenolytic enzyme. [00105] Embodiment 7. The method of Embodiments 3 or 4, wherein the one or more enzymes comprises a proteolytic enzyme and a collagenolytic enzyme.

[00106] Embodiment 8. The method of any one of Embodiments 1 to 7, further comprising: comparing the sequenced DNA to parental DNA to remove inherited genomic variants from being labeled as de novo mutations.

[00107] Embodiment 9. The method of any one of Embodiments 1 to 8, wherein the genomic variants are copy number variants.

[00108] Embodiment 10. The method of any one of Embodiments 1 to 9, wherein the threshold percentage of partitions is 50%.

[00109] Embodiment 11. The method of any one of Embodiments 1 to 10, wherein at least four cells are obtained from the tissue sample.

[00110] Embodiment 12. The method of any one of Embodiments 1 to 11, wherein the sequencing is whole exome sequencing.

[00111] Embodiment 13. The method of any one of Embodiments 1 to 11, wherein the sequencing is whole genome sequencing.

[00112] Embodiment 14. The method of any one of Embodiments 1 to 13, further comprising: applying a filter to the identified genomic variants to remove genomic variants selected from the group consisting of: amplification artifacts, inherited variants, sequencing error variants, mapping error variants, polymerase error variants, and combinations thereof.

[00113] Embodiment 15. A method for disaggregating a plurality of cells obtained from a tissue sample, comprising: providing a plurality of cells in a vessel, immersing the plurality of cells in one or more enzymes, incubating the immersed plurality of cells for a time period necessary for cells to disaggregate, dispersing individual cells disaggregated by the one or more enzymes, and selecting individual cells for transporting to a respective one of a plurality of partitions for analysis. [00114] Embodiment 16. The method of Embodiment 15, further comprising: inhibiting the one or more enzymes after incubating the immersed plurality of cells.

[00115] Embodiment 17. The method of Embodiments 15 or 16, wherein the time period is at least 5 minutes.

[00116] Embodiment 18. The method of Embodiments 15 or 16, wherein the time period is at least 10 minutes.

[00117] Embodiment 19. The method of Embodiments 15 or 16, wherein the time period is at least 50 minutes.

[00118] Embodiment 20. The method of Embodiments 15 or 16, wherein the time period is at least 100 minutes.

[00119] Embodiment 21. The method of Embodiments 15 or 16, wherein the time period is between 100 and 120 minutes.

[00120] Embodiment 22. The method of Embodiments 15 or 16, wherein the time period is up to 150 minutes.

[00121] Embodiment 23. The method of any one of Embodiments 15 to 22, wherein the one or more enzymes comprises a proteolytic enzyme.

[00122] Embodiment 24. The method of any one of Embodiments 15 to 23, wherein the one or more enzymes comprises a collagenolytic enzyme.

[00123] Embodiment 25. The method of any one of Embodiments 15 to 22, wherein the one or more enzymes comprises a proteolytic enzyme and a collagenolytic enzyme.

[00124] Embodiment 26. A method for identifying de novo mutations in human embryos indicative of an autism spectrum disorder, comprising: obtaining a plurality of cells from a human embryo, separating the plurality of cells into a plurality of partitions, wherein each partition includes a single cell, amplifying the DNA in the partitions, sequencing the DNA in the partitions, identifying genomic variants in the partitions, labeling a genomic variant as a de novo mutation when it is present in at least a threshold percentage of partitions, and determining that the human embryo is at high risk for developing an autism spectrum disorder based on a threshold number of genes having de novo mutations occurring at specific chromosomal locations.

[00125] Embodiment 27. The method of Embodiment 26, further comprising: enzymatically disaggregating the plurality of cells into individual cells before separating the cells into the plurality of partitions.

[00126] Embodiment 28. The method of Embodiment 27, further comprising:

adding one or more enzymes in sufficient amount to immerse the plurality of cells;

dispersing the individual cells disaggregated by the one or more enzymes; and

selecting individual cells for transporting to a respective one of the plurality of partitions.

[00127] Embodiment 29. The method of Embodiment 28, further comprising: inhibiting the one or more enzymes prior to transporting the individual cells.

[00128] Embodiment 30. The method of Embodiments 28 or 29, wherein the one or more enzymes comprises a proteolytic enzyme.

[00129] Embodiment 32. The method of any one of Embodiments 28 to 30, wherein the one or more enzymes comprises a collagenolytic enzyme.

[00130] Embodiment 32. The method of Embodiments 28 or 29, wherein the one or more enzymes comprises a proteolytic enzyme and a collagenolytic enzyme.

[00131] Embodiment 33. The method of any one of Embodiments 26 to 32, further comprising: comparing the sequenced DNA to parental DNA to remove inherited genomic variants from being labeled as de novo mutations.

[00132] Embodiment 34. The method of any one of Embodiments 26 to 33, wherein the genomic variants are copy number variants. [00133] Embodiment 35. The method of any one of Embodiments 26 to 34, wherein the threshold percentage of partitions is 50%.

[00134] Embodiment 36. The method of any one of Embodiments 26 to 35, wherein at least four cells are obtained from the embryo.

[00135] Embodiment 37. The method of any one of Embodiments 26 to 36, wherein the sequencing is whole exome sequencing.

[00136] Embodiment 38. The method of any one of Embodiments 26 to 36, wherein the sequencing is whole genome sequencing.

[00137] Embodiment 39. The method of any one of Embodiments 26 to 38, further comprising: applying a filter to the identified genomic variants to remove genomic variants selected from the group consisting of: amplification artifacts, inherited variants, sequencing error variants, mapping error variants, polymerase error variants, and combinations thereof.

[00138] Embodiment 40. The method of any one of Embodiments 26 to 39, wherein the threshold number of genes is greater than one.

[00139] Embodiment 41. The method of any one of Embodiments 26 to 39, wherein the threshold number of genes is greater than three.

[00140] Embodiment 42. The method of any one of Embodiments 26 to 39, wherein the threshold number of genes is greater than ten.

[00141] Embodiment 43. The method of any one of Embodiments 26 to 39, wherein the threshold number of genes is greater than twenty.