Simple methods for analyzing DNA and cell-free DNA and their uses

Abstract

Non-amplified methods for preimplantation genetic testing (PGT) of in vitro fertilization (IVF) embryos, non-amplified methods for creating libraries for PGT, and methods for identifying the genetic background of IVF embryos are provided. Also provided are methods for determining the degree of non-embryonic DNA contamination and non-amplified methods for creating cell-free DNA (cfDNA) libraries from biological materials. Similarly, methods for enriching methylated fragments from non-amplified libraries and methods for identifying methylation profiles and / or changes therein in samples from various sources are provided.

Description

[Technical Field]

[0001] The present invention relates to technologies for DNA genetic testing. In particular, the present invention relates to a non-amplified method for conveniently analyzing small amounts of DNA and cell-free DNA, such as those found in preimplantation genetic testing (PGT) of in vitro fertilized (IVF) embryos, microbiopsies, and human body fluids. [Background technology]

[0002] background During the natural proliferation of cells, small amounts of DNA can be released into the cellular environment. This DNA is usually in a degraded form of tens to thousands of base pairs. All cells have a natural lifespan, at the end of which a cellular degradative process fragments chromosomes into fragments, usually hundreds of base pairs, and multimers representative of nucleosome structures—often called apoptosis. When cellular tissue is damaged, internal processes trigger the chromosomal degradation process, again breaking down DNA into smaller, less well-defined fragments. Any or all of these processes can occur in cells in culture, such as tissue or embryo culture, or in complex biological systems such as the human body.

[0003] Culture systems are closed environments, typically in culture dishes, where cells reside in a nutrient-rich medium. Within this medium, nucleic acids released from the cell line accumulate and / or slowly degrade. Even after release from the original culture, the DNA likely represents the genetic makeup of the remaining living cells. The total amount of degraded DNA is generally very small—too small to measure directly. Because harvesting large numbers of cultured cells for genetic analysis is not always possible or feasible, some form of amplification process is usually considered. Most current common amplification processes (often called whole genome amplification) can actually fail to amplify some types of DNA present and over-amplify other DNA, potentially introducing imbalances into the subsequent chromosomal profile. While advanced bioinformatics techniques can be used to correct some of these imbalances, this is not always successful and can lead to erroneous interpretations of the cytogenetics. Furthermore, some important secondary information, such as fragment length and DNA modifications present within the fragments, which may be important for interpretation, is lost during the amplification process. A method or approach that allows for the analysis of small amounts of DNA without altering the chromosomal profile or losing secondary information could be advantageous for researchers wanting detailed information about cultured cell lines.

[0004] Complex natural cellular systems, such as those in humans, contain many different cell types, each with a common chromosomal profile but each with its own unique lifespan and DNA modification profile. They are part of a multicellular system interconnected by the circulatory system, which provides nutrients to these cells and removes waste products, including extracellular DNA (cfDNA), associated with natural growth and death processes. Circulating blood therefore carries many distinct DNA signatures from each of the various interconnected cell types. Similarly, excretory systems, such as urine, may also carry some of these degraded DNA fragments with associated signatures. At any given time, the body contains many cells undergoing the death process and shedding degraded DNA. However, this DNA is continually cleared by the liver and excreted via the bladder and urinary system, resulting in a relatively small amount of DNA. While the chromosomal and genetic profile of this DNA is generally consistent throughout the body, each cell type and each organ may possess unique patterns of secondary DNA modifications, which can be used, for example, to identify its contribution to the overall cell-free DNA profile in the plasma component of blood or in urine. Changes in the relative amount of cell-free DNA or individual organ contributions can be used to indicate disease states or cellular damage caused by injury or infection. Typically, large amounts of plasma are used as a source of cell-free DNA, which can then be purified and quantified. A method that can easily quantify the relative amount of cfDNA in cell-free plasma could help identify underlying tissue damage or infection that may be present. Identifying which organs are shedding this DNA could aid in diagnosing potential sites of injury. Such injury may be the result of physical or chemical processes or may be related to underlying disease processes, such as cancer or metabolic imbalances such as diabetes, other chronic disease states, or simply the aging process.Currently, the most common approach to identifying which organs or tissues are the source of cfDNA involves purifying relatively large amounts of DNA and then chemically modifying it so that the underlying DNA signature can be analyzed by some method, such as DNA sequencing. This process requires large amounts of DNA starting material, much of which is destroyed during DNA processing. Furthermore, subsequent analysis of this modified / processed DNA is complex and requires specialized interpretive bioinformatics. The potential for simpler methods that require less starting DNA, fewer processing steps, less intervention, and reduced need for complex and specialized bioinformatics could be beneficial for routine use in chronic disease identification and health management, cancer testing, noninvasive prenatal testing, organ-of-origin research, and many other tests.

[0005] Genomes are complex mixtures of various DNA fragments, many of which may not be of direct interest for analytical purposes. This high complexity can mean inefficient use of resources such as DNA sequencing if only a limited target range is examined. Several methods are available to reduce this complexity and make subsequent analysis more efficient and cost-effective. These include directed amplification of specific target sites within the DNA mixture (e.g., PCR - polymerase chain reaction, LAMP - loop-mediated isothermal amplification, MLPA - multiplex ligation-dependent probe amplification, and bacterial and viral recombination and repair-based systems such as CRISPR). However, these methods require detailed knowledge of each target site, primer design and development, and typically require intact DNA targets for the regions of interest. Furthermore, some elements of the original target information (e.g., fragment start / end points as well as internal DNA base modifications) are lost during amplification approaches. Furthermore, accessing multiple targets in a single reaction can be limited by the amplification method used and may require extensive development work to ensure compatibility with each specific probe / target combination. While some DNA, such as chromosomal DNA from tissue and cell preparations, is ostensibly intact for most targets, other preparations, such as cfDNA, can be significantly damaged, preventing standard amplification methods from amplifying some or even all of the desired targets, especially when the starting amount of DNA is very low. A method that allows selective DNA enrichment from the starting source, is less sensitive to DNA condition in terms of DNA degradation profiles, and does not require extensive upfront work with multiple targets, would be useful for analyzing many different DNA sources for genetic profiling. If such a method also preserved original information, including original fragment features such as start and end points, or secondary aspects such as DNA base modifications, it could be advantageous in many different applications.Methods that can preserve the identity of the original target strand would be useful for eliminating target representation bias if subsequent analysis requires an amplification process. An alternative to directional amplification is the capture of desired regions of DNA with homologous probes that exploit specific DNA base-pairing interactions. Such systems are commercially available and can be liquid / solution-based (e.g., Agilent or IDT probes) or liquid / solid-based (e.g., arrays from Agilent or other suppliers). These systems typically require large amounts of DNA as input—a potential problem when only limited starting DNA is available. Methods that can reduce the complexity of DNA sources through enrichment while simultaneously preserving hidden but desired features of DNA could be useful in many fields, including cancer diagnosis, prenatal testing, and chronic disease assessment. Specific application areas include, but are not limited to, embryo evaluation to improve assisted reproductive technologies, cancer detection and treatment monitoring, chromosomal profiling, allelic imbalance determination, and cell-based DNA analysis.

[0006] The same problem exists in other application fields for analyzing cfDNA or very small amount of genomic DNA.Therefore, instead of current approach, the simple analysis method of the present disclosure can be similarly applicable and useful for analyzing the samples from many different biological sources. Summary of the Invention

[0007] The present disclosure provides an approach to prepare limited amounts of DNA for sequencing libraries suitable for next generation sequencing or for selective enrichment of desired fragments.

[0008] In a first aspect, the present disclosure provides a non-amplification method for analyzing a biological sample, the method comprising the steps of: i) providing a sample containing cell-free DNA (cfDNA); ii) repairing the cfDNA fraction to obtain single-base overhang fragments; iii) adding directional barcoded sequencing-compatible adapters to each end of the repaired cfDNA, thereby creating a barcoded library for sequencing; and iv) sequencing the barcoded library.

[0009] In a second aspect, the present disclosure provides a non-amplification method for generating a library for preimplantation genetic testing (PGT) of in vitro fertilized (IVF) embryos, the method comprising the steps of: i) providing a sample containing cell-free DNA (cfDNA) from spent medium in the culture of IVF embryos; ii) repairing the cfDNA to obtain single-base overhang fragments; and iii) adding directional barcoded adapters to each end of the repaired cfDNA, thereby creating a barcoded library of spent media cfDNA for sequencing.

[0010] In a third aspect, the present disclosure provides a method for identifying the genetic background of an in vitro fertilized (IVF) embryo, the method comprising the steps of: i) providing a sample containing cell-free DNA (cfDNA) from spent medium in the culture of IVF embryos; ii) repairing the cfDNA to obtain single-base overhang fragments; iii) adding directional barcoded adapters to each end of the repaired cfDNA, thereby creating a barcoded library; and iv) determining a 24-chromosome copy number profile, a genome-wide heterozygous SNP profile, or a mitochondrial sequence by sequencing the barcoded library.

[0011] In a fourth aspect, the present disclosure provides a non-amplified method for determining the extent of non-embryonic DNA contamination in spent medium in the culture of IVF embryos, the method comprising the steps of: i) providing a sample containing cell-free DNA (cfDNA) from spent medium in the culture of IVF embryos; ii) repairing the cfDNA to obtain single-base overhang fragments; iii) adding directional barcoded adapters to each end of the repaired cfDNA, thereby creating a barcoded library for sequencing; iv) sequencing the library; and v) calculating the mitochondrial DNA (mtDNA) to chromosome ratio, A higher mtDNA to chromosome ratio indicates a lower degree of non-embryonic DNA contamination, and a lower mtDNA to chromosome ratio indicates a higher degree of non-embryonic DNA contamination.

[0012] In a fifth aspect, the present disclosure provides a non-amplification method for generating a library of cell-free DNA (cfDNA) from a biological material, the method comprising the steps of: i) providing a sample containing cell-free DNA (cfDNA) from a biological material; ii) repairing the cfDNA to obtain single-base overhang fragments; and iii) adding directional barcoded adapters to each end of the repaired cfDNA, thereby creating a barcoded library for sequencing.

[0013] In a sixth aspect of the present invention, there is provided a non-amplification method for preparing a sample for methylation profile analysis, the method comprising the steps of: i) providing a sample containing cell-free DNA (cfDNA) from a biological material; ii) repairing the cfDNA to obtain single-base overhang fragments; and iii) Adding directional barcoded adapters to each end of the repaired cfDNA, thereby creating a barcoded library and avoiding PCR amplification, which destroys methylation patterns by converting methyl-C to C.

[0014] In a seventh aspect of the present disclosure, there is provided a non-amplification method for examining a selected genomic region on a chromosome associated with a gene, the method comprising the steps of: i) providing a sample containing cell-free DNA (cfDNA); ii) repairing the cfDNA fraction to obtain single-base overhang fragments; iii) adding directional barcoded sequencing-compatible adapters to each end of the repaired cfDNA, thereby creating a barcoded library; iv) enriching the library for selected genomic regions; and v) sequencing the barcoded library.

[0015] In an eighth aspect of the present disclosure, there is provided a non-amplification method for examining a selected genomic region on a chromosome associated with a polymorphic site, the method comprising the steps of: i) providing a sample containing cell-free DNA (cfDNA); ii) repairing the cfDNA fraction to obtain single-base overhang fragments; iii) adding directional barcoded sequencing-compatible adapters to each end of the repaired cfDNA, thereby creating a barcoded library; iv) enriching the library for selected genomic regions; and v) sequencing the barcoded library. [Brief explanation of the drawings]

[0016] [Figure 1]A schematic diagram of how the Simplified Analysis Method can be used for direct analysis or further processed (cf) to obtain more specific information about the DNA sample is shown. [Figure 2] 1 shows a comparison of the non-amplification method for generating libraries of the present disclosure with a WGA-based method for analyzing culture medium. [Figure 3] The size of native cfDNA in the spent medium of embryos is shown, with panels A, B, C, and D representing four separate samples. [Figure 4] Analysis of different DNA size fractions, <200 bp (A) and >200 bp (B), for reads corresponding to chromosomes 21, 22, and X are shown. [Figure 5] Determination of sex chromosome balance is shown, specifically the depth of reads mapped to autosomes and the X chromosome in a 46,XY sample (A) and a 46,XX sample (B), as well as mapping of Y chromosome-specific sequences in two 46,XY samples and two 46,XX samples (C). [Figure 6] The ploidy of the embryos is shown, including a 46,XY euploid embryo (A), a 46,XX euploid embryo (B), and an aneuploid embryo lacking one copy of chromosome 15 (C). [Figure 7] Possible sources of non-embryonic DNA contamination are indicated. [Figure 8] Analysis of mitochondrial DNA is shown. [Figure 9] Sequencing methylation plots from various cfDNA and tissue sources are shown, showing changes in methylation capture across genomic sites. [Figure 10] Gene probe capture plots for various (cf) DNA sources showing changes in meDNA capture at different genes, representing changes in the methylation status of the gene. [Figure 11] SNP probe proportions from model systems showing different minor allele abundances (4%–8%). DETAILED DESCRIPTION OF THE INVENTION

[0017] Detailed Description of the Invention General Techniques and Definitions Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., molecular genetics, bioinformatics, developmental biology, and IVF).

[0018] Unless otherwise indicated, the techniques utilized in this disclosure are standard procedures well known to those skilled in the art. Such techniques are described and explained in the literature, for example, in the following sources: J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984); J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989); T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991); D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996); and F.M. Ausubel et al., (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date); Ed Harlow and David Lane (editors), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988); and J.E. Coligan et al., (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates to date).

[0019] As used herein, the term "about" refers to ±10%, more preferably ±5% of the specified value, unless otherwise specified.

[0020] As used herein, the word "comprise" or variations thereof is understood to mean the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of other elements, integers or steps, or groups of elements, integers or steps.

[0021] The term "or" as used herein is intended to mean an inclusive "or" rather than an exclusive "or." That is, unless otherwise specified or clear from the context, "X uses A or B" is intended to mean any of the natural inclusive permutations. That is, if X uses A; if X uses B; or if X uses both A and B, each of the foregoing satisfies "X uses A or B." Furthermore, at least one of A and B and / or the like generally refers to either A or B, or both A and B. Additionally, the articles "a" and "an," as used in this application and the appended claims, can be construed generally to mean "one or more," unless otherwise specified or clear from the context to refer to the singular form.

[0022] The term "screening" as used herein refers to the process of evaluating embryos. Such a process can be used, for example, to select suitable embryos for implantation into a woman's uterus.Similarly, the term "screening" can also refer to the evaluation of DNA samples to determine whether the DNA samples are at risk of having aneuploid genomes, pathogenic genetic mutations, or polymorphisms of interest to researchers.

[0023] As used herein, the term "euploid" or "euploid" means a cell, embryo, or DNA sample that contains one or more complete genomes (two in the case of humans) without any redundant chromosomes, and the term "aneuploid" or "aneuploid" means that the cell, embryo, or DNA sample contains at least one incomplete genome or at least one redundant chromosome or portion thereof.

[0024] The term "pathogenic" is used herein to refer to a genetic variation known or predicted to be associated with disease. The association of one or more genetic variations with a disease may result in the disease or may represent a genetic predisposition or risk for developing the disease.

[0025] As used herein, the term "aligned", "alignment" or "align" refers to one or more sequences that are identified as matching known sequences from a reference genome in terms of the order of nucleic acid molecules. Such alignment can be performed manually or by computer algorithms, examples of which include the Efficient Local Alignment of Nucleotide Data (ELAND) computer program distributed as part of the Illumina Genomics Analysis pipeline. The alignment of sequence reads can be 100% sequence match or less than 100% (not perfect match).

[0026] The term "allele" as used herein refers to a sequence variant of a genetic sequence. For the purposes of this application, an allele may be located within a genetic sequence, but is not necessarily required to be. An allele may be identified with respect to one or more polymorphic positions, such as SNPs, while the remainder of the genetic sequence may remain unspecified. For example, an allele may be defined by the nucleotide present in a single SNP, or by the nucleotide present in multiple SNPs.

[0027] The term "sequencing" as used herein refers to the method of determining the nucleotide sequence of polynucleotides, such as genomic DNA.Preferably, sequencing methods include, but are not limited to, NGS (i.e., high-throughput sequencing), in which clonally amplified DNA templates or single DNA molecules are sequenced in a massively parallel manner (Volkerding et al., 2009; Metzker et al., 2010).

[0028] The term "sequencing read" or "read" refers to a DNA sequence of sufficient length (e.g., at least about 30 bp) that can be used to identify a larger sequence or region, e.g., that can be aligned and specifically assigned to a chromosome or genomic region or gene.

[0029] As used herein, the term "whole genome amplification" refers to the process of amplifying genomic DNA sequences present in a sample to provide multiple copies of the genome represented by the sequences.

[0030] The term "haplotype" refers to a DNA sequence containing one or more genetic variations of interest contained within a subregion of a single chromosome in an individual. The genetic variations in a haplotype may be of the same type, e.g., all SNPs, or a combination of two or more types of genetic variations, e.g., a combination of SNPs and STRs. A haplotype can also refer to a set of genetic variations in a single gene, an intergenic sequence, or a larger sequence containing both genes and intergenic sequences, e.g., a collection of genes or a collection of genes and intergenic sequences. For example, a haplotype can refer to a set of genetic variations at the complement activation regulatory (RCA) locus, which includes the gene sequences for complement factor H (CFH), FHR3, FHR1, FHR4, FHR2, FHR5, and F13B, as well as intergenic sequences (i.e., intergenic sequences, upstream sequences, and downstream sequences in linkage disequilibrium with the genetic variations within the gene region). A haplotype can be, for example, a set of maternally inherited alleles or a set of paternally inherited alleles at a given locus.

[0031] The term "haplotyping" as used herein refers to the process of determining one or more haplotypes in an individual, and includes the use of pedigrees, molecular techniques, and / or statistical inference. Preferably, haplotypes are determined by sequencing using next-generation sequencing technology.

[0032] The term "adapter" as used herein refers to a compatible nucleotide fragment that can be ligated or joined to a repaired DNA fragment. Such adapters can be double-stranded DNA or DNA analogs, or RNA or RNA analogs, and have a defined sequence that is compatible with subsequent processes such as DNA sequencing and DNA amplification. Y adapters have single-stranded and double-stranded regions and can be directionally added to the fragment of interest.

[0033] In vitro fertilization (IVF) The methods described herein can be used to screen IVF embryos to identify those at risk of having aneuploid genomes or pathogenic genetic mutations, thereby allowing the selection of embryos suitable for implantation. IVF is a fertilization process in which an egg and sperm are combined outside the body, i.e., in vitro. This process involves monitoring and, in some cases, stimulating the female's ovulation process, retrieving one or more ova (eggs) from the female's ovaries, and fertilizing them in an appropriate fluid in a laboratory setting. The fertilized eggs (zygotes) undergo embryo culture for approximately two to seven days and then are transferred into the uterus of the same or another female (e.g., a surrogate mother) with the intention of achieving a successful pregnancy. Parents typically undergo IVF when they are having difficulty conceiving naturally or when there is a risk of transmitting a genetic disease to the embryo.

[0034] The method described herein is suitable for the IVF embryo of any animal, provided that suitable reference genome is available, and thus sequencing data can be aligned to identify potential genetic variations.For example, embryo can be human or other non-human animal embryo.In some embodiments, embryo is human embryo.In other embodiments, embryo is cow, sheep, horse, pig, dog, cat or other non-human animal embryo.

[0035] As described herein, obtaining sequencing data for an "embryo" includes sequencing cfDNA from spent medium of an embryo, such as a blastocyst, that was fertilized at least about 40 hours prior to sampling (typically an embryo on day 4, 5, 6, or 7 after fertilization). Thus, in some embodiments, the embryo is a 2-day-old, 3-day-old, 4-day-old, 5-day-old, 6-day-old, or 7-day-old embryo. As used herein, the term "embryo" includes the plural form of the term, as it is intended that multiple embryos or blastocysts may be simultaneously screened or implanted according to the methods of the present disclosure.

[0036] "Implanting" an IVF embryo refers to the process of placing an IVF embryo into a female subject with the intent of the IVF embryo implanting in the uterus and resulting in a viable pregnancy. The female subject may be the embryo's female parent or another female suitable for embryo transfer, for example, in the case of a gestational surrogate.

[0037] As intended herein, the method of the present disclosure can be used to simultaneously screen one or more embryos, so that multiple IVF embryos that are deemed to be free from the risk of having an aneuploid genome or a pathogenic genetic mutation can be identified and implanted.The number of such embryos that may be suitable for implantation can be determined by those skilled in the art according to conventional methods.

[0038] The terms "genetic variation," "polymorphism," and "variant" are used interchangeably herein to refer to the presence of variation in the genetic sequence of an embryo's genome (or the genome of either parent) compared to a reference genome.

[0039] Similarly, any DNA samples can be screened simultaneously in parallel. The number of samples to be screened can be determined by one skilled in the art according to conventional methods.

[0040] The terms "genetic variation," "polymorphism," and "variant" are used interchangeably herein and refer to the presence of variation in the genetic sequence of a DNA sample compared to a reference genome.

[0041] Genetic variations include sequence differences such as single nucleotide polymorphisms (SNPs), tandem SNPs, small multi-base deletions or insertions called "indels" (also called deletion-insertion polymorphisms or DIPs), multinucleotide polymorphisms (MNPs), short sequence repeats (STRs), restriction fragment length polymorphisms (RFLPs), deletions such as microdeletions, insertions such as microinsertions, duplications, inversions, translocations, multiplications, complex multisite variants, copy number variations (CNVs), and other structural variations including other sequence changes in chromosomes.

[0042] The term "single nucleotide polymorphism (SNP)" refers to a variation of a single base (nucleotide) in a DNA sequence among individuals within a population.

[0043] An "indel" is an insertion or deletion of bases in an organism's genome, which is classified as a small genetic variation, typically ranging in length from one base pair to 10,000 base pairs.

[0044] As used herein, the term "structural variation" refers to any variation in the structure of an organism's chromosome. This includes many types of genomic variation, typically including microscopic and submicroscopic types, deletions, duplications, copy number variations, insertions, inversions, and translocations. Generally, structural variations affect a larger portion of the sequence than SNPs, but less than chromosomal abnormalities (although there is some overlap between these definitions).

[0045] As used herein, the term "copy number variation" refers to a type of structural variation, which is a variation in the copy number of a nucleic acid sequence, typically of about 1 kb or more, present in a test sample compared to the copy number of the nucleic acid sequence present in a qualified sample.

[0046] Examples of specific application fields of the present invention 1. Embryo evaluation to improve assisted reproductive technologies It has been observed that for any couple undergoing IVF, a significant proportion of embryos generated during the process do not have a balanced set of chromosomes. Implantation of such embryos usually results in a non-initiated pregnancy, or, in those that do, in a miscarriage, abnormal fetal development, or, in some cases, a live birth with a syndromic phenotype. The proportion of chromosomally unbalanced embryos generally increases with maternal age; however, even in younger women, some or all of their embryos may have an unbalanced set of chromosomes. Therefore, all women undergoing IVF can derive some benefit from the identification and selection of euploid embryos. Furthermore, patients with genetic predispositions can also benefit from the selection of euploid, disease-free embryos for implantation.

[0047] Currently, PGT cycles performed worldwide involve the collection of one or a few embryonic cells using invasive biopsy procedures to test for chromosomal and / or underlying genetic disorders. Invasive biopsy, followed by analysis using whole genome amplification (WGA) and array or next-generation sequencing, has become the standard for final embryonic diagnosis. While this technique is well-developed and widely applied in many PGT laboratories, concerns exist in some circles that this invasive approach may result in significant harm to the embryo. These secondary embryo manipulations appear to have varying outcomes depending on clinical expertise, and the harm sustained by the embryo may outweigh any benefits of PGT. Furthermore, many countries have laws prohibiting any manipulation of embryos, including biopsies, for religious and / or philosophical reasons.

[0048] Therefore, a non-invasive technique that allows testing of embryonic DNA without invasive steps could greatly benefit all women undergoing IVF in identifying euploid embryos for transfer. Such an approach would also allow other couples at genetic risk to have the option of selecting embryos in a non-invasive manner.

[0049] Recently, low-molecular-weight cell-free DNA (cfDNA), derived from both chromosomal and mitochondrial sources, has been reported to be present in small amounts in spent embryo culture medium and blastocyst cavity fluid. Using paired-end sequencing of cfDNA released from the blastocyst cavity fluid of expanded embryos, the predominant size of cfDNA was shown to be approximately 156 bp. Other analyses focusing on the source of cfDNA in spent embryo culture medium have found significant contamination in up to 60% of samples from different sources, including cumulus cells, first and second polar bodies, and sperm, as well as from human serum albumin (HSA), a major component of most embryo culture media. Additionally, the presence of cells or microcells that normally contain DNA may also contribute to this background DNA by releasing their DNA using sample processing steps involving heating or extraction processes.

[0050] All current non-invasive methods for testing this cfDNA in spent embryo media employ a PCR-based WGA step to amplify the cfDNA, followed by next-generation sequencing or array copy number analysis to interpret the embryo's chromosomal ploidy status. All current WGA approaches use heat as a primary denaturation step and a thermal cycling protocol for enzymatic amplification. This heating step not only destroys the target cfDNA molecules, but also destroys extraneous cells in the sample, such as cells associated with the embryo, embryo-associated structures, or operator-introduced cellular contamination. Such cell-based contamination can result in amplification of non-embryonic DNA in preference to the desired embryonic target DNA molecules, potentially confounding or even invalidating subsequent embryonic DNA-based genomic analysis.

[0051] One of the physical limitations of most WGA methods is the size restriction on the target DNA that can be adequately amplified. The lower size limit for amplified DNA fragments is typically greater than 200 bp. Therefore, WGA methods are inherently biased toward targeting and amplifying larger fragments present in spent culture medium, thereby introducing bias at the earliest stages of analysis and potentially resulting in amplified libraries that are not representative of the original cfDNA and, ultimately, the embryonic genome. A further limitation of WGA, based on the PCR amplification process, is the inherent amplification bias due to over- and under-representation of specific genomic sequences in the final amplified DNA preparation. This inherent bias in WGA toward larger targets can result in preferential amplification of large fragments of non-embryonic DNA contaminants present in or introduced into the spent culture medium, potentially creating DNA libraries not only from the original embryonic cfDNA but also from contaminating and introduced sources of both non-embryonic cfDNA and cell-based DNA.

[0052] WGA-based approaches are not only relatively costly but also time-consuming, requiring up to 3 hours to create a DNA library; many steps require operator intervention, followed by additional procedures to purify and quantify the amplified DNA and measure the quality of the library for sequencing. Furthermore, after WGA, it is not possible to estimate the original size of the cfDNA present in the culture medium or to determine how much was originally present in the spent medium sample. Therefore, it is not surprising that to date, many published non-invasive PGT-A results using various WGA assay methods have high amplification failure rates, are variable, often contain ploidy discrepancies compared to invasive trophectoderm results, and produce variable quality chromosomal profiles, often resulting in uninterpretable results. In many cases, WGA completely fails to create DNA libraries suitable for sequencing analysis.

[0053] As an alternative to current invasive and non-invasive approaches to PGT, there is a need for non-invasive techniques that are simple and rapid in sample preparation, reliable, and accurate.

[0054] Accordingly, the present disclosure overcomes or at least mitigates some of the problems of the prior art and improves PGT outcomes for patients using a non-invasive approach for embryo testing.

[0055] 2. Cancer detection and treatment monitoring Methylation is an important epigenetic DNA modification that regulates the fundamental cellular processes of transcription and gene expression. Studies have shown that alterations in methylation patterns due to hypomethylation or hypermethylation can lead to altered gene expression and the development and propagation of various human pathologies. Therefore, changes in DNA methylation patterns can be an indicator of underlying changes in organs and tissues and can be used as a test for early detection or evaluation of diseases.

[0056] Circulating cfDNA in the plasma fraction is fragmented DNA, with the majority of fragment sizes ranging from 166 to 176 base pairs. This cfDNA represents the genomic degradation products of tissue cells from many different parts of the body. Approximately 1–3% of chromosomal DNA is methylated, which translates to a similar proportion of cfDNA being similarly methylated. This methylated cfDNA found in plasma reflects the general methylation status of normal somatic cells from various organs connected through the circulatory system. Different organs each produce their own unique methylation DNA subsignatures, sharing many of the same methylation modifications with other cell lineages, but also display tissue-of-origin specific patterns in some chromosomal regions that are unique or specific to that cell lineage. Changes in methylation patterns reflect changes in tissue gene expression and, therefore, alterations in mRNA profiles. Therefore, potential alterations in normal or typical cellular function can be analyzed by changes in the normal methylation profile of that cell type. For example, in cancer, normal cellular functions are disrupted with uncontrolled cell proliferation and the acquisition of invasive capabilities, often associated with concomitant changes in specific DNA methylation sites. DNA methylation patterns in tumor tissue typically differ from those in the tissue of origin, at least in some chromosomal regions, and these altered methylation signatures can subsequently appear in cfDNA fractions from plasma, serum, or urine.

[0057] There are a wide variety of methods for detecting DNA methylation, including gene-specific assays that utilize methylation-specific modifications to effectively enable specific targeting by PCR, genome-wide assays utilizing chemical DNA modifications (bisulfite sequencing), and methylation-specific arrays. More recently, allele-specific DNA sequencing on third-generation nanopore-based DNA sequencing platforms has demonstrated the ability to detect and map methylation sites throughout the genome. However, genome-wide assays developed to date are generally cumbersome, laborious, time-consuming, and prone to various errors. As a result, current methods are technically complex and not cost-effective for routine use in analyzing methylated cfDNA.

[0058] There is a need for simple, rapid, reliable, and accurate alternatives to current methylation detection approaches. Therefore, a simple, noninvasive test targeting the methylated cfDNA fraction for early disease detection and treatment response monitoring would be a major advance in disease detection and monitoring.

[0059] Accordingly, one aspect of the present disclosure is to overcome or at least mitigate some of the problems of the prior art and to facilitate genome-wide methylation detection as a means of assessing an individual's general health and well-being with respect to early signs of cancer and other chronic diseases.

[0060] 3. Chromosomal Profile Many cancer cells harbor altered chromosomal regions, observed as the loss and / or gain of entire chromosomes or chromosomal regions. While these changes in chromosomal profiles were traditionally observed using classical cytogenetic karyotyping, in recent years, the more commonly used platforms for chromosomal profile assessment are next-generation sequencing (CNVseq) or array-based hybridization systems. These array systems can be simple sequence-tagged sites and / or single-nucleotide polymorphism probes. In the early stages of cancer growth, there tends to be an evolving and constantly changing set of chromosomal profiles between different subclones of tumors and their metastases. This is often reflected in a combination of apoptosis (cell death) and necrosis (necrosis) in the tumor mass—each of these death processes has its own unique fragment profile. Any changes in tissue growth dynamics can be reflected in changes in cfDNA—not only DNA quantity but also fragment and chromosomal profiles. Any method for easily visualizing these features could potentially aid in the underlying cancer detection and progression and facilitate treatment monitoring.

[0061] The placenta of pregnant women releases fragmented fetal cfDNA into the maternal circulation – the basis of current non-invasive prenatal screening (NIPS). While the majority of cfDNA is maternal, 4–20% is fetal – a relatively small amount of fetal DNA in a large maternal background, but sufficient to identify major chromosomal imbalances of fetal origin. Purifying cfDNA from approximately 1 milliliter of blood typically yields approximately 20 ng of cfDNA (this amount of DNA is equivalent to 3,000 diploid cells, or 10 10This purified cfDNA is then used to create libraries for array or next-generation sequencing (NGS)-based analysis. However, array analysis requires more DNA and may require an amplification step. NGS analysis requires less DNA but requires library preparation, which typically involves several intermediate PCR-type amplification processes. Bioinformatics is used to reduce the potential for bias in chromosomal profiles associated with amplification and multiplex analysis of the original library fragments. However, only approximately 3 × 10 fragments of the final library are used for final sequencing analysis. 6 ~8×10 6 Only a small portion contains only fragments of

[0062] Techniques that can reduce the number of intermediate steps and require less starting cfDNA can facilitate the manipulation and processing of NIPS samples. Similarly, avoiding the possibility of daughter strand analysis would simplify bioinformatics and simultaneously improve the efficiency of sequencing resources.

[0063] 4. Determination of Allelic Imbalance Similar to chromosomal profiles, changes in the allelic ratios of the SNPs present are indicative of altered chromosomes and / or chromosomal segments that are often associated with the development of cancer. Current approaches involving directional PCR amplification are not only limited in scope, but also unable to provide clarity for many of the SNP targets due to the degradation profiles of the individual SNP targets.

[0064] The placenta of a pregnant woman releases cfDNA representative of the fetus—this contains both maternal SNP contributions and a new set of paternal halves. When these paternal alleles differ from maternal alleles, minor alleles can be observed. Therefore, the ratio of major to minor alleles can be used to assess the fetal cfDNA fraction in a predominant maternal cfDNA background. Similar to chromosomal profiles, perturbations in the SNP ratio profile of fetal cfDNA across different chromosomes can be used as an indicator of unbalanced chromosomal ratios in the developing fetus.

[0065] Organ transplant patients receive tissue with different allele combinations across many regions of different chromosomes. Once the organ is accepted by the recipient's system, cells within the transplanted organ's tissue will begin the same life cycle of cell proliferation / death as the original organ. This cycle introduces unique SNPs present in the donor tissue into the recipient's cfDNA. Evaluating these SNPs as a ratio relative to recipient SNPs can be a useful indicator of whether the transplant is adapting to its new location or failing. An increase in the ratio after the initial settlement period may indicate organ rejection, while an early decrease in the ratio may indicate early graft failure.

[0066] A simple method that can help estimate these allele ratios from cfDNA samples and provide total relative cfDNA levels may be useful for monitoring cancer development or assessing treatment progress, assessing fetal genomic complement in pregnancy, and potentially for monitoring the success of tissue / organ transplants.

[0067] 5. Cell-based DNA analysis In many cases, the amount of sample available for genomic screening is more than sufficient to use state-of-the-art platforms. However, sometimes the amount of tissue available for DNA analysis is limited. These can be fine-needle biopsies from suspected tumors, slide-mounted tissue sections, embryonic biopsies, or small tissue biopsies. DNA can be extracted from these samples using conventional methods, but is then typically subjected to some kind of whole-genome amplification process—which, like cfDNA amplification, can result in the loss of some of the original information or the introduction of initial targeting bias. Alternative approaches to fragmenting DNA can be performed using any of the currently available methods, such as sonication or nuclease digestion. Whether it is amplified or native DNA, sequencing libraries can be prepared after fragmentation using the simplified analysis methods described herein. If secondary information elements are not required for analysis, sequencing libraries can be amplified and used for techniques requiring more DNA (e.g., arrays) or as source material for enrichment procedures (e.g., SNP or selected gene targets). While this amplification may introduce some bias into the final DNA profile, it is unlikely that this bias is due to a combination of early amplification target initiation and DNA sequence amplification variability. Methods that can identify the original fragments present at the start of library construction could be useful for analyzing the original DNA fragments present. Similarly, methods that can mitigate the variable imbalances introduced by current PCR-based whole genome amplification could potentially be useful in aiding the study of both simple and more complex cells and tissues.

[0068] Creating a Library The present disclosure provides a non-amplification method for generating a library of cell-free DNA (cfDNA) from biological material, the method comprising the steps of: i) providing a sample containing cell-free DNA (cfDNA) from a biological material; ii) repairing the cfDNA and preferably obtaining single-base overhang fragments; and iii) adding barcoded adapters to each end of the repaired cfDNA, thereby creating a barcoded library for sequencing.

[0069] Similarly, this method can be performed with DNA from a variety of cell samples, not just spent medium from the culture of IVF embryos. Accordingly, the present disclosure provides a non-amplification method for generating libraries for preimplantation genetic testing (PGT) of in vitro fertilization (IVF) embryos, the method comprising the steps of: i) providing a sample containing cell-free DNA (cfDNA) from spent medium in the culture of IVF embryos; ii) repairing the cfDNA and preferably obtaining single-base overhang fragments; and iii) adding directional barcoded adapters to each end of the repaired cfDNA, thereby creating a barcoded library for sequencing.

[0070] The present disclosure provides libraries produced by the methods of the invention.

[0071] Methods for obtaining DNA, such as cfDNA, from spent medium in biological samples, such as IVF embryo cultures, are known in the art.

[0072] Advantageously, the spent medium can be used directly without an isolation step. For example, embryos are grown to the blastocyst stage in a drop of medium to allow genetic analysis of individual embryos. In some embodiments, the sample in step i) is spent medium from the culture of IVF embryos.

[0073] cfDNA may contain 5'- and / or 3'-overhanging ends, or internal nicks. Thus, in some embodiments, repairing cfDNA comprises converting the 5'- and / or 3'-overhanging ends to blunt ends and / or repairing the internal nicks.

[0074] Usually, repairing step can be achieved by using methods or kits known in the art.The repaired fragment can be phosphorylated by enzymatic treatment, for example, using polynucleotide kinase.In some embodiments, a single deoxynucleotide, for example, deoxyadenosine (A), is added to the 3' end of the fragment by the activity of certain DNA polymerases, for example, Taq polymerase or Klenow exo minus polymerase.In some embodiments, this method further comprises adding 3'dA overhangs to each end of the repaired fragment.

[0075] The dA-tailed products correspond to the "T" overhangs present at the 3'-end of each double-stranded region of the adapter to which they will be ligated in a subsequent step. The dA-tailing prevents self-ligation of both blunt-ended polynucleotides, thereby biasing the formation of adapter-ligated sequences. The dA-tailed fragments are ligated to double-stranded adapter polynucleotide sequences. The same adapters may be used on both ends of the fragment, or two sets of adapters may be utilized. Ligation methods are known in the art and utilize a ligase enzyme, such as DNA ligase, to covalently link the adapters to the dA-tailed polynucleotides. The adapters may contain a 5'-phosphate moiety to facilitate ligation to the target 3'-OH. The dA-tailed fragments contain a 5'-phosphate moiety (either remnant from the shearing process or added using an enzymatic treatment step), are end-repaired, and may optionally be extended with one or more overhanging bases to provide a 3'-OH suitable for ligation.

[0076] In some embodiments of the methods of the present disclosure, the directional barcoded adapter is a Y adapter. In some embodiments, the Y adapter comprises a barcode in a double-stranded region. In some embodiments, the Y adapter comprises a barcode in a single-stranded region.

[0077] The products of the ligation reaction can be purified to remove unligated adapters and / or adapters that may have been ligated to each other. Purification of the ligation products can be performed by methods such as gel electrophoresis and solid-phase reversible immobilization (SPRI). Purification can also remove enzymes, buffers, salts, etc. to provide favorable reaction conditions for subsequent steps. In some embodiments, the methods of the present disclosure include a purification step.

[0078] Libraries with different barcodes can be combined and sequenced.Therefore, it is possible to prepare a combination of libraries.It should be noted that purification may result in the loss of cfDNA of interest, which is detrimental to subsequent steps.Because the DNA level in the combination is much higher than that of a single library, the loss of DNA during purification will not be a significant detriment.In some embodiments, this method comprises creating multiple barcoded libraries with different barcodes.In some embodiments, this method comprises combining multiple barcoded libraries prior to purification.

[0079] cfDNA may be bound to protein, and this protein will affect sequencing.In some embodiments, this method comprises removing the protein that is bound to cfDNA.In some embodiments, cfDNA is treated with protease and / or detergent to remove nucleosome or heterochromatin structure from cfDNA fragment.

[0080] As previously mentioned, cfDNA is obtained from spent medium from embryos fertilized at least about 40 hours prior to sampling, such as blastocysts (usually embryos on day 4, 5, 6, or 7 post-fertilization). In some embodiments, spent medium is collected from day 5-7 blastocyst cultures.

[0081] In some embodiments, sample comprises the cfDNA extracted from cell or tissue.In some embodiments, cfDNA is derived from the tissue that is extracted from embryo by biopsy.cfDNA can be further processed to be suitable for subsequent library preparation.

[0082] IVF embryo screening The present inventors have surprisingly found that libraries generated by the above established methods can be directly sequenced for screening of IVF embryos suitable for transfer.

[0083] The present disclosure provides a non-amplified method for preimplantation genetic testing (PGT) of in vitro fertilization (IVF) embryos, the method comprising the steps of: i) providing a sample containing cell-free DNA (cfDNA) from spent medium in the culture of IVF embryos; ii) repairing the cfDNA fraction and preferably obtaining single-base overhang fragments; iii) adding directional barcoded adapters to each end of the repaired cfDNA, thereby creating a barcoded library for sequencing; and iv) sequencing the barcoded library.

[0084] In some embodiments, repairing the cfDNA includes converting 5' and / or 3' overhangs to blunt ends and / or repairing internal nicks.

[0085] In some embodiments, the single-base overhang fragment comprises a 3' dA overhang at each end of the repaired fragment. In some embodiments, the Y adaptor comprises a barcode in the double-stranded region. In some embodiments, the Y adaptor comprises a barcode in the single-stranded region.

[0086] In some aspects, the methods of the present disclosure include a purification step.

[0087] In some embodiments, the method includes generating multiple barcoded libraries with different barcodes. In some embodiments, the barcoded adapters can be further modified to include random nucleotide sequences that provide unique sequences for the adapters. In some embodiments, the method includes combining multiple of the barcoded libraries prior to purification.

[0088] In some embodiments, the method includes removing proteins bound to the cfDNA. In some embodiments, the cfDNA is treated with a protease and / or a detergent to remove nucleosomes or heterochromatin structures from the cfDNA fragments.

[0089] In some embodiments, spent medium is collected from day 5-7 blastocyst cultures.

[0090] Any nucleic acid sequencing platform is suitable for sequencing genomic DNA, including high-throughput DNA sequencing (commonly referred to as "next-generation sequencing" or "NGS"). Thus, in some embodiments, the barcoded library is sequenced using high-throughput sequencing. In some embodiments, the library is sequenced using DNA nanoball sequencing. In some embodiments, the DNA nanoball sequencing is performed using combinatorial probe anchor ligation (cPAL). Sequencing can be paired-end or unpaired-end sequencing, preferably paired-end sequencing.

[0091] NGS method provides different sequence read sizes from tens to hundreds of base pairs.In some embodiments of the method described herein, sequence read is about 20bp, about 25bp, about 30bp, about 35bp, about 40bp, about 45bp, about 50bp, about 55bp, about 60bp, about 65bp, about 70bp, about 75bp, about 80bp, about 85bp, about 90bp, about 95bp, about 100bp, about 110bp, about 120bp, about 130bp, about 140bp, about 150bp, about 200bp, about 250bp, about 300bp, about 350bp, about 400bp, about 450bp, or about 500bp.With the advancement of technology, it is expected that single-end reads of more than 500bp will be possible, and when paired-end reads are generated, reads of more than about 1000bp will be possible.In one embodiment, sequence reads are 36bp. Other sequencing methods that can be employed in the methods of the present invention include single-molecule sequencing, which can sequence nucleic acid molecules larger than 5000 bp. The massive sequence output is routed through an analysis pipeline that converts the primary imaging output from the sequencer into a string of bases. An integrated algorithm package performs the core primary data conversion steps of image analysis, intensity scoring, base calling, and alignment.

[0092] In some embodiments, the sequencing data covers at least 10,000 mapped sequencing reads, or at least 0.02% of the human genome.

[0093] This method may include one or more additional steps for analyzing the data obtained from sequencing. In some embodiments, this method further comprises analyzing the sequencing data to obtain a chromosome profile, such as a 24-chromosome profile of a human, to determine the chromosome ploidy state of the embryo. In some embodiments, this method further comprises analyzing the sequencing data to obtain an X and Y chromosome profile, for example, of a human embryo, to determine the balance of sex chromosomes. In some embodiments, this method further comprises calculating the copy number of the X chromosome by comparison with the autosomal region.

[0094] To assess sex chromosome balance, software is designed to first calculate the copy number of the X chromosome relative to the autosomal region. This identifies whether one or two copies of the X chromosome are present relative to the autosomes. To verify against the possible background of low levels of male DNA in the HSA component of the culture medium, Y-specific gene sequences, or other Y-specific sequences from multicopy genes such as TSPY (17–30 copies) or heterochromatin, are then analyzed for relative abundance. 46,XX embryos will reveal a background of Y-specific sequences from HSA male DNA contamination. Therefore, designated 46,XY embryos are confirmed by higher levels of Y-specific sequences than female embryos.

[0095] The sequencing data can be analyzed by aligning it to a reference genome, so that any differences between the embryo's genome sequence (and the parent) and the reference can be identified as potential genetic variations. In some embodiments, the reference genome is a human reference genome. In some embodiments, the reference genome is a Genome Reference Consortium Human Build. In some embodiments, the reference genome is Genome Reference Consortium Human Build 37 (GRCh37) or Genome Reference Consortium Human Build 38 (GRCh38), or a future build (i.e., Build 39 or later).

[0096] cfDNA size profile can be used to bioinformatically separate or enrich embryonic DNA fraction with respect to non-embryonic DNA fraction.In this way, the embryonic fraction or enriched embryonic fraction of sequencing data can be selected for analysis, thereby improving the reliability and accuracy of embryonic genetic diagnosis.This is similar to non-invasive prenatal diagnosis, where it is known that the fetal cfDNA fragments in maternal plasma are generally smaller than the maternal cfDNA fragments, which can then be used to determine the fetal fraction of the total cfDNA.Therefore, in some embodiments, this method includes a step of fractionating reads based on size profile.

[0097] The present disclosure also provides a method for identifying the genetic background of an in vitro fertilized (IVF) embryo, the method comprising the steps of: i) providing a sample containing cell-free DNA (cfDNA) from spent medium in the culture of IVF embryos; ii) repairing the cfDNA, preferably obtaining single-base overhang fragments; iii) adding directional barcoded adapters to each end of the repaired cfDNA, thereby creating a barcoded library; and iv) determining the genome-wide heterozygous SNP profile or mitochondrial sequence by sequencing the barcoded library.

[0098] In some embodiments, repairing the cfDNA includes converting 5' and / or 3' overhangs to blunt ends and / or repairing internal nicks.

[0099] In some embodiments, the single-base overhang fragment comprises a 3'dA overhang at each end of the repaired fragment. In some embodiments, the Y adapter comprises a barcode in the double-stranded region. In some embodiments, the barcoded adapter can be further modified to include a random nucleotide sequence that provides a unique sequence for the adapter. In some embodiments, the Y adapter comprises a barcode in the single-stranded region.

[0100] In some embodiments, the methods of the present disclosure include a purification step. Standard techniques for isolating and purifying nucleic acids are known and are described, for example, in Miller (ed.) 1972 Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; Old and Primrose, 1994 Principles of Gene Manipulation, 5th ed., University of California Press, Berkeley; Schleif and Wensink, 1982 Practical Methods in Molecular Biology; Glover (Ed.) 1985 DNA Cloning: Vols. I AND II, IRL Press, Oxford, UK; Harnes and Higgins (Eds.) 1985 Nucleic Acid Hybridization, IRL Press, Oxford, UK; and Setlow and Hollaender 1979 Genetic Engineering: Principles and Methods, Vols. 1-4, Plenum Press, New York City.

[0101] In some embodiments, the method includes generating multiple barcoded libraries with different barcodes. In some embodiments, the barcoded adapters can be further modified to include random nucleotide sequences that provide unique sequences for the adapters. In some embodiments, the method includes combining multiple of the barcoded libraries prior to purification.

[0102] In some embodiments, the method includes removing proteins bound to the cfDNA. In some embodiments, the cfDNA is treated with a protease to remove nucleosomes or heterochromatin structures from the cfDNA fragments.

[0103] In some embodiments, spent medium is collected from day 5-7 blastocyst cultures.

[0104] In some embodiments, the method comprises haplotyping to determine a genome-wide heterozygous SNP profile.

[0105] Sequencing data can be analyzed by aligning it to a reference genome, so that any differences between the genomic sequence of the embryo (and parent) and the reference can be identified as potential genetic variations. In some embodiments, the reference genome is a human reference genome. In some embodiments, the reference genome is a Genome Reference Consortium Human Build. In some embodiments, the reference genome is Genome Reference Consortium Human Build 37 (GRCh37) or Genome Reference Consortium Human Build 38 (GRCh38), or a future build (i.e., Build 39 or later). Reference genomes include nuclear genomes and mitochondrial genomes, for example, the mitochondrial genome of the female parent.

[0106] The genetic background includes genetic variations, particularly pathogenic variations, such as single nucleotide polymorphisms (SNPs), insertions or deletions (indels), copy number variations (CNVs), and / or structural variations.

[0107] In single-gene analysis, software can be designed to search for heterozygous single nucleotide polymorphisms (SNPs) for linkage analysis and genetic disease prediction.

[0108] For SNP analysis and potentially mitochondrial disease diagnosis, the cfDNA in the libraries of the present invention, generated by non-amplification methods, may need to be amplified to generate sufficient material for subsequent approaches, such as further enrichment of selected DNA targets, arrays, sequencing, or real-time PCR. Nucleic acid amplification methods are also well known, including polymerase chain reaction (PCR) (PCR Protocols, A Guide to Methods and Applications, ed. Innis, Academic Press, NY 1990; PCR: A Practical Approach, MJ McPherson, et al., IRL Press (1991)); ligase chain reaction (LCR) (Landegren et al., 1988); transcription amplification (Kwoh et al., 1989); self-sustained sequence replication (Guatelli et al., 1990); Qβ replicase amplification (Smith et al., 1997), and other RNA polymerase-mediated techniques, such as nucleic acid sequence-based amplification (NASBA) (US 4,683,195 and US 4683202); 3SR (self-sustained sequencing reaction); RACE-PCR (rapid amplification of cDNA ends); PLCR (combination of polymerase chain reaction and ligase chain reaction); SDA (strand displacement amplification); and SOE-PCR (splice overlap extension PCR).

[0109] Pathogenic genetic variants can be identified, for example, by querying databases of known genetic variants annotated with their pathogenicity level. Alternatively, for genetic variants that are not present in such databases or whose pathogenicity level is uncertain, pathogenicity prediction algorithms can be used to determine whether the variant is likely to be pathogenic.

[0110] Appropriate databases of known pathogenic genetic variants include: ClinVar (https: / / www.ncbi.nlm.nih.gov / clinvar / ); CLINVITAE (http: / / clinvitae.invitae.com / ); Leiden Open Variant Database (LOVD; http: / / www.lovd.nl / ); Human Genetic Variation Database (HGVD; http: / / www.hgvd.genome.med.kyoto-u.ac.jp / ); Online Mendelian Inheritance in Man (OMIM; https: / / www.omim.org / ); EGL's Variant Classification Catalog (EmVClass; http: / / www.egl-eurofins.com / emvclass / emvclass.php); the ARUP mutation database (http: / / www.arup.utah.edu / database / ); or the Carver Allele-Specific Mutation Database (https: / / www.carverlab.org / database).

[0111] Many computer algorithms are available for aligning sequences, including, but not limited to, BLAST, BLITZ, FASTA, BOWTIE, ELAND (Illumina, Inc., San Diego, Calif., USA), Burrows-Wheeler Aligner (Li and Durbin, 2010), or GATK (DePristo et al., 2011; McKenna et al., 2010). Analysis of sequencing information to identify polymorphic sequences allows for small mismatches (0–2 mismatches per sequence tag) to account for minor polymorphisms that may exist between the reference genome and the embryonic or parental genome.

[0112] To avoid misdiagnosis due to amplification errors such as allele dropout or locus dropout, it is understood that sequencing of the allele of interest herein may include sequencing of the nucleic acids surrounding the allele to ensure amplification accuracy. For example, a disease-causing allele may be physically linked (close) to a nearby non-causing allele in the DNA sequence. These two sites in the DNA are highly likely to be inherited together unless recombination occurs between the sites during meiosis. Sites close to each other are less likely to undergo recombination. As a result, the non-causing allele can be used as a confirmatory marker for the disease-causing allele to avoid misdiagnosis due to PCR dropout of the disease allele. Such techniques are well known to those skilled in the art, such as "haplotyping." Suitable methods include those described in WO 14145820 and WO 15051006.

[0113] In some embodiments, sample comprises the cfDNA extracted from cell or tissue.In some embodiments, cfDNA is derived from the tissue that is extracted from embryo by biopsy.cfDNA can be further processed to be suitable for subsequent library preparation.

[0114] Determination of non-embryonic DNA in samples It is known that culture medium can contain significant maternal DNA contamination compared to embryonic DNA, and this medium is subsequently used without any separate or distinct cfDNA purification step.

[0115] Thus, the present disclosure provides a non-amplified method for determining the extent of non-embryonic DNA contamination in spent medium in the culture of IVF embryos, the method comprising the steps of: i) providing a sample containing cell-free DNA (cfDNA) from spent medium in the culture of IVF embryos; ii) repairing the cfDNA, preferably obtaining single-base overhang fragments; iii) adding directional barcoded adapters to each end of the repaired cfDNA, thereby creating a barcoded library for sequencing; iv) sequencing the library; and v) calculating the mtDNA to chromosome ratio, A higher mtDNA to chromosome ratio indicates a lower degree of non-embryonic DNA contamination, and a lower mtDNA to chromosome ratio indicates a higher degree of non-embryonic DNA contamination.

[0116] In some embodiments, repairing the cfDNA includes converting 5' and / or 3' overhangs to blunt ends and / or repairing internal nicks.

[0117] In some embodiments, the single-base overhang fragment comprises a 3'dA overhang at each end of the repaired fragment. In some embodiments, the Y adapter comprises a barcode in the double-stranded region. In some embodiments, the barcoded adapter can be further modified to include a random nucleotide sequence that provides a unique sequence for the adapter. In some embodiments, the Y adapter comprises a barcode in the single-stranded region.

[0118] In some aspects, the methods of the present disclosure include a purification step.

[0119] In some embodiments, the method includes generating multiple barcoded libraries with different barcodes. In some embodiments, the barcoded adapters can be further modified to include random nucleotide sequences that provide unique sequences for the adapters. In some embodiments, the method includes combining multiple of the barcoded libraries prior to purification.

[0120] In some embodiments, the method includes removing proteins bound to the cfDNA. In some embodiments, the cfDNA is treated with a protease to remove nucleosomes or heterochromatin structures from the cfDNA fragments.

[0121] In some embodiments, spent medium is collected from day 5-7 blastocyst cultures.

[0122] In some embodiments, the mtDNA to chromosome ratio is the mtDNA to autosome ratio.

[0123] Sequencing data can be analyzed by aligning it to a reference genome, so that any differences between the genomic sequence of the embryo (and parent) and the reference can be identified as potential genetic variations. In some embodiments, the reference genome is a human reference genome. In some embodiments, the reference genome is a Genome Reference Consortium Human Build. In some embodiments, the reference genome is Genome Reference Consortium Human Build 37 (GRCh37) or Genome Reference Consortium Human Build 38 (GRCh38), or a future build (i.e., Build 39 or later). Reference genomes include nuclear genomes and mitochondrial genomes, for example, the mitochondrial genome of the female parent.

[0124] The level of maternal DNA contamination in a 46,XY embryo can be determined by assessing the copy number of the X chromosome. In some embodiments, the embryo is a human 46,XY embryo, and the method further comprises assessing the copy number of the X chromosome. A copy number increase between 1 and 2 indicates maternal contamination. Therefore, the absolute copy number of the X can predict the percentage of maternal DNA contamination. As an extension of this analysis, if a specific chromosome is aneuploid, its copy number will also be affected in the same way as the X chromosome and should show an intermediate copy number between 1 and 2, reflecting non-embryonic X chromosome contamination.

[0125] In some embodiments, sample comprises the cfDNA extracted from cell or tissue.In some embodiments, cfDNA is derived from the tissue that is extracted from embryo by biopsy.cfDNA can be further processed to be suitable for subsequent library preparation.

[0126] Methylation profile analysis The present disclosure provides a non-amplification method for preparing a sample for methylation profile analysis, the method comprising the steps of: i) providing a sample containing cell-free DNA (cfDNA) from a biological material; ii) repairing the cfDNA and preferably obtaining single-base overhang fragments; and iii) Adding directional barcoded adapters to each end of the repaired cfDNA, thereby creating a barcoded library and avoiding PCR amplification, which destroys methylation patterns by converting methyl-C to C.

[0127] In a preferred embodiment, the adapters contain unique Y-adapters with embedded barcodes. This allows for directional addition of adapters and allows for more samples to be sequenced per individual run. Avoiding PCR overlaps results in more effective reads per sample, significantly increasing the information available for data analysis. Alternative sequencing primers / adapters can be utilized to perform sequencing on different NGS or third-generation sequencing platforms.

[0128] In a preferred embodiment, barcoded libraries from different samples can be combined into a single sample during preparation, which is cost-effective for enrichment of multiple methylated cfDNA fractions.

[0129] In a preferred embodiment, the method includes paired-end sequencing of the Y adapter library fragments to provide the full-length sequence of each fragment, which improves mapping of methylated fragments and maximizes the methylation data available for analysis.

[0130] In a preferred embodiment, the Y adapter can be modified to have a random sequence in the single-stranded region that is added to each primer strand, thereby giving each strand a unique and distinguishable 5' and 3' portion. Such tagging allows for direct identification of all original, unique targets containing a particular methylation site.

[0131] The resulting library contains both methylated and unmethylated cfDNA. In a preferred embodiment, the method includes enriching the methylated fraction of the combined cfDNA library of the present disclosure, for example, by specifically capturing methylated cfDNA fragments.

[0132] In a preferred embodiment, specific capture is performed using antibodies with specificity for double-stranded or single-stranded methylated DNA fragments, such as anti-mC monoclonal or polyclonal antibodies, or anti-ds antibodies from patients with SLE disease.

[0133] In a preferred embodiment, an antibody, such as an anti-mC antibody, is bound to or captured by an enrichment matrix such as beads to efficiently enrich for methylated fragments; unmethylated cfDNA is washed away, and the methylated cfDNA fraction is eluted into a single tube for NGS, further prepared for nanopore sequencing, or subjected to further enrichment, such as capturing selected genes or intergenic regions. For example, a cfDNA library can be enriched by hybridization to a gene chip, or in a solution containing DNA probes designed from known methylated regions, or using other matrices that preferentially bind methylated DNA (mDNA), and the methylated DNA can then be differentially eluted and sequenced.

[0134] In a preferred embodiment, the bound methylated DNA fragments are released from the enrichment matrix and sequenced without further modification to reveal a global methylation profile of the DNA fragment library, which can be generated by simple mapping and binning against a standard chromosomal reference.

[0135] In a preferred embodiment, the method further comprises simply amplifying the enriched DNA fragments using primers homologous to the directional adapter sequences originally added prior to mDNA capture. Because enrichment of methylated fragments has already occurred, no information is lost in the subsequent amplification process. The exact methylation signature (only the methylated regions) that was missed will be identified.

[0136] In a preferred embodiment, the method includes a step of improving DNA recovery using a protease. The protease can digest proteins bound to DNA or other protein structures in plasma. This step is designed to remove proteins, preferably all protein structures (nucleosomes and heterochromatin) bound to DNA fragments, to expose hidden methylation sites and improve the efficiency of DNA library preparation and methylation detection.

[0137] In a preferred embodiment, the protease is known to be effective in digesting and removing protein structures bound to DNA, for example, it is selected from the group consisting of trypsin and proteinase K.

[0138] In preferred embodiments, the protease is heat-labile proteinase K or membrane-bound trypsin. Such proteases can be inactivated or removed to avoid interference with sample preparation, e.g., downstream molecular processing of the DNA, including DNA repair, A addition, and ligation.

[0139] In some embodiments, the method further comprises sequencing the methylated DNA, for example by NGS.

[0140] In a preferred embodiment, the entire DNA sequence of the methylated DNA fragment is obtained by NGS using a sequencing primer for the Y adapter.

[0141] In a preferred embodiment, NGS is performed in paired-end NGS sequencing mode to derive the full-length sequence of each enriched methylated DNA fragment and maximize the sequencing information of the methylated regions.

[0142] In a preferred embodiment, nanopore sequencing is used to obtain the entire DNA sequence of long stretches of enriched methylated DNA fragments in terms of bases A, G, C, T and methylated C.

[0143] In a preferred embodiment, the method further comprises analyzing sequencing data derived from the methylated DNA fragments with a novel algorithm. In a preferred embodiment, the method comprises analyzing the NGS data to map methylated regions to chromosomal bins and generate a methylation profile for analysis. In a preferred embodiment, the method comprises analyzing nanopore sequencing data to plot methylation sites across the genome for methylation analysis.

[0144] In some embodiments, the method identifies hypermethylated and hypomethylated genomic regions. In some embodiments, the method identifies specific genomic sites of hypermethylation and hypomethylation.

[0145] This method can be used to predict disease states (such as cancer or other chronic diseases) depending on changes in the methylation profile of the genome. This method can also be used to enrich for other selected regions of the genome, such as polymorphic sites or known mutation sites. Such information can be used to assess the relative contribution of different tissues to the final cfDNA profile. Furthermore, this method allows for multiple different enrichment steps of cfDNA.

[0146] In some embodiments, sample comprises the cfDNA extracted from cell or tissue.In some embodiments, cfDNA is derived from the tissue that is extracted from embryo by biopsy.cfDNA can be further processed to be suitable for subsequent library preparation.

[0147] Testing selected genomic regions The present disclosure provides a non-amplification method for examining a selected genomic region on a chromosome associated with a gene, the method comprising the steps of: i) providing a sample containing cell-free DNA (cfDNA); ii) repairing the cfDNA fraction and preferably obtaining single-base overhang fragments; iii) adding directional barcoded sequencing-compatible adapters to each end of the repaired cfDNA, thereby creating a barcoded library; iv) enriching the library for selected genomic regions; and v) sequencing the barcoded library.

[0148] In a preferred embodiment, the library is enriched by selectively binding cfDNA homologous to selected genomic regions with hybridization capture probes, including exon or intron sites of known genes, or chromosomal regions associated with mutations, methylated regions, or other regions of interest to researchers.

[0149] Preferably, sequence can be evaluated for the relative ratio of any polymorphism identified in the DNA fragments that are captured.This information is useful for evaluating the contribution of different tissues, such as transplanted organs, the spontaneous chromosomal changes associated with cancer, pregnancy, etc., and distinguishable sequence is useful for evaluating the genetic characteristics of tissues, and can also be useful for evaluating the general health of different tissues.

[0150] In preferred embodiments, the cfDNA may be cell-associated, but has been extracted and processed into a form compatible with library preparation and subsequent manipulation.

[0151] The present disclosure provides a non-amplification method for examining a selected genomic region on a chromosome associated with a polymorphic site, the method comprising the steps of: i) providing a sample containing cell-free DNA (cfDNA); ii) repairing the cfDNA fraction and preferably obtaining single-base overhang fragments; iii) adding directional barcoded sequencing-compatible adapters to each end of the repaired cfDNA, thereby creating a barcoded library; iv) enriching the library for selected genomic regions; and v) sequencing the barcoded library.

[0152] In a preferred embodiment, the library is enriched by selectively binding cfDNA homologous to selected genomic regions with hybridization capture probes, which include polymorphic single base sites or other polymorphic regions of interest to researchers.

[0153] In a preferred embodiment, selected genomic regions can be sequenced and analyzed for mutations such as single base substitutions or small indels.

[0154] Preferably, sequence can be evaluated for the relative ratio of any polymorphism identified in the DNA fragments that are captured.This information is useful for evaluating the contribution of different tissues, such as transplanted organs, the spontaneous chromosomal changes associated with cancer, pregnancy, etc., and distinguishable sequence is useful for evaluating the genetic characteristics of tissues, and can also be useful for evaluating the general health of different tissues.

[0155] In preferred embodiments, the cfDNA may be cell-associated, but has been extracted and processed into a form compatible with library preparation and subsequent manipulation.

[0156] In some embodiments, sample comprises the cfDNA extracted from cell or tissue.In some embodiments, cfDNA is derived from the tissue that is extracted from embryo by biopsy.cfDNA can be further processed to be suitable for subsequent library preparation.

[0157] Aspects 1. A non-amplified method for preimplantation genetic testing (PGT) of in vitro fertilization (IVF) embryos, comprising the steps of: i) providing a sample containing cell-free DNA (cfDNA) from spent medium in the culture of IVF embryos; ii) repairing the cfDNA fraction and preferably obtaining single-base overhang fragments; iii) adding directional barcoded adapters to each end of the repaired cfDNA, thereby creating a barcoded library for sequencing; and iv) sequencing the barcoded library; and optionally, v) fractionating the reads based on their size profile. 2. The method of embodiment 1, wherein the step of repairing the cfDNA comprises converting 5' and / or 3' overhangs to blunt ends and / or repairing internal nicks. 3. The method of embodiment 1, wherein the single-base overhang fragment comprises a 3'dA overhang at each end of the repaired fragment. 4. The method of embodiment 1, wherein the barcoded library is sequenced by paired-end sequencing. 5. The method of embodiment 1, wherein the barcoded adapter is a Y adapter. 6. The method of embodiment 5, wherein the Y adapter comprises a barcode in the double-stranded region. 7. The method of embodiment 6, wherein the Y adaptor comprises a random nucleotide sequence of 1 to 10 bases in the single-stranded region of each arm. 8. The method of any one of aspects 1 to 7, further comprising a step of removing proteins bound to the cfDNA, e.g., treating the cfDNA with a protease to remove nucleosomes or heterochromatin structures from the cfDNA fragments. 9. Multiple barcoded libraries with different barcodes The method according to any one of embodiments 1 to 8, comprising the step of forming: 10. The method of any one of aspects 1 to 9, further comprising a purification step. 11. The method of embodiment 10, comprising combining a plurality of said barcoded libraries prior to purification. 12. The method of any one of aspects 1 to 11, further comprising analysis of the sequencing data to obtain a 24-chromosome profile to determine the chromosomal ploidy state of the embryo. 13. The method of any of aspects 1-11, further comprising analysis of the sequencing data to obtain X and Y chromosome profiles to determine sex chromosome balance. 14. The method of embodiment 13, further comprising calculating the copy number of the X chromosome by comparison with autosomal regions. 15. The method of any one of aspects 1-14, wherein the spent medium is collected from day 5-7 blastocyst cultures. 16. A non-amplification method for generating libraries for preimplantation genetic testing (PGT) of in vitro fertilization (IVF) embryos, comprising the steps of: i) providing a sample containing cell-free DNA (cfDNA) from spent medium in the culture of IVF embryos; ii) repairing the cfDNA and preferably obtaining single-base overhang fragments; and iii) adding directional barcoded adapters to each end of the repaired cfDNA, thereby creating a barcoded library for sequencing. 17. The method of embodiment 16, wherein repairing the cfDNA comprises converting 5' and / or 3' overhangs to blunt ends and / or repairing internal nicks. 18. The method of embodiment 16, wherein the single-base overhang fragment comprises a 3'dA overhang at each end of the repaired fragment. 19. The method of embodiment 16, wherein the barcoded adapter is a Y adapter. 20. The method of embodiment 19, wherein the Y adapter comprises a barcode in the double-stranded region. 21. The method of embodiment 20, wherein the Y adaptor comprises a random sequence of 1 to 10 nucleotide bases in the single-stranded region of each arm. 22. The method of any one of aspects 15 to 21, further comprising a step of removing proteins bound to the cfDNA, e.g., treating the cfDNA with a protease to remove nucleosomes or heterochromatin structures from the cfDNA fragments. 23. Multiple barcoded libraries with different barcodes 23. The method of any one of embodiments 15 to 22, comprising creating: 24. The method of any one of aspects 15 to 23, further comprising a purification step. 25. The method of embodiment 24, comprising combining a plurality of said barcoded libraries prior to purification. 26. The method of any one of aspects 15-25, wherein the spent medium is collected from day 5-7 blastocyst cultures. 27. A method for identifying the genetic background of an in vitro fertilization (IVF) embryo, comprising the steps of: i) providing a sample containing cell-free DNA (cfDNA) from spent medium in the culture of IVF embryos; ii) repairing the cfDNA and preferably obtaining single-base overhang fragments; iii) adding directional barcoded adapters to each end of the repaired cfDNA, thereby creating a barcoded library; and iv) determining the genome-wide heterozygous SNP profile or mitochondrial sequence by sequencing the barcoded library. 28. The method of embodiment 27, wherein repairing the cfDNA comprises converting 5' and / or 3' overhangs to blunt ends and / or repairing internal nicks. 29. The method of embodiment 27, wherein the single-base overhang fragment comprises a 3'dA overhang at each end of the repaired fragment. 30. The method of embodiment 27, wherein the barcoded library is sequenced by paired-end sequencing. 31. The method of embodiment 27, wherein the barcoded adapter is a Y adapter. 32. The method of embodiment 31, wherein the Y adapter comprises a barcode in the double-stranded region. 33. The method of embodiment 32, wherein the Y adaptor comprises a random sequence of 1 to 10 bases in the single-stranded region of each arm. 34. The method of any one of aspects 27 to 33, further comprising a step of removing proteins bound to the cfDNA, for example, treating the cfDNA with a protease to remove nucleosomes or heterochromatin structures from the cfDNA fragments. 35. Multiple barcoded libraries with different barcodes 35. The method of any one of embodiments 27 to 34, comprising creating: 36. The method of any one of aspects 27 to 34, further comprising a purification step. 37. The method of embodiment 36, comprising combining a plurality of said barcoded libraries prior to purification. 38. The method of any one of aspects 27-37, wherein the spent medium is collected from day 5-7 blastocyst cultures. 39. A non-amplified method for determining the extent of non-embryonic DNA contamination in spent medium from the culture of IVF embryos, comprising the steps of: i) generating a library by a method according to any one of embodiments 15 to 34; ii) sequencing the library; and iii) calculating the mitochondrial DNA (mtDNA) to chromosomal ratio, a higher mtDNA to chromosome ratio indicates a lower degree of non-embryonic DNA contamination, and a lower mtDNA to chromosome ratio indicates a higher degree of non-embryonic DNA contamination; and optionally, iv) assessing the X chromosome copy number if the embryo is a human 46,XY embryo. 40. The method of embodiment 39, wherein the ratio of mtDNA to chromosomes is the ratio of mtDNA to autosomes. 41. A non-amplification method for generating a library of cell-free DNA (cfDNA) from biological material, comprising the steps of: i) providing a sample containing cell-free DNA (cfDNA) from a biological material; ii) repairing the cfDNA and preferably obtaining single-base overhang fragments; and iii) adding barcoded adapters to each end of the repaired cfDNA, thereby creating a barcoded library for sequencing. 42. The method of embodiment 41, wherein the step of repairing the cfDNA comprises converting 5' and / or 3' overhangs to blunt ends and / or repairing internal nicks. 43. The method of embodiment 41, wherein the single-base overhang fragment comprises a 3'dA overhang at each end of the repaired fragment. 44. The method of embodiment 41, wherein the directional barcoded adapter is a Y adapter. 45. The method of embodiment 44, wherein the Y adapter comprises a barcode in the double-stranded region. 46. ​​The method of embodiment 44, wherein the Y adaptor comprises a random sequence of 1 to 10 bases in the single-stranded region of each arm. 47. The method of any one of aspects 41 to 46, further comprising a step of removing proteins bound to the cfDNA, for example, treating the cfDNA with a protease to remove nucleosomes or heterochromatin structures from the cfDNA fragments. 48. Multiple barcoded libraries with different barcodes 47. The method of any one of embodiments 41 to 46, comprising creating: 49. The method of any one of aspects 41 to 47, further comprising a purification step. 50. The method of embodiment 48, comprising combining a plurality of said barcoded libraries prior to purification. 51. A sequencing library produced by a method according to any one of aspects 16 to 26 and 41 to 50. 52. A method for genetic testing of a sample, comprising sequencing the library of embodiment 51, and analyzing the sequencing data to determine a genome-wide heterozygous SNP profile or mitochondrial sequence. 53. A non-amplification method for preparing samples for methylation profile analysis, comprising the steps of: i) providing a sample containing cell-free DNA (cfDNA) from a biological material; ii) repairing the cfDNA and preferably obtaining single-base overhang fragments; and iii) Adding directional barcoded adapters to each end of the repaired cfDNA, thereby creating a barcoded library and avoiding PCR amplification, which destroys methylation patterns by converting methyl-C to C. 54. The method of embodiment 53, wherein the adapter comprises a unique Y adapter incorporating a barcode. 55. The method of embodiment 53 or 54, wherein barcoded libraries from different samples are combined into a single sample during preparation. 56. The method of any one of aspects 53 to 55, comprising paired-end sequencing of the Y adapter library fragments to provide the full-length sequence of each fragment. 57. The method of any one of aspects 53 to 56, wherein the Y adaptor is modified to have a random sequence in the single-stranded region that is added to each primer strand. 58. A method according to any one of aspects 53 to 57, comprising enriching the methylated fraction of the combined cfDNA library, e.g., by specific capture of methylated cfDNA fragments. 59. The method of embodiment 58, wherein specific capture is performed using an antibody with specificity for double-stranded or single-stranded methylated DNA fragments, or using a matrix that preferentially binds methylated DNA (mDNA), which can then be differentially eluted and sequenced. 60. The method of embodiment 59, wherein the antibody is selected from an anti-mC monoclonal or polyclonal antibody, or an anti-ds antibody from a patient with SLE disease. 61. The method of embodiment 59, wherein the antibody is bound to or captured by an enrichment matrix, such as a bead, to efficiently enrich for methylated fragments. 62. The method of any one of aspects 58 to 61, wherein the captured methylated DNA fragments are released from the enrichment matrix and sequenced without further modification. 63. The method of any one of aspects 58 to 62, further comprising simply amplifying the enriched DNA fragments using primers homologous to the directional adapter sequences originally added prior to mDNA capture. 64. The method of any one of aspects 53 to 63, further comprising the addition of a protease, such as trypsin and proteinase K. 65. The method of embodiment 64, wherein the protease is heat-labile proteinase K or membrane-bound trypsin. 66. The method of any one of aspects 53 to 65, further comprising sequencing the methylated DNA, for example by NGS. 67. The method described in embodiment 66, wherein the entire DNA sequence of the methylated DNA fragment is obtained by NGS using a sequencing primer for the Y adapter. 68. The method of embodiment 66 or 67, wherein NGS is performed in paired-end NGS sequencing mode. 69. The method of any of aspects 53 to 65, further comprising sequencing the methylated DNA by nanopore sequencing to obtain the entire DNA sequence of long concatenated enriched methylated DNA fragments, e.g., in terms of bases A, G, C, T and methylated C. 70. The method of any one of aspects 53 to 69, further comprising analyzing sequencing data derived from the methylated DNA fragments. 71. The method of embodiment 70, comprising analyzing the NGS data to map methylated regions to chromosomal bins and generate a methylation profile for analysis. 72. The method of embodiment 70, comprising analyzing nanopore sequencing data to plot methylation sites across the genome for methylation analysis. 73. A non-amplification method for examining a selected genomic region on a chromosome associated with a gene or polymorphic site, comprising the steps of: i) providing a sample containing cell-free DNA (cfDNA); ii) repairing the cfDNA fraction and preferably obtaining single-base overhang fragments; iii) adding directional barcoded sequencing-compatible adapters to each end of the repaired cfDNA, thereby creating a barcoded library; iv) enriching the library for selected genomic regions; and v) sequencing the barcoded library. 74. The method of embodiment 73, wherein the library is enriched by selectively binding any cfDNA homologous to the selected genomic region with a hybridization capture probe. 75. The method of embodiment 73 or 74, wherein the sequence can be evaluated for the relative proportion of any polymorphisms identified in the captured DNA fragments. 76. The method of any one of aspects 73 to 75, wherein the cfDNA may be cell-associated but has been extracted and processed into a form compatible with library preparation and subsequent manipulation. 77. A method according to any one of aspects 73 to 76, wherein the selected genomic region can be sequenced and analyzed for mutations such as single base substitutions or small indels. 78. The method of any one of claims 1 to 77, wherein the sample comprises cfDNA extracted from a cell or tissue. 79. The method of any one of claims 1 to 78, wherein the cfDNA is derived from tissue removed from an embryo by biopsy and optionally processed to make it compatible with subsequent library preparation.

[0158] Advantages of the Invention The present disclosure provides a method for sample preparation, such as library generation, that is simple, rapid, reliable, and accurate, and does not require whole genome amplification, at least for library generation. [Example]

[0159] Example 1. Analysis of spent embryo medium Sample Spent medium following blastocyst culture up to day 5 onwards, which is normally discarded once embryo development to the required blastocyst stage is complete, was collected for analysis.

[0160] 1.2. Library preparation Ten microliters of spent embryo culture medium was mixed with 10 μl of Repair Mix (0.8 μl T4 DNA polymerase (Hunan Yearthbio) (3 U / μl), 0.4 μl rTaq (Hunan Yearthbio) (5 U / μl), 2 μl 10× T4 ligase buffer (500 mM Tris-HCl, 100 mM MgCl2, 10 mM ATP, 100 mM DTT), 1.5 μl 10 mM dNTPs), and 5.3 μl water to obtain a total volume of 20 μl. For end repair and "A addition," the mixture was incubated at 37°C for 20 minutes and then at 72°C for 30 minutes.

[0161] This product was then mixed with 10 μl of Adapter Mix (0.6 μL ADT-FL (1 μM, Hunan Yearthbio), 1 μL T4 ligase (600 U / μL), 1 μL 10× T4 ligase buffer (500 mM Tris-HCl, 100 mM MgCl2, 10 mM ATP, 100 mM DTT), PEG 6000 (Sigma) (final concentration: 25–27%), and water to a total volume of approximately 30 μl. The barcoded Y adapter ligation mix was incubated at 20°C for 15 min to obtain the barcoded library and then heated at 65°C for 10 min to inactivate the ligase.

[0162] Libraries with different barcodes were then combined, and the resulting modified cfDNA library was purified using 0.8X Agencourt AMPure beads according to the manufacturer's instructions. After washing with 80% fresh ethanol to remove unused reagents and Y-adapters, the modified cfDNA was eluted from the beads using 20–40 μl of DB pH 8.

[0163] 1.3. Sequencing The combined barcoded library was sequenced on an NGS platform (Illumina NovaSeq, Applied Biosystems S5, BGI MGI-T7).

[0164] To obtain the relative sequencing reads for each library, the libraries were pooled and purified. The pooled libraries were adjusted to 10 pmoles per μL in resuspension buffer (RSB) and injected into an Illumina NovaSeq flow cell. Sequencing was performed in 2x150 paired-end mode.

[0165] 1.4. Read Processing Reads were processed using Redis (for data storage), Burrow-Wheeler-Aligner (for mapping), fastq count (for raw data QC), GCcorrect (for GC correction), and CNVcalling.R (for profile calculation) or their derivatives.

[0166] Example 2. Size analysis of native cfDNA in spent embryo medium The size of native cfDNA in the spent medium of embryos was analyzed by paired-end sequencing of the libraries followed by mapping to the reference genome and fragment size estimation.

[0167] As shown in Figure 2, a major peak of approximately 170 bp fragments was consistently observed in all four cfDNA samples from embryo culture media, along with smaller peaks of apparent multimers of approximately 340 bp and 510 bp.

[0168] It should be noted that the majority of cfDNA molecules, which are approximately 170 bp or less in length, cannot be easily amplified by current PCR-based WGA methods.

[0169] Example 3. Copy number analysis Paired-end sequencing reads were aligned to the reference genome and divided into two size fractions: <200 bp and >200 bp. These reads were mapped to the human genome; the results are shown in Figure 3 for chromosomes 21, 22, and X (a 46,XX sample). Fragments from the p-arms of chromosomes 21 and 22 cannot be mapped because they contain only repetitive DNA structures.

[0170] Copy number results were similar from the two size fractions.

[0171] Example 4. Determining sex chromosome balance Sex chromosome balance was determined by the copy number of the X chromosome.

[0172] Figure 4A shows the identification of a 46,XY sample, indicating that the embryo contains two copies of each autosome and one copy of the X chromosome.

[0173] Figure 4B shows the identification of a 46,XX sample, indicating that the embryo contains two copies of each autosome and X chromosome.

[0174] Figure 4C shows the identification of Y-chromosome-specific sequences from the 46,XY and 46,XX samples, demonstrating that reads corresponding to the Y chromosome are present in the 46,XY sample data but absent in the 46,XX sample data. This figure shows fragments plotted only on the Y chromosome. Similarly, other fragments plotted against other autosomes are unique to that chromosome. Sequences shared across chromosomes are removed during initial analysis because they cannot be assigned to a single location.

[0175] Example 5. Detection of euploidy and aneuploidy in embryos Sequencing data were analyzed to detect euploidy or aneuploidy of the embryos. Chromosomes are present as one, two, or more copies, with zero copies of Y in females. Because autosomes are conventionally two copies, sex chromosomes can be compared to autosomes, giving a relative copy number of one or two (zero Y in females).

[0176] A 24-chromosome plot is shown in Figure 5. Figures 5A and 5B show results from a 46,XY euploid embryo and a 46,XY euploid embryo, respectively; whereas Figure 5C shows results from an aneuploid embryo, in which the read corresponding to chromosome 15 showed a copy number of 1, indicating that only one chromosome 15 is present in this embryo.

[0177] Example 6. Predicting non-embryonic DNA contamination by calculating mtDNA:autosomal ratios mtDNA levels (totaling less than 48 million base pairs) typically represent only 1% or less of cellular DNA content. Comparing large numbers to very small numbers can be insensitive. Because chromosomes vary in size, from 250 million base pairs in the largest to approximately 60 million base pairs in the smallest, comparing mtDNA to chromosomes that are closer in DNA content can be more sensitive. It does not have to be a single chromosome; multiple independent comparisons can provide more robust estimates.

[0178] Therefore, relative mtDNA reads were compared with autosomal reads (all chromosomes or selected chromosomes) to predict non-embryonic DNA contamination.

[0179] Example 7. Analysis of mitochondrial DNA Mitochondrial DNA data from the mother was used as a reference.

[0180] Figure 8A shows the mapping and read coverage of the mitochondrial DNA sequence. The mtDNA reads were aligned to the mtDNA reference genome. The number of reads at any given position indicates the depth and copy number of the mtDNA relative to the autosomal fragment.

[0181] Figure 8B shows the differentiation of embryos in different patient cohorts by mitochondrial DNA polymorphisms. In particular, in one embryo group, Embryos 2, 5, and 9 showed the same polymorphisms in mitochondrial DNA reads as Patient A, and were therefore identified as belonging to the same cohort. In another embryo group, Embryos 1 and 5 showed the same polymorphisms in mitochondrial DNA reads as Patient B, and were therefore identified as belonging to the same cohort.

[0182] Example 8. Enrichment of methylated DNA fragments Sample Samples included human genomic DNA and cfDNA isolated from isolated cell fractions of blood.

[0183] Genomic DNA was isolated and purified according to standard protocols. Portions of the DNA were sonicated to obtain an average fragment size of approximately 200 bp. Sources included individuals with known malignancies and individuals without any known underlying disease.

[0184] cfDNA was prepared from the serum / plasma fraction of blood from patients, including those with known colorectal cancer and those without any known malignancies, according to standard protocols.

[0185] 8.2. Library Preparation Libraries were prepared as follows using 160 ng of sheared genomic DNA, or purified cfDNA (approximately 10–20 ng) from 1 ml of plasma.

[0186] Ten microliters of spent embryo culture medium was mixed with 10 μl of Repair Mix (0.8 μl T4 DNA polymerase (Hunan Yearthbio) (3 U / μl), 0.4 μl rTaq (Hunan Yearthbio) (5 U / μl), 2 μl 10× T4 ligase buffer (500 mM Tris-HCl, 100 mM MgCl2, 10 mM ATP, 100 mM DTT), 1.5 μl 10 mM dNTPs), and 5.3 μl water to obtain a total volume of 20 μl. For end repair and "A addition," the mixture was incubated at 37°C for 20 minutes and then at 72°C for 30 minutes.

[0187] This product was then mixed with 10 μl of Adapter Mix (0.6 μL ADT-FL (1 μM, Hunan Yearthbio), 1 μL T4 ligase (600 U / μL), 1 μL 10× T4 ligase buffer (500 mM Tris-HCl, 100 mM MgCl2, 10 mM ATP, 100 mM DTT), PEG 6000 (Sigma) (final concentration: 25–27%), and water to a total volume of approximately 30 μl. The barcoded Y adapter ligation mix was incubated at 20°C for 15 min to obtain the barcoded library and then heated at 65°C for 10 min to inactivate the ligase.

[0188] 8.3. Enrichment of methylated DNA A concentrated mDNA fraction was prepared using a Methylated-DNA IP kit (Zymo Research, Cat. No. D5101) according to the manufacturer's instructions.

[0189] Essentially, the fragments prepared in 8.2 were combined with DNA denaturation buffer to a total volume of 50 μl. This was heated at 98°C for 5 minutes. Next, 15 μl of ZymoMag Protein A and 0.8 μl of anti-methylcytosine antibody were added to prepare 250 μl of MIP buffer. The denatured DNA was added, and the mixture was incubated at 37°C for 0.5–1 hour. The beads were separated on a magnetic rack, and the supernatant was discarded. The beads were then washed twice with 500 μl of MIP buffer. The DNA was eluted with 500 μl of DNA elution buffer. The tube was then incubated at 75°C for 5 minutes and then spun in a microcentrifuge for 2 minutes. The enriched DNA was contained in the supernatant fraction.

[0190] 8.4. Sequencing The combined barcoded library was sequenced on an NGS platform (Illumina NovaSeq, Applied Biosystems S5, BGI MGI-T7).

[0191] 8.5. Read Operations Reads were processed using Redis (for data storage), Burrow-Wheeler-Aligner (for mapping), fastq count (for raw data QC), GCcorrect (for GC correction), and CNVcalling.R (for profile calculation) or their derivatives.

[0192] The results are shown in Figures 9 and 10. In particular, Figure 9 shows randomly selected regions of chromosome 4 compared for captured fragments across three different samples, demonstrating the overall reliability of this process across samples. Figure 10 shows regions of both similarity and difference in the mDNA profiles from different samples, demonstrating the utility of this approach in examining commonalities and differences in methylation across samples.

[0193] Example 9. Detection and quantification of minor alleles Sample The samples included: Plasma from pregnant women known to be male, and plasma from women. Plasma from men. cfDNA was prepared from the serum / plasma fraction of blood according to standard protocols.

[0194] 9.2. Library Preparation cfDNA was purified from 1 ml of plasma (approximately 10–20 ng) and used for library preparation as follows.

[0195] Ten μL of DNA was mixed with 0.8 μL of T4 DNA polymerase (3 U / μL), 0.4 μL of rTaq (Hunan Yearthbio) (5 U / μL), 3.5 μL of Buffer 1 (2 μL of 10× T4 ligase buffer (500 mM Tris-HCl, 100 mM MgCl2, 10 mM ATP, 100 mM DTT) and 1.5 μL of 10 mM dNTPs), and 5.3 μL of water (total 20 μL). For end repair and "A addition," the mixture was incubated at 37°C for 20 min and then at 72°C for 30 min.

[0196] This product was then mixed with 0.6 μL of ADT-FL (1 μM), 1 μL of T4 ligase (600 U / μL), 8.7 μL of buffer 2 (1 μL of 10× T4 ligase buffer (500 mM Tris-HCl, 100 mM MgCl2, 10 mM ATP, 100 mM DTT), and PEG 6000 (Sigma) (add water to 8.7 μL for a final concentration of 6% in the ligation mix). The mixture was incubated at 20°C for 15 minutes to ligate the barcoded Y adapters, thereby obtaining a barcoded library, and then incubated at 65°C for 10 minutes to inactivate the ligase enzyme.

[0197] 9.3. Enrichment of Polymorphic SNPs SNP enrichment was performed using the cfDNA Library NanoID Panel Capture kit according to the manufacturer's instructions (DeepL).

[0198] Briefly, the purified cfDNA library was dried after adding 5 μl of human Cot DNA and 2 μl of NadPrep NanoBlockers. The dried probe was redissolved in a hybridization mix containing 8.5 μl of Hyb#1, 2.7 μl of Hyb#2, and 6 μl of NanoID Panel Probe. After denaturing at 95°C for 30 seconds, the mix was hybridized overnight at 65°C. Streptavidin beads (50 μl) were prepared according to the manufacturer's instructions and added to the hybridization mix. This was then held at 65°C for 45 minutes, with mixing every 10–12 minutes. 100 μl of Wash Buffer 1 was added to the library / bead mix, and the beads were separated on a magnetic rack. The beads were washed twice with Wash Buffer (65°C x 5 min), once with Wash Buffer 1 (2 min at room temperature), once with Wash Buffer 2 (2 min at room temperature), and once with Wash Buffer 3 (2 min at room temperature), and then resuspended in 23 μl of HO. The bead mix was added to a PCR reaction mix containing 25 μl of HiFi HotStart Ready Mix and 2 μL of P5+P7 primer mix (25 μM), and cycled 1× 98°C x 45 s; 15× (98°C x 15 s / 60°C x 30 s / 72°C x 30 s); and 1× 72°C x 1 min. The library was then purified using 54 μl of VAHTS DNA Clean Beads according to the manufacturer's instructions.

[0199] 9.4. Sequencing The combined barcoded library was sequenced on an NGS platform (Illumina NovaSeq, Applied Biosystems S5, BGI MGI-T7).

[0200] 9.5. Read Operations Reads were processed using Redis (for data storage), Burrow-Wheeler-Aligner (for mapping), fastq count (for raw data QC), GCcorrect (for GC correction), and CNVcalling.R (for profile calculation) or their derivatives.

[0201] The results are shown in Figure 11. In particular, Figure 11 shows the SNP profile of minor alleles in mixed genetic samples, demonstrating their utility in other applications such as transplant monitoring, pregnancy monitoring, or mixed biological samples (samples where different genetic contributions are present and estimation of relative abundance can be a useful analysis).

Claims

1. A non-amplification method for analyzing cell-free DNA (cfDNA) in a biological sample, comprising: i) providing a sample containing cell-free DNA (cfDNA); ii) repairing the cfDNA fraction to obtain single-base overhang fragments; iii) adding barcoded adapters to each end of the repaired cfDNA, thereby creating a barcoded library for sequencing; and iv) sequencing the barcoded library or processing it through further enrichment steps prior to sequencing.

2. A non-amplified method for preimplantation genetic testing (PGT) of in vitro fertilization (IVF) embryos, comprising the steps of: i) providing a sample containing cell-free DNA (cfDNA) from spent medium in the culture of IVF embryos; ii) repairing the cfDNA fraction to obtain blunt-ended fragments; iii) adding barcoded adapters to each end of the repaired cfDNA, thereby creating a barcoded library for sequencing; and iv) sequencing the barcoded library.

3. 3. The method of claim 1 or 2, wherein the step of repairing the cfDNA comprises converting 5' and / or 3' overhangs to blunt ends and / or repairing internal nicks.

4. 3. The method of claim 1 or 2, wherein the single-base overhang fragment comprises a 3' dA overhang at each end of the repaired fragment.

5. 3. The method of claim 1 or 2, wherein the barcoded library is sequenced by paired-end sequencing.

6. The method of claim 1 or 2, wherein the barcoded adapter is a Y adapter.

7. 7. The method of claim 6, wherein the Y adapter comprises a barcode in the double-stranded region.

8. 7. The method of claim 6, wherein the Y adaptor comprises a random nucleotide sequence of 1 to 10 bases on each arm of the single-stranded region.

9. The method of any one of claims 1 to 8, further comprising removing proteins bound to cfDNA.

10. Multiple barcoded libraries with different barcodes The method of any one of claims 1 to 9, comprising the step of creating

11. The method of any one of claims 1 to 10, further comprising a purification step.

12. 12. The method of claim 11, comprising combining a plurality of said barcoded libraries prior to purification.

13. 13. The method of any one of claims 1 to 12, further comprising analysis of the sequencing data to obtain a 24-chromosome profile to determine the chromosomal ploidy state of the embryo.

14. 13. The method of any one of claims 1 to 12, further comprising analysis of the sequencing data to obtain X and Y chromosome profiles to determine sex chromosome balance.

15. 15. The method of claim 14, further comprising calculating the copy number of the X chromosome by comparison with autosomal regions.

16. 16. The method of any one of claims 1 to 15, wherein the spent medium is collected from day 5 to day 7 blastocyst cultures.

17. A non-amplification method for generating libraries for preimplantation genetic testing (PGT) of in vitro fertilization (IVF) embryos, comprising the steps of: i) providing a sample containing cell-free DNA (cfDNA) from spent medium in the culture of IVF embryos; ii) repairing the cfDNA to obtain blunt-ended fragments; and iii) adding barcoded adapters to each end of the repaired cfDNA, thereby creating a barcoded library for sequencing.

18. 18. The method of claim 17, wherein repairing the cfDNA comprises converting 5' and / or 3' overhangs to blunt ends and / or repairing internal nicks.

19. 16. The method of claim 15, wherein the single-base overhang fragment comprises a 3' dA overhang at each end of the repaired fragment.

20. 16. The method of claim 15, wherein the barcoded adapter is a Y adapter.

21. 19. The method of claim 18, wherein the Y adapter comprises a barcode in the double-stranded region.

22. 22. The method of claim 21, wherein the Y adaptor comprises a random nucleotide sequence of 1 to 10 bases on each arm of the single-stranded region.

23. The method of any one of claims 17 to 22, further comprising removing proteins bound to cfDNA.

24. Multiple barcoded libraries with different barcodes 24. The method of any one of claims 17 to 23, comprising the step of creating

25. 25. The method of any one of claims 17 to 24, further comprising a purification step.

26. 26. The method of claim 25, comprising combining a plurality of said barcoded libraries prior to purification.

27. 27. The method of any one of claims 17 to 26, wherein the spent medium is collected from day 5 to day 7 blastocyst cultures.

28. A method for identifying the genetic background of an in vitro fertilization (IVF) embryo, comprising the steps of: i) providing a sample containing cell-free DNA (cfDNA) from spent medium in the culture of IVF embryos; ii) repairing the cfDNA to obtain blunt-ended fragments; iii) adding barcoded adapters to each end of the repaired cfDNA, thereby creating a barcoded library; and iv) determining the genome-wide heterozygous SNP profile or mitochondrial sequence by sequencing the barcoded library.

29. 29. The method of claim 28, wherein repairing the cfDNA comprises converting 5' and / or 3' overhangs to blunt ends and / or repairing internal nicks.

30. 29. The method of claim 28, wherein the single-base overhang fragment comprises a 3' dA overhang at each end of the repaired fragment.

31. 29. The method of claim 28, wherein the barcoded library is sequenced by paired-end sequencing.

32. 29. The method of claim 28, wherein the barcoded adapter is a Y adapter.

33. 33. The method of claim 32, wherein the Y adaptor comprises a barcode in the double-stranded region.

34. 34. The method of claim 33, wherein the Y adaptor comprises a random nucleotide sequence of 1 to 10 bases on each arm of the single-stranded region.

35. The method of any one of claims 28 to 34, further comprising removing proteins bound to cfDNA.

36. Multiple barcoded libraries with different barcodes 35. The method of any one of claims 28 to 34, comprising the step of creating

37. 37. The method of any one of claims 28 to 36, further comprising a purification step.

38. 37. The method of claim 36, comprising combining a plurality of said barcoded libraries prior to purification.

39. 39. The method of any one of claims 28 to 38, wherein the spent medium is collected from day 5 to day 7 blastocyst cultures.

40. A non-amplified method for determining the extent of non-embryonic DNA contamination in spent medium in the culture of IVF embryos, comprising the steps of: i) generating a library by the method according to any one of claims 17 to 27; ii) sequencing the library; and iii) calculating the mitochondrial DNA (mtDNA) to chromosomal ratio, A higher mtDNA to chromosome ratio indicates a lower degree of non-embryonic DNA contamination, and a lower mtDNA to chromosome ratio indicates a higher degree of non-embryonic DNA contamination.

41. 41. The method of claim 40, wherein the mtDNA to chromosome ratio is the mtDNA to autosome ratio.

42. A non-amplification method for generating a library of cell-free DNA (cfDNA) from biological material, comprising the steps of: i) providing a sample containing cell-free DNA (cfDNA) from a biological material; ii) repairing the cfDNA to obtain blunt-ended fragments; and iii) adding barcoded adapters to each end of the repaired cfDNA, thereby creating a barcoded library for sequencing.

43. 43. The method of claim 42, wherein repairing the cfDNA comprises converting 5' and / or 3' overhangs to blunt ends and / or repairing internal nicks.

44. 43. The method of claim 42, wherein the single-base overhang fragment comprises a 3' dA overhang at each end of the repaired fragment.

45. 43. The method of claim 42, wherein the barcoded adapter is a Y adapter.

46. 43. The method of claim 42, wherein the Y adaptor comprises a barcode in the double-stranded region.

47. 43. The method of claim 42, wherein the Y adaptor comprises a random nucleotide sequence of 1 to 10 bases on each arm of the single-stranded region.

48. The method of any one of claims 42 to 47, further comprising removing proteins bound to cfDNA.

49. Multiple barcoded libraries with different barcodes 49. The method of any one of claims 42 to 48, comprising the step of:

50. 50. The method of any one of claims 42 to 49, further comprising a purification step.

51. 51. The method of Claim 50, comprising combining a plurality of said barcoded libraries prior to purification.

52. 1. A non-amplification method for preparing samples for methylation profile analysis, comprising the steps of: i) providing a sample containing cell-free DNA (cfDNA) from a biological material; ii) repairing the cfDNA to obtain single-base overhang fragments; and iii) Adding directional barcoded adapters to each end of the repaired cfDNA, thereby creating a barcoded library and avoiding PCR amplification that destroys methylation patterns by converting methyl-C to C.

53. 53. The method of Claim 52, wherein the library comprises both methylated and unmethylated cfDNA.

54. 54. The method of Claim 52 or 53, further comprising enriching the methylated fraction of said library.

55. 55. The method of any one of claims 1 to 54, wherein the sample comprises cfDNA extracted from a cell or tissue.

56. 55. The method of any one of claims 1 to 54, wherein the cfDNA is derived from tissue removed from an embryo by biopsy.

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