Simplified analysis method of DNA and free DNA and application thereof

Abstract

The present application provides an amplification-free method for gene detection (PGT) before implantation of an in vitro fertilization (IVF) embryo, an amplification-free method for generating a library of PGTs, and a method for identifying a genetic context of an IVF embryo. The invention also provides a method for determining the pollution degree of non-embryonic DNA, and an amplification-free method for generating a free DNA (cfDNA) library from a biological material. The invention also provides a method for enriching methylation fragments from an amplification-free library, and a method for identifying methylation maps and / or changes thereof in samples of different sources.

Description

Technical Field

[0001] The present invention relates to DNA gene detection technology, and in particular to a non-amplification method for simplified analysis of small amounts of DNA and free DNA in in vitro fertilization (IVF) embryo preimplantation genetic testing (PGT), microbiopsy, and human body fluids. Background Art

[0002] During the natural course of cell growth, trace amounts of DNA are released into the cellular environment, usually in the form of degraded fragments of tens to thousands of base pairs. All cells have a natural lifespan, at the end of which a cellular degradation process breaks down chromosomes into fragments of typically a few hundred base pairs and polymers representing nucleosome structures, a process often referred to as apoptosis. When cellular tissue is damaged, internal processes trigger chromosome degradation processes that again break down DNA into smaller fragments, but in a more disordered manner, with less well-defined fragments. These processes can occur in cultured cells, such as tissue culture or embryo culture, but can also occur in complex living systems, such as the human body.

[0003] A culture system is a closed environment, usually a dish, where cells are surrounded by a nutrient-rich medium. Any nucleic acids released by a cell system will accumulate and / or slowly degrade further in this medium. After release from the original cultured cells, the DNA may represent the genetic information of the remaining viable cells. However, the total amount of this degraded DNA is usually so small that its quantity cannot be directly measured. Since it is usually not possible or appropriate to take a large number of cultured cells for genetic analysis, some kind of amplification process is usually considered. The most common amplification process currently used, often referred to as whole genome amplification (WGA), may actually fail to amplify certain types of DNA and may over-amplify other types, thereby introducing imbalances in the subsequent chromosome map. While this imbalance can be partially corrected using advanced bioinformatics methods, this is not always successful and can lead to incorrect interpretations of the cytogenetics. In addition, some secondary information that may be important for certain interpretations, such as fragment length and any DNA modifications present in the fragments, is lost during the amplification process. For researchers who need detailed information about a cultured cell system, a method that can analyze small amounts of DNA without altering the map or losing secondary information may be advantageous.

[0004] Complex cellular systems in nature, such as the human body, contain many different cell types, each of which shares a general chromosomal profile, but also has individual lifespan and DNA modification profiles. They are part of a multicellular system and are interconnected by a circulatory system that both supplies them with nutrients and removes waste products, including extracellular DNA (cfDNA), associated with natural growth and death processes. As a result, circulating blood carries many different DNA signatures of different cell types that are interconnected. Similarly, excretory systems, such as urine, can also carry some of these degraded DNA fragments and their associated signatures. While the body has a large number of cells undergoing death processes and releasing degraded DNA at any one time, continuous clearance by the liver and excretion through the bladder and urinary system means that the amount of this DNA is relatively small. In general, the chromosomal and genetic profile of this DNA is common throughout the body, but each cell type and each organ may have unique secondary DNA modification patterns that can be used to identify their contribution to the profile of cell-free DNA in plasma or urine. Changes in the relative amounts of cell-free DNA or contributions from individual organs 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 simply quantify the relative amount of cfDNA in cell-free plasma may help identify potential tissue damage or infection that may be present. The ability to identify which organ(s) is releasing DNA can help diagnose potential sites of damage. This damage may be the result of physical or chemical processes, or it may be related to underlying disease processes such as cancer or metabolic imbalances such as diabetes, other chronic disease conditions, or simply the aging process. Currently, the most common method to identify the organ or tissue of origin of cfDNA is to purify a relatively large amount of DNA and then chemically modify it in some way so that the underlying DNA features can be analyzed, such as DNA sequencing. This process requires a large amount of DNA starting material, much of which is destroyed during DNA processing. In addition, subsequent analysis of this modified / processed DNA is complex and requires specialized bioinformatics to interpret. A simple process that requires less starting DNA, fewer manipulation steps, fewer interactions, and reduces the need for complex and specialized bioinformatics may be beneficial in routine applications such as chronic disease identification and health management, cancer research, non-invasive prenatal testing, organ source research, and many other studies.

[0005] Genomes are complex combinations of different DNA fragments, many of which may not be directly relevant for analytical purposes. High complexity can mean that resources (such as DNA sequencing) are used less efficiently when only a limited range of targets is interrogated. There are several approaches that can reduce this complexity, making subsequent analysis more efficient and cost-effective. These approaches may involve directed amplification of specific target sites contained in DNA complexes (e.g., PCR-polymerase chain reaction, LAMP-loop-mediated isothermal amplification, MLPA-multiplex ligation-dependent probe amplification, and systems based on bacterial and viral recombination and repair such as CRISPR), but these methods require detailed knowledge of each target site, primer design and development, and often require intact DNA targets for the region of interest. In addition, due to the amplification method, some elements of the original target information are lost (e.g., fragment start / end points and internal modifications of DNA bases). In addition, access to multiple targets in a single reaction may also be limited by the amplification procedure used, or may require extensive development work to ensure compatibility between each specific probe / target combination. While some DNA, such as chromosomal DNA from tissue and cell preparations, is apparently intact for most targets, other preparations, such as cfDNA, may be severely damaged, which may preclude amplification of some or even all desired targets using standard amplification methods, especially when the DNA input amount is very small. A method that can selectively enrich DNA from a starting source, is less sensitive to the degradation state of the DNA, and does not require extensive pretreatment for multiple targets would facilitate the analysis of many different DNA sources for genotyping. If this method can also preserve original information, including original fragment characteristics such as start and end points or minor aspects such as DNA base modifications, it may be advantageous in many different applications. If subsequent analysis requires the amplification process, a method that can maintain the identity of the original target strand will help eliminate bias in target representation. An alternative to targeted amplification is to capture the target region of DNA by homologous probes that utilize specific DNA base pairing interactions. Such systems are commercially available and may be liquid / solution-based (such as Agilent or IDT probes) or liquid / solid-based (such as arrays from Agilent or other suppliers). These systems generally require large amounts of DNA as input, which may be a problem if only limited starting DNA is available. A method that can simultaneously preserve the otherwise hidden but desirable features of DNA and reduce the complexity of the DNA source through enrichment could be useful in many areas, including cancer analysis, prenatal analysis, and chronic disease assessment. Examples of specific application areas include, but are not limited to, embryo assessment for improved assisted reproductive technology, cancer detection and treatment monitoring, chromosome mapping, determination of allelic imbalance, and cell-based DNA analysis.

[0006] In other application fields, similar problems also exist in analyzing cfDNA or very small amounts of genomic DNA. Therefore, the simplified analysis method disclosed in the present invention can replace the current method and is also applicable to analyzing samples from many different biological sources and has advantages. Summary of the invention

[0007] The present application provides a method for preparing a sequencing library of a limited amount of DNA for next generation sequencing or for selectively enriching desired fragments.

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

[0009] In a second aspect, the present disclosure provides a method for generating a library without amplification for in vitro fertilization (IVF) preimplantation genetic testing (PGT), comprising:

[0010] i) Providing a sample containing cell-free DNA (cfDNA) from spent culture medium from IVF embryos;

[0011] ii) repairing the cfDNA to obtain single-base overhanging end fragments; and

[0012] iii) Adding directional barcode adapters to each end of the repaired cfDNA to generate a barcoded library of spent culture medium cfDNA for sequencing.

[0013] In a third aspect, the present disclosure provides a method for identifying the genetic background of an in vitro fertilization (IVF) embryo, comprising:

[0014] i) Providing a sample containing cell-free DNA (cfDNA) from spent culture medium from IVF embryos;

[0015] ii) repairing cfDNA to obtain single-base overhanging end fragments;

[0016] iii) adding directional barcode adapters to each end of the repaired cfDNA, thereby generating a barcoded library; and

[0017] iv) Determine 24 chromosome copy number profiles, genome-wide heterozygous SNP profiles, or mitochondrial sequences by sequencing barcoded libraries.

[0018] In a fourth aspect, the present disclosure provides a method for determining the degree of non-embryonic DNA contamination in spent culture medium of IVF embryo culture without amplification, comprising:

[0019] i) Providing a sample containing cell-free DNA (cfDNA) from spent culture medium from IVF embryos;

[0020] ii) repairing cfDNA to obtain single-base overhanging end fragments;

[0021] iii) adding directional barcode adapters to each end of the repaired cfDNA to generate a barcoded library for sequencing;

[0022] iv) sequencing the library; and

[0023] v) Calculate the ratio of mitochondrial DNA (mtDNA) to chromosomes;

[0024] A higher mtDNA to chromosome ratio indicates a lower level of non-embryonic DNA contamination, whereas a lower mtDNA to chromosome ratio indicates a higher level of non-embryonic DNA contamination.

[0025] In a fifth aspect, the present disclosure provides a method for generating a cell-free DNA (cfDNA) library from biological material without amplification, the method comprising:

[0026] i) providing a sample containing cell-free DNA (cfDNA) from biological material;

[0027] ii) repairing the cfDNA to obtain single-base overhanging end fragments; and

[0028] iii) Adding directional barcode adapters to each end of the repaired cfDNA to generate a barcoded library for sequencing.

[0029] In a sixth aspect of the present invention, a method for sample preparation for methylation profiling without amplification is provided, the method comprising:

[0030] i) providing a sample containing cell-free DNA (cfDNA) from biological material;

[0031] ii) repairing the cfDNA to obtain single-base overhanging end fragments; and

[0032] iii) adding directional barcoded adapters to each end of the repaired cfDNA,

[0033] This allows the generation of barcoded libraries and avoids PCR amplification, which can disrupt methylation patterns by converting methyl-C to C.

[0034] In a seventh aspect of the present disclosure, there is provided a method for examining a selected genomic region on a chromosome associated with a gene without requiring amplification, the method comprising:

[0035] i) Providing samples containing cell-free DNA (cfDNA);

[0036] ii) repairing cfDNA fragments to obtain single-base overhanging end fragments;

[0037] iii) adding directional barcode sequencing-compatible adapters to each end of the repaired cfDNA to generate a barcoded library;

[0038] iv) enriching the library for selected genomic regions; and

[0039] v) Sequencing the barcoded library.

[0040] In an eighth aspect of the present disclosure, there is provided a method for examining a selected genomic region on a chromosome associated with a polymorphic site without requiring amplification, the method comprising:

[0041] i) Providing samples containing cell-free DNA (cfDNA);

[0042] ii) repairing cfDNA fragments to obtain single-base overhanging end fragments;

[0043] iii) adding directional barcode sequencing-compatible adapters to each end of the repaired cfDNA to generate a barcoded library;

[0044] iv) enriching the library for selected genomic regions; and

[0045] v) Sequencing the barcoded library. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 Schematic diagram showing how the simplified analytical method can be used for direct analysis or can be further processed to obtain more specific information about a (cf)DNA sample.

[0047] Figure 2 A comparison of the amplification-free library generation method of the present disclosure with a whole genome amplification (WGA)-based culture medium analysis method is shown.

[0048] Figure 3 The sizes of native cfDNA in embryonic waste media are shown, with panels A, B, C, and D representing four individual samples.

[0049] Figure 4Analysis of different DNA size fragments (<200 bp (panel A) and >200 bp pairs (panel B)) corresponding to reads from chromosome 21, chromosome 22, and chromosome X are shown.

[0050] Figure 5 Determination of sex chromosome balance is shown, specifically the depth of reads mapped to autosomes and chromosome X in a 46,XY sample (Panel A) and a 46,XX sample (Panel B), and the mapping of Y chromosome-specific sequences in two 46,XY samples and two 46,XX samples (Panel C).

[0051] Figure 6 The ploidy of the embryos is shown, including a 46,XY euploid embryo (Panel A), a 46,XX euploid embryo (Panel B), and an aneuploid embryo lacking a copy of chromosome 15 (Panel C).

[0052] Figure 7 Possible sources of non-embryonic DNA contamination are shown.

[0053] Figure 8 Analysis of mitochondrial DNA is shown.

[0054] Fig. 9 Sequencing methylation profiles from various cfDNA and tissue sources are shown, depicting changes in methylation capture across genomic loci.

[0055] Fig.10 Gene probe capture profiles of various (cf) DNA sources are shown. Changes in meDNA capture at different genes are depicted, indicating changes in the methylation status of the genes.

[0056] Fig.11 Shown are SNP probe ratios from model systems depicting different numbers of minor alleles (4%-8%). DETAILED DESCRIPTION

[0057] General techniques and definitions

[0058] Unless specifically defined otherwise, all technical and scientific terms used herein shall have the same meanings as commonly understood by one of ordinary skill in the art (eg, molecular genetics, bioinformatics, developmental biology, and IVF).

[0059] Unless otherwise indicated, the techniques used in this disclosure are standard procedures, well known to those skilled in the art. These techniques are described and explained in such references as J. Perbal, Molecular Cloning: A Practical Guide (John Wiley and Sons, 1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbour Laboratory Press, 1989), TA Brown (ed.), Basic Molecular Biology: A Practical Approach, Volumes 1 and 2 (IRL Press, 1991), DM Glover and BD Hames (eds.), DNA Cloning: A Practical Approach, Volumes 1-4 (IRL Press, 1995 and 1996), and FM Ausubel et al. (eds.), Current Protocols in Molecular Biology (Greene Pub. Associates and Wiley-Interscience, 1988, including all updates to date), Ed Harlow and David Lane (eds.), Antibodies: A Laboratory Manual (Cold Spring Harbour Laboratory, 1988), and JE Coligan et al. (eds.), Current Protocols in Immunology (John Wiley & Sons, including all updates to date).

[0060] As used herein, the term "about", unless otherwise indicated to the contrary, means + / - 10%, more preferably + / - 5%, of the specified value.

[0061] As used herein, the word "comprise" or variations thereof will be understood to imply the inclusion of stated elements, integers or steps, or groups of elements, integers or steps, but not the exclusion of any other elements, integers or steps, or groups of elements, integers or steps.

[0062] As used herein, the term "or" is intended to mean an inclusive "or" rather than an exclusive "or". That is, unless specified otherwise or clear from the context, "X employs A or B" is intended to mean any of the natural inclusive permutations. That is, if X employs A; X employs B; or X employs both A and B, then "X employs A or B" satisfies in any of the foregoing cases. In addition, at least one of A and B and / or similar terms generally mean A or B or both A and B. Moreover, the articles "a" and "an" used in this application and the appended claims may generally be construed to mean "one or more" unless specified otherwise or clear from the context to be directed to the singular form.

[0063] As used herein, the term "screening" refers to the process of evaluating an embryo. For example, such a process can be used to select an embryo suitable for implantation into a woman's uterus. Similarly, the term "screening" can refer to evaluating any DNA sample to determine whether it has a risk of an aneuploid genome, a pathogenic genetic variation, or a polymorphism of interest to a researcher.

[0064] As used herein, the term "euploidy" or "euploid" refers to a cell, embryo or DNA sample that contains one or more complete genomes (two for humans) and without any redundant chromosomes, while the term "aneuploidy" or "aneuploidy" refers to a cell, embryo or DNA sample that contains at least one incomplete genome or at least one redundant chromosome or a portion thereof.

[0065] The term "pathogenic" is used herein to refer to genetic variations that are known or predicted to be associated with a disease. The association of one or more genetic variations with a disease may cause the disease or may represent a genetic predisposition, ie, risk, for developing the disease.

[0066] As used herein, the term "alignment", "alignment" or "alignment" refers to the matching of one or more sequences with the known sequence of a reference genome in terms of the order of its nucleic acid molecules. This alignment can be done manually or by a computer algorithm, such as the Efficient Local Nucleotide Data Alignment (ELAND) computer program distributed as part of the Illumina genome analysis process. The matching of sequence reads in the alignment can be 100% sequence matching or less than 100% (non-perfect match).

[0067] The term "allele" herein refers to a sequence variant of a genetic sequence. For the purposes of this application, an allele may be, but not necessarily, located within a gene sequence. An allele may be identified relative to one or more polymorphic positions such as SNPs, and the remainder of a gene sequence may not be specified. For example, an allele may be defined by the nucleotides present at a single SNP, or by the nucleotides present at a plurality of SNPs.

[0068] The term "sequencing" herein refers to a method for determining the nucleotide sequence of a polynucleotide (e.g., genomic DNA). Preferably, the sequencing method includes, but is not limited to, an NGS method (i.e., a high-throughput sequencing method), in which cloned amplified DNA templates or single DNA molecules are sequenced in a massively parallel manner (Volkerding et al., 2009; Metzker et al., 2010).

[0069] 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, such as a sequence that can be aligned and specifically assigned to a chromosome or genomic region or gene.

[0070] The term "whole genome amplification" as used herein refers to the process of amplifying a genomic DNA sequence present in a sample to provide multiple copies of the genome represented by the sequence.

[0071] The term "haplotype" refers to a DNA sequence of one or more interested genetic variations contained in the subregion comprising an individual single chromosome. The genetic variation of a haplotype can be of the same type, such as all SNPs, or can be a combination of two or more types of genetic variations, such as a combination of SNPs and STRs. A haplotype can refer to a group of genetic variations in a single gene, an intergenic sequence, or a larger sequence (including gene and intergenic sequence, such as a gene set or a gene and intergenic sequence). For example, a haplotype can refer to a group of genetic variations in a complement activation regulation (RCA) site, including complement factor H (CFH), FHR3, FHR1, FHR4, FHR2, FHR5, and F13B gene sequences and intergenic sequences (i.e., the middle intergenic sequence, upstream sequence, and downstream sequence that are in linkage disequilibrium with the genetic variation in the gene region). For example, a haplotype can be a group of maternal alleles or a group of paternal alleles at any site.

[0072] The term "haplotype analysis" herein refers to the process of determining one or more haplotypes in an individual, including the use of family pedigrees, molecular techniques and / or statistical inference. Preferably, the haplotype is determined by sequencing using next generation sequencing technology.

[0073] The term "joint" herein refers to a compatible nucleotide fragment that can be connected or attached to a repaired DNA fragment. Such a joint can be a double-stranded DNA or a DNA analog or an RNA or an RNA analog, and has a defined sequence compatible with any subsequent process (such as DNA sequencing or DNA amplification). The Y joint has single-stranded and double-stranded regions, enabling it to be directed to be added to the fragment of interest.

[0074] In vitro fertilization (IVF)

[0075] The methods described herein can be used to screen IVF embryos to identify embryos at risk for an aneuploid genome or pathogenic genetic variation, thereby allowing the selection of appropriate embryos for transplantation. IVF is a fertilization process in which an egg is combined with sperm in vitro. The process involves monitoring and in some cases stimulating the woman's ovulation process, removing eggs from the woman's ovaries, and fertilizing them with sperm in a suitable fluid in a laboratory environment. The fertilized egg (zygote) is implanted in the uterus of the same or another woman after about 2-7 days of embryo culture, with the goal of establishing a successful pregnancy. Typically, parents undergo IVF if they have difficulty conceiving naturally or are at risk of passing a genetic disease to the embryo.

[0076] Method described herein is applicable to the IVF embryo of any animal, as long as suitable reference genome is available, so that sequencing data can be compared to identify potential genetic variation. For example, embryo can be human or other non-human animal embryos. In some embodiments, embryo is human embryo. In other embodiments, embryo is cattle, sheep, horse, pig, dog, cat or other non-human animal embryo.

[0077] As described herein, obtaining sequencing data for "embryos" includes sequencing cfDNA in discarded culture medium of embryos fertilized no less than about 40 hours before sampling, including blastocysts (typically embryos on days 4, 5, 6, or 7 after fertilization). Therefore, in some embodiments, the embryo is an embryo of 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days. The plural form of the term is also included, and therefore, the term "an embryo" used herein takes into account that multiple embryos or blastocysts can be screened or transplanted simultaneously according to the methods disclosed herein.

[0078] "Transferring" an IVF embryo refers to the process of placing an IVF embryo into a female subject with the goal of implanting the embryo and resulting in a viable pregnancy. The female subject can be the female parent of the embryo or any other woman suitable for transfer of the embryo, such as in the case of pregnancy.

[0079] As contemplated herein, the methods disclosed herein can be used to simultaneously screen one or more embryos so that multiple IVF embryos that are considered to have no risk of aneuploid genomes or pathogenic genetic variations can be identified and transplanted. The number of such embryos that can be transplanted can be determined by those skilled in the art according to conventional methods.

[0080] As used herein, the terms "genetic variation," "polymorphism," and "variant" are used interchangeably to refer to changes in the genetic sequence of the embryo's genome (or the genome of either parent) relative to a reference genome.

[0081] Similarly, any DNA samples can be screened simultaneously. The number of samples screened can be determined by those skilled in the art according to routine methods.

[0082] Genetic variation includes sequence differences such as single nucleotide polymorphisms (SNPs), tandem SNPs, small-scale multi-base deletions or insertions (called "indels" (also called deletion-insertion polymorphisms or DIPs), multiple nucleotide polymorphisms (MNPs), short tandem repeats (STRs), restriction fragment length polymorphisms (RFLPs), deletions (including microdeletions), insertions (including microinsertions), duplications, inversions, translocations, doublings, complex multilocus variants, copy number variations (CNVs), and other structural variations including any other sequence changes in a chromosome.

[0083] The term "single nucleotide polymorphism (SNP)" refers to a single base (nucleotide) polymorphism in a DNA sequence between individuals in a population.

[0084] An "indel" is an insertion or deletion of bases in the genome of an organism. It is usually classified as a small genetic variation, ranging from 1 to 10,000 base pairs in length.

[0085] As used herein, the term "structural variation" refers to any variation in the structure of an organism's chromosomes. It includes a variety of variations in the genome, typically including microscopic and submicroscopic types, deletions, duplications, copy number variations, insertions, inversions, and translocations. Typically, structural variation affects a larger portion of the sequence than SNPs but smaller than chromosomal abnormalities (although there is some overlap in the definitions).

[0086] The term "copy number variation" herein refers to a structural variation, which is a change in the number of copies of a nucleic acid sequence, usually about 1 kb or larger, in a test sample compared to the number of copies of the nucleic acid sequence in a qualified sample.

[0087] Examples of specific application fields of the present invention

[0088] 1. Embryo assessment for improving assisted reproductive technology

[0089] It has been observed that for any couple undergoing IVF, a significant proportion of embryos generated during the IVF process do not have a balanced set of chromosomes. Transfer of such embryos generally results in a pregnancy that fails to initiate, but for those that do initiate, there may be miscarriage or fetal developmental abnormalities, and in some cases, live births with syndromic phenotypes. The proportion of chromosomally unbalanced embryos generally increases with maternal age, but even young women may have some or even all of their embryos with unbalanced chromosome sets. Therefore, all women undergoing IVF may benefit from embryo euploidy identification and selection. In addition, patients with a genetic predisposition may also benefit from the selection of euploid and disease-free embryos for transfer.

[0090] Currently, in PGT cycles performed worldwide, an invasive biopsy procedure is used to obtain one or a few embryonic cells to examine chromosomes and / or potential genetic conditions. Invasive biopsy using whole genome amplification (WGA) and analysis using array or next-generation sequencing methods has become the standard for making a final embryonic diagnosis. Although this procedure is well developed and widely used in many PGT laboratories, there is concern in some circles that this invasive method can cause significant harm to the embryo. Based on clinical expertise, these secondary embryo manipulations appear to have variable outcomes and the harm caused to the embryo may outweigh any benefits of PGT. In addition, some countries have legislation prohibiting any manipulation of the embryo, such as biopsy, for religious and / or philosophical reasons.

[0091] Therefore, a non-invasive technique that allows testing of embryonic DNA without invasive procedures could greatly help all women undergoing IVF to identify euploid embryos suitable for transfer. Such an approach could also allow other couples at risk for genetic disease to opt for a non-invasive embryo selection process.

[0092] Recently, small amounts of low-molecular-weight free DNA (cfDNA) of chromosomal and mitochondrial origin have been reported in embryo waste media and blastocoel fluid. Paired-end sequencing of cfDNA from blastocoel fluid of expanded embryos showed that the predominant size of cfDNA was approximately 156 bp. Other analyses of the sources of cfDNA in embryo waste media have found significant contamination in up to 60% of samples, with sources including cumulus cells, first polar bodies, second polar bodies, sperm, and human serum albumin (HSA), a major component of most embryo culture media. In addition, any cells or microcells that normally contain DNA will also contribute to this background DNA by releasing their DNA through the use of any sample processing steps involving heating or extraction processes.

[0093] All current non-invasive methods for studying cfDNA in embryonic spent culture media employ a PCR-based WGA step to amplify cfDNA followed by next-generation sequencing or array copy number analysis to interpret the embryo’s chromosomal ploidy status. All current WGA methods use heat as the primary denaturation step and cyclic heat protocols for enzymatic amplification. This heating step destroys not only the target cfDNA molecules, but also any foreign cells in the sample, including embryo-associated cells, embryo-associated structures, or operator-introduced cellular contamination. These cell-based contaminations may cause non-embryonic DNA to be amplified preferentially over the desired embryonic target DNA molecules and may confound or even overwhelm any subsequent embryonic DNA-based genomic analyses.

[0094] A physical limitation of most WGA methods is the size limit on the target DNA that can be properly amplified. The lower limit of amplified DNA fragment size is typically greater than 200 bp. Therefore, WGA methods are inherently biased towards targeting and amplifying larger fragments present in the spent culture medium, thereby introducing bias at the earliest stages of analysis and potentially rendering the amplified library non-representative of the original cfDNA and, in turn, the fetal genome. Another limitation of WGA is that, based on the PCR amplification process, there is an inherent amplification bias for specific genomic sequences in the final amplified DNA preparation, i.e., over- and under-representation. Due to the inherent bias of WGA towards larger targets, large fragments of any non-embryonic DNA contamination present or introduced in the spent culture medium may be preferentially amplified, thereby generating DNA libraries from both the original fetal cfDNA as well as any contaminating and introduced non-embryonic cfDNA and cell-based DNA sources.

[0095] WGA-based methods are relatively expensive and time-consuming, with the generation of DNA libraries potentially taking up to three hours, numerous steps requiring operator intervention, and subsequent additional procedures to purify and quantify the amplified DNA and to measure the quality of the library for sequencing. Furthermore, after WGA, there is no way to estimate the original size of any cfDNA present in the culture medium, nor is it possible to determine how much cfDNA was originally present in the discarded culture medium sample. Not surprisingly, therefore, many of the non-invasive PGT-A results published to date using different WGA assays have high rates of amplification failure, high variability, often inconsistent ploidy compared to invasive trophectoderm results, variable quality of chromosome maps, and often fail to produce interpretable results. In many cases, WGA simply fails to generate DNA libraries suitable for sequencing analysis.

[0096] As an alternative to the current invasive and noninvasive methods for PGT, a noninvasive technique is needed with simple, rapid, reliable, and accurate sample preparation.

[0097] Thus, the present disclosure overcomes or at least alleviates some of the problems of the prior art and improves PGT outcomes for patients using non-invasive embryo detection methods.

[0098] 2. Cancer detection and treatment monitoring

[0099] Methylation is a key epigenetic DNA modification that regulates the fundamental cellular processes of transcription and gene expression. Studies have shown that altered methylation patterns through hypomethylation or hypermethylation can lead to altered gene expression and the onset and progression of various human disease conditions. Therefore, changes in DNA methylation patterns may indicate underlying changes in organs and tissues and can be used as a test for early disease detection or assessment.

[0100] The cfDNA circulating in plasma is fragmented DNA with a major fragment size of 166-167bp. This cfDNA represents the genomic degradation product of tissue cells from many different parts of the body. About 1-3% of chromosomal DNA is methylated, so a similar proportion of cfDNA will also be methylated. This methylated cfDNA seen in plasma reflects the general methylation status of normal somatic cells in different organs connected by the circulatory system. Different organs will each produce their own methylated DNA sub-signatures, sharing many of the same methylation modifications with other cell lineages, but also showing tissue-origin-specific patterns in some chromosomal regions that are unique or specific to that cell lineage. Changes in methylation patterns will reflect changes in tissue gene expression and therefore changes in the mDNA profile. Therefore, possible changes in normal or typical cell function can be analyzed by changes in the usual methylation profile of that cell type. For example, in cancer, normal cell function is disrupted by uncontrolled cell growth and often accompanied by changes in specific DNA methylation sites to gain invasive potential. DNA methylation patterns of tissue tumors often differ from those of the tissue of origin in at least some chromosomal regions, and these altered methylation signatures can subsequently be seen in plasma, serum, or urine cfDNA fractions.

[0101] There are multiple methods for detecting DNA methylation, including: gene-specific detection methods using methylation-specific modifications to efficiently achieve specific targeting by PCR; genome-wide detection methods using chemical DNA modification (bisulfite sequencing) and methylation-specific arrays. Recently, allele-specific DNA sequencing performed on third-generation nanopore-based DNA sequencing platforms has been shown to be able to detect and map methylation sites throughout the genome. However, in general, the genome-wide detection methods developed to date are cumbersome, laborious, and time-consuming, and are prone to many different errors. This results in current methods being technically complex and not cost-effective for routine application of methylated cfDNA analysis.

[0102] As an alternative to current methylation detection methods, a simple, rapid, reliable, and accurate technology is needed. Therefore, a simple, non-invasive test for the methylated cfDNA fraction would be a major advance in disease detection and monitoring for early disease detection and monitoring of treatment effects.

[0103]

[0013] Accordingly, one aspect of the present disclosure is to overcome or at least alleviate some of the problems of the prior art and to simplify genome-wide methylation testing as a means of assessing an individual's general health and well-being relative to early signs of cancer or other chronic diseases.

[0104] 3. Chromosome map

[0105] Many cancer cells have altered chromosomal regions, manifested as deletions and / or gains of entire chromosomes or chromosomal regions. Traditionally, classical cytogenetic karyotyping has been used to observe these changes in chromosomal profiles, but in recent years, more common platforms for chromosomal profile assessment have been next-generation sequencing (CNVseq) or array-based hybridization systems. These array systems can be simple sequence tag sites and / or single nucleotide polymorphism probes. In the early stages of cancer growth, there is often an evolving and changing set of chromosomal profiles between different subclones of the tumor and its metastases. This is often reflected in a combination of apoptosis and necrosis of the mass - each of these death processes has its own fragment profile. Any changes in the dynamics of tissue growth can be reflected in changes in cfDNA - including the amount of DNA, the fragment profile, and the chromosomal profile. Any method that can easily view these features can help detect potential cancers and facilitate treatment monitoring.

[0106] The placenta of pregnant women releases fragmented cfDNA from the fetus into the maternal circulation - this has become the basis of modern non-invasive prenatal screening (NIPS). Most cfDNA is of maternal origin, but 4-20% is of fetal origin - although this is relatively small in the context of a large maternal background, it is sufficient to identify major chromosomal imbalances of fetal origin. Typically, purification of cfDNA from about 1 ml of blood yields about 20 ng of cfDNA (this amount of DNA is equivalent to 3000 diploid cells and represents >1010 160-200 bp fragments). This purified cfDNA is then used to prepare libraries for array or next-generation sequencing (NGS) analysis. However, array analysis may require more DNA and therefore requires an amplification step. NGS analysis requires only smaller amounts of DNA but requires library preparation, which usually involves some intermediate PCR-type amplification. Bioinformatics is used to reduce possible biases in chromosome maps associated with amplification and multiple analyses of any original library fragments. However, only a small fraction of the final library is used for the final sequence analysis, involving only about 3x10 6 -8x10 6 fragments.

[0107] Any technology that reduces the number of intermediate steps and requires less cfDNA as a starting point could facilitate the processing of NIPS samples. Likewise, the possibility of avoiding the analysis of daughter strands would simplify bioinformatics while increasing the efficiency of sequencing resources.

[0108] 4. Determine allelic imbalance

[0109] As with the chromosome profile, changes in the allele ratios of any SNPs present are also indicators of altered chromosomes and / or chromosome segments associated with cancer development. Current methods involving targeted PCR amplification are limited in scope and fail to account for many SNP targets due to degradation profiles of individual SNP targets.

[0110] The placenta of a pregnant woman releases cfDNA representing the fetus - this will be the maternal SNP contribution as well as the new paternal half set. When these paternal alleles differ from the 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 the background of major maternal cfDNA. Similar to the chromosome profile, the disturbance of the fetal cfDNA SNP ratio profile on different chromosomes can be used as an indicator of imbalance in the chromosome ratios of the developing fetus.

[0111] An organ transplant patient will receive tissue with different allele combinations in many regions of different chromosomes. As the organ is accepted by the recipient system, the cells within the transplanted organ tissue will begin to undergo the same cell growth / death life cycle as the original organ. This life cycle will introduce any unique SNPs present in the donor tissue into the recipient's cfDNA. Assessing the ratio of these SNPs to the recipient's SNPs can be a useful indicator of whether the transplant has adapted to its new location or if it has failed. An elevated ratio after an initial adaptation period may indicate organ rejection, while an initially decreased ratio may indicate initial transplant failure.

[0112] A simple method that can help estimate these allele ratios from cfDNA samples and indicate total relative cfDNA levels could be useful for monitoring cancer development or evaluating treatment progress, assessing fetal genomic complement in pregnancy, or for monitoring tissue / organ transplant success.

[0113] 5. Cell-based DNA analysis

[0114] Typically, the amount of sample available for genomic screening is sufficient for use with most current technology platforms. However, occasionally the amount of tissue available for DNA analysis is limited. These may be fine needle biopsies from suspected tumors, tissue sections on glass slides, embryo biopsies, or small tissue biopsies. This DNA can be extracted in the usual way, but it is usually then subjected to some kind of whole genome amplification process - so there is also a loss of some original information or the introduction of the same initial targeting bias as cfDNA amplification. Another approach is to fragment the DNA using any currently available method (such as sonication or nuclease digestion). Whether amplified DNA or native DNA, sequence libraries can be prepared after fragmentation using the simplified analysis methods described here. If the secondary information elements are not required in the analysis being performed, the sequencing library can be amplified and used in procedures that require more DNA (such as arrays) or as source material for enrichment procedures (such as SNP or selected gene targets). This amplification may introduce some degree of bias in the final DNA profile, but it is unlikely to be a combined bias from the initial amplification target priming and the variability of DNA sequence amplification. A method that can identify the original fragments present at the beginning of library construction may be useful for analyzing the original DNA fragments present. Likewise, a method that could reduce the variable imbalances produced by current PCR-type whole-genome amplifications could potentially aid both simple and more complex studies of cells and tissues.

[0115] Library generation

[0116] The present disclosure provides a method for generating a cell-free DNA (cfDNA) library from biological material without amplification, the method comprising:

[0117] i) providing a sample containing cell-free DNA (cfDNA) from biological material;

[0118] ii) repairing cfDNA and preferably obtaining single-base overhanging end fragments; and

[0119] iii) Adding barcode adapters to each end of the repaired cfDNA to generate a barcoded library for sequencing.

[0120] Similarly, the method can be performed using DNA from various cell samples, not limited to waste culture medium of IVF embryo culture. Therefore, the present disclosure provides a method for generating a library without amplification for in vitro fertilization (IVF) embryo preimplantation genetic testing (PGT), comprising:

[0121] i) Providing a sample containing cell-free DNA (cfDNA) from spent culture medium from IVF embryos;

[0122] ii) repairing cfDNA and preferably obtaining single-base overhanging end fragments; and

[0123] iii) Adding directional barcode adapters to each end of the repaired cfDNA to generate a barcoded library for sequencing.

[0124] The present disclosure provides libraries generated by the methods of the present invention.

[0125] Methods for obtaining DNA (eg, cfDNA) from spent culture medium of a biological sample (eg, IVF embryo culture) are known in the art.

[0126] More conveniently, the waste culture medium can be used directly without a separation step. For example, an embryo can be grown to the blastocyst stage in a single culture medium drop so that genetic analysis can be performed on a single embryo. In some embodiments, the sample in step i) is the waste culture medium of an IVF embryo culture.

[0127] The cfDNA may contain 5'- and / or 3'-overhanging ends, or internal nicks. Thus, in some embodiments, repairing the cfDNA comprises converting the 5' and / or 3' overhanging ends to blunt ends, and / or repairing internal nicks.

[0128] Typically, repair can be achieved using methods or kits known in the art. The repaired fragments can be phosphorylated by enzymatic treatment, such as using polynucleotide kinase. In some embodiments, a single deoxynucleotide, such as deoxyadenosine (A), is added to the 3' end of the fragment by the activity of certain types of DNA polymerases (such as Taq polymerase or Klenowexominus polymerase). In some embodiments, the method also includes the step of adding a 3'dA protruding end to each end of the repaired fragment.

[0129] The dA tail product is compatible with the 'T' protruding end present on the 3' end of each double-stranded region of the adapter connected thereto in the subsequent step. The dA tail prevents the polynucleotide of the flat end from connecting itself, thereby facilitating the formation of a sequence connected to the adapter. The dA tail fragment is connected to the double-stranded adapter polynucleotide sequence. The same adapter can be used for both ends of the fragment, or two sets of adapters can be used. The connection method is known in the art, and the adapter is covalently connected to the dA tail polynucleotide using a ligase (such as DNA ligase). The adapter may include a 5'-phosphate portion to facilitate connection to the target 3'-OH. The dA tail fragment includes a 5'-phosphate portion, which may be residual during the shearing process, or added by an enzyme treatment step, and has been end-repaired, and optionally extended by one or more protruding bases to provide a 3'-OH suitable for connection.

[0130] In some embodiments of the present disclosure, the directional barcode 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.

[0131] The product of the ligation reaction can be purified to remove unconnected joints and / or joints that may be connected to each other. The purification of the ligation product can be obtained by methods including 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 method of the present disclosure includes a purification step.

[0132] Libraries with various barcodes can be combined for sequencing. Thus, a combination of libraries can be prepared. It should be noted that purification may result in the loss of cfDNA of interest, which is disadvantageous for subsequent steps. Since the DNA level in the combination is significantly higher than that in a single library, the loss of DNA during purification will be less disadvantageous. In some embodiments, the method includes generating a plurality of barcode libraries with different barcodes. In some embodiments, the method includes the step of combining a plurality of barcode libraries before purification.

[0133] cfDNA may be bound to proteins, which can affect sequencing. In some embodiments, the method includes a step of removing proteins bound to cfDNA. In some embodiments, cfDNA is treated with a protease and / or a detergent to remove nucleosomes or heterochromatin structures from the cfDNA fragments.

[0134] As previously described, cfDNA is derived from spent culture medium from embryos fertilized no less than about 40 hours prior to sampling, including blastocysts (typically embryos on days 4, 5, 6, or 7 after fertilization). In some embodiments, spent culture medium is collected from blastocyst cultures from days 5 to 7.

[0135] In some embodiments, the sample comprises cfDNA extracted from cells or tissues. In some embodiments, the cfDNA is from tissue removed from an embryo by biopsy. The cfDNA can be further processed in a manner that makes it compatible with subsequent library preparation.

[0136] IVF Embryo Screening

[0137] The inventors unexpectedly discovered that the library generated by the above-established method can be directly sequenced and used to screen IVF embryos suitable for implantation.

[0138] The present disclosure provides an amplification-free method for preimplantation genetic testing (PGT) of in vitro fertilization (IVF) embryos, comprising:

[0139] i) Providing a sample containing cell-free DNA (cfDNA) from spent culture medium from IVF embryos;

[0140] ii) repairing cfDNA fragments and preferably obtaining single-base overhanging end fragments;

[0141] iii) adding directional barcode adapters to each end of the repaired cfDNA, thereby generating a barcoded library for sequencing; and

[0142] iv) Sequencing the barcoded library.

[0143] In some embodiments, repairing the cfDNA comprises converting 5' and / or 3' overhangs to blunt ends, and / or repairing internal nicks.

[0144] In some embodiments, the single base overhang end fragments include adding a 3' dA overhang end to each end of the repaired fragment. In some embodiments, the Y adapter comprises a barcode in the double stranded region. In some embodiments, the Y adapter comprises a barcode in the single stranded region.

[0145] In some embodiments, the methods of the present disclosure include a purification step.

[0146] In some embodiments, the method includes generating a plurality of barcode libraries having different barcodes. In some embodiments, the barcode adapter can be further modified to include a random nucleotide sequence that imparts a unique sequence to the adapter. In some embodiments, the method includes the step of combining the plurality of barcode libraries prior to purification.

[0147] In some embodiments, the method includes a step of removing proteins associated with cfDNA. In some embodiments, cfDNA is treated with a protease and / or a detergent to remove nucleosomes or heterochromatin structures from the cfDNA fragments.

[0148] In some embodiments, the spent culture medium is collected from day 5 to day 7 blastocyst culture.

[0149] Any nucleic acid sequencing platform is suitable for sequencing genomic DNA, including high-throughput DNA sequencing methods (also commonly referred to as "next generation sequencing" or "NGS"). Therefore, in some embodiments, the barcode library is sequenced using a high-throughput sequencing method. In some embodiments, the library is sequenced using DNA nanoball sequencing. In some embodiments, DNA nanoball sequencing is performed using combined probe anchor ligation (cPAL). Sequencing can be paired end sequencing or unpaired end sequencing, preferably paired end sequencing.

[0150] The sequence read sizes provided by NGS methods vary from tens to hundreds of base pairs. In some embodiments described herein, the sequence read is about 20 bp, about 25 bp, about 30 bp, about 35 bp, about 40 bp, about 45 bp, about 50 bp, about 55 bp, about 60 bp, about 65 bp, about 70 bp, about 75 bp, about 80 bp, about 85 bp, about 90 bp, about 95 bp, about 100 bp, about 110 bp, about 120 bp, about 130 bp, about 140 bp, about 150 bp, about 200 bp, about 250 bp, about 300 bp, about 350 bp, about 400 bp, about 450 bp, or about 500 bp. It is expected that technological advances will enable single-end reads greater than 500 bp, and when paired-end reads are generated, reads greater than about 1000 bp can be achieved. In one embodiment, the sequence read is 36 bp. Other sequencing methods that can be used by the method of the present invention include single molecule sequencing methods that can sequence nucleic acid molecules greater than 5000 bp. A large amount of sequence output is transmitted through an analysis pipeline that converts the raw imaging output of the sequencer into base strings. A set of integrated algorithms performs the core primary data conversion steps: image analysis, intensity scoring, base recognition and alignment.

[0151] In some embodiments, the sequencing data covers at least 10,000 mapped sequencing reads or at least 0.02% of the human genome.

[0152] The method may include one or more additional steps for analyzing sequencing data. In some embodiments, the method also includes analyzing sequencing data to obtain chromosome maps, such as 24 chromosome maps of the human race, for determining the chromosome ploidy state of the embryo. In some embodiments, the method also includes analyzing sequencing data to obtain X and Y chromosome maps, such as maps of human embryos, for determining sex chromosome balance. In some embodiments, the method also includes calculating the copy number of chromosome X by comparing with the autosomal region.

[0153] To assess sex chromosome balance, the software is designed to first calculate the copy number of the X chromosome by comparison to the autosomal region. This determines whether one or two copies of the X chromosome are present relative to the autosomes. To confirm the relative amount of Y-specific gene sequences or other Y-unique sequences from multi-copy genes such as TSPY (17-30 copies) or heterochromatin against a background of low-level male DNA that may be present in the HSA component of the culture medium, the 46,XX embryo will show a background of Y-specific sequences from HSA male DNA contamination. Therefore, a designated 46,XY embryo is confirmed by a higher level of Y-specific sequences than a female embryo.

[0154] Sequencing data can be analyzed by comparing with reference genome, so as to identify any difference between embryo genome sequence (and parents') and reference as potential genetic variation.In some embodiments, reference genome is human reference genome.In some embodiments, reference genome is genome reference alliance mankind build.In some embodiments, reference genome is genome reference alliance mankind build 37 (GRCh37) or genome reference alliance mankind build 38 (GRCh38) or any future build (i.e. build 39 or higher versions).

[0155] The cfDNA size map can be used to separate or enrich the embryonic DNA portion relative to the non-embryonic DNA portion by bioinformatics methods. Therefore, the embryonic or enriched embryonic portion of the sequencing data can be selected for analysis to improve the reliability and accuracy of embryonic genetic diagnosis. This is similar to non-invasive prenatal diagnosis, in which it is known that the fetal cfDNA fragments in maternal plasma are generally smaller than the maternal cfDNA fragments, which can help determine the fetal portion of the overall cfDNA. Therefore, in some embodiments, the method includes a step of fractionating the reads according to the size map.

[0156] The present disclosure also provides a method for identifying the genetic background of an in vitro fertilization (IVF) embryo, comprising:

[0157] i) Providing a sample containing cell-free DNA (cfDNA) from spent culture medium from IVF embryos;

[0158] ii) repairing cfDNA and preferably obtaining single-base overhanging end fragments; and

[0159] iii) adding directional barcode adapters to each end of the repaired cfDNA, thereby generating a barcoded library; and

[0160] iv) Determine the whole-genome heterozygous SNP profile or mitochondrial sequence by sequencing the barcoded library.

[0161] In some embodiments, repairing the cfDNA comprises converting 5' and / or 3' overhangs to blunt ends, and / or repairing internal nicks.

[0162] In some embodiments, the single base overhang end fragments include adding a 3'dA overhang end to each end of the repaired fragment. In some embodiments, the Y adapter comprises a barcode in the double-stranded region. In some embodiments, the barcode adapter can be further modified to include a random nucleotide sequence that imparts a unique sequence to the adapter. In some embodiments, the Y adapter comprises a barcode in the single-stranded region.

[0163] In some embodiments, the method of the present disclosure includes a purification step. Standard techniques for nucleic acid separation and purification are known and described in, for example, Miller (ed.) 1972 Experiments in Molecular Genetics (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY); Old and Primrose, 1994 Principles of Genetic Manipulation, 5th edition (University of California Press, Berkeley); Schleif and Wensink, 1982 Practical Methods in Molecular Biology; Glover (ed.) 1985 DNA Cloning: Volumes 1 and 2 (IRL Press, Oxford, UK); Harnes and Higgins (ed.) 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).

[0164] In some embodiments, the method includes generating a plurality of barcode libraries having different barcodes. In some embodiments, the barcode adapter can be further modified to include a random nucleotide sequence that imparts a unique sequence to the adapter. In some embodiments, the method includes the step of combining the plurality of barcode libraries prior to purification.

[0165] In some embodiments, the method includes a step of 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.

[0166] In some embodiments, the spent culture medium is collected from day 5 to day 7 blastocyst culture.

[0167] In some embodiments, the method comprises haplotype analysis to determine a genome-wide heterozygous SNP profile.

[0168] Sequencing data can be analyzed by comparing with reference genome, so as to identify any difference between embryo genome sequence (and parents') and reference as potential genetic variation.In some embodiments, reference genome is human reference genome.In some embodiments, reference genome is genome reference alliance mankind build.In some embodiments, reference genome is genome reference alliance mankind build 37 (GRCh37) or genome reference alliance mankind build 38 (GRCh38) or any future construction (i.e. build 39 or higher versions). Reference genome includes nuclear genome and mitochondrial genome, for example the mitochondrial genome of female parents.

[0169] Genetic background includes genetic variants, particularly pathogenic variants, such as single nucleotide polymorphisms (SNPs), insertions or deletions (indels), copy number variations (CNVs), and / or structural variants.

[0170] For single gene analysis, software can be designed to search for heterozygous single nucleotide polymorphisms (SNPs) for linkage analysis and genetic disease prediction.

[0171] For SNP analysis and possible mitochondrial disease diagnosis, it may be necessary to amplify the cfDNA in the libraries of the invention (which are generated by methods that do not require amplification) to generate sufficient material for subsequent methods, such as further enrichment of selected DNA targets, arrays, sequencing, or real-time PCR. Nucleic acid amplification methods are also well known and include the polymerase chain reaction (PCR) (PCR Protocol: A Guide to Methods and Applications, Innis ed., 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); QBeta replicase amplification (Smith et al., 1997), as well as other RNA polymerase-mediated techniques such as nucleic acid sequence-based amplification, NASBA (U.S. Pat. Nos. 4,683,195 and 4,683,202); 3SR (self-sustained sequence reaction); RACE-PCR (rapid amplification of cDNA ends); PLCR (a combination of polymerase chain reaction and ligase chain reaction); SDA (strand displacement amplification); and SOE-PCR (splicing overlap extension PCR).

[0172] Pathogenic genetic variants can be identified by querying a database of known genetic variants that have been annotated with their pathogenicity level. Alternatively, for genetic variants that are not in such a database, or for which the pathogenicity level is uncertain, pathogenicity prediction algorithms can be used to determine whether the variant is likely to be pathogenic.

[0173] Suitable databases of known pathogenic genetic variants include ClinVar (https: / / www.ncbi.nlm.nih.gov / clinvar / ), CLINVITAE (http: / / clinvitae.invitae.com / ), the Leiden Open Variation Database (LOVD; http: / / www.lovd.nl / ), the 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 Classification of Variants (EmVClass; http: / / www.egl-eurofins.com / emvclass / emvclass.php), the ARUP Mutation Database (http: / / www.arup.utah.edu / database / ), or the Carver Allele-Specific Variant Database (https: / / www.carverlab.org / database).

[0174] There are many computer algorithms for aligning sequences, including but not limited to BLAST, BLITZ, FASTA, BOWTIE, ELAND (Illumina, San Diego, California, USA), Burrows-Wheeler Aligner (Li and Durbin, 2010) or GATK (DePristo et al., 2011; McKenna et al., 2010). When analyzing the sequencing information to identify polymorphic sequences, a certain degree of mismatch (0-2 mismatches per sequence tag) can be allowed to take into account small polymorphisms that may exist between the reference genome and the embryo or parental genome.

[0175] In order to avoid the misdiagnosis caused by amplification errors (such as allele or site deletion), it is understood herein that sequencing the allele of interest can include sequencing the nucleic acid around the allele to ensure amplification accuracy. For example, the pathogenic allele may be physically connected (close to) with the nearby non-pathogenic allele in the DNA sequence. These two sites in DNA are likely to be inherited together, unless meiotic recombination occurs between these sites. Recombination is unlikely to occur in sites close to each other. Therefore, the non-pathogenic allele can be used as a confirmation marker for the pathogenic allele, to avoid the misdiagnosis caused by the disease allele PCR deletion. Such technology is familiar to those skilled in the art, including "haplotype analysis". Suitable methods include the method described in WO14145820 and WO15051006.

[0176] In some embodiments, the sample comprises cfDNA extracted from cells or tissues. In some embodiments, the cfDNA is from tissue removed from an embryo by biopsy. The cfDNA can be further processed in a manner that makes it compatible with subsequent library preparation.

[0177] Determination of non-fetal DNA in samples

[0178] It is known that the culture medium may contain significant maternal DNA contamination relative to fetal DNA. The culture medium is then used without any separate cfDNA purification step.

[0179] Therefore, the present disclosure further provides a method for determining the degree of non-embryonic DNA contamination in spent culture medium of IVF embryo culture without amplification, comprising:

[0180] i) Providing a sample containing cell-free DNA (cfDNA) from spent culture medium from IVF embryos;

[0181] ii) repairing cfDNA and preferably obtaining single-base overhanging end fragments; and

[0182] iii) adding directional barcode adapters to each end of the repaired cfDNA to generate a barcoded library for sequencing;

[0183] iv) sequencing the library; and

[0184] v) Calculate the ratio of mitochondrial DNA (mtDNA) to chromosomes,

[0185] A higher mtDNA to chromosome ratio indicates a lower level of non-embryonic DNA contamination, whereas a lower mtDNA to chromosome ratio indicates a higher level of non-embryonic DNA contamination.

[0186] In some embodiments, repairing the cfDNA comprises converting 5' and / or 3' overhangs to blunt ends, and / or repairing internal nicks.

[0187] In some embodiments, the single base overhang end fragments include adding a 3'dA overhang end to each end of the repaired fragment. In some embodiments, the Y adapter comprises a barcode in the double-stranded region. In some embodiments, the barcode adapter can be further modified to include a random nucleotide sequence that imparts a unique sequence to the adapter. In some embodiments, the Y adapter comprises a barcode in the single-stranded region.

[0188] In some embodiments, the methods of the present disclosure include a purification step.

[0189] In some embodiments, the method includes generating a plurality of barcode libraries having different barcodes. In some embodiments, the barcode adapter can be further modified to include a random nucleotide sequence that imparts a unique sequence to the adapter. In some embodiments, the method includes the step of combining the plurality of barcode libraries prior to purification.

[0190] In some embodiments, the method includes a step of 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.

[0191] In some embodiments, spent culture medium is collected from day 5 to day 7 blastocyst cultures.

[0192] In some embodiments, the ratio of mitochondrial DNA to chromosomes is a ratio of mitochondrial DNA to autosomes.

[0193] Sequencing data can be analyzed by comparing with reference genome, so that any difference between embryonic genome sequence (and parents') and reference is identified as potential genetic variation.In some embodiments, reference genome is human reference genome.In some embodiments, reference genome is genome reference alliance human construction.In some embodiments, reference genome is genome reference alliance human construction 37 (GRCh37) or genome reference alliance human construction 38 (GRCh38) or any future construction (i.e. construction 39 or higher versions). Reference genome includes nuclear genome and mitochondrial genome, for example the mitochondrial genome of female parents.

[0194] In 46, XY embryos, the level of maternal DNA contamination can be determined by assessing the number of copies of chromosome X. In some embodiments, the embryo is a human 46, XY embryo, and the method further comprises a step of assessing the number of copies of chromosome X. An increase in the number of copies of chromosome X between 1 and 2 indicates maternal contamination. Therefore, the absolute number of copies of X can predict the percentage of maternal DNA contamination. In an extension of this analysis, if aneuploidy occurs in a specific chromosome, its copy number should be similarly affected, showing an intermediate copy number between 1 and 2, reflecting non-embryonic X chromosome contamination.

[0195] In some embodiments, the sample comprises cfDNA extracted from cells or tissues. In some embodiments, the cfDNA is from tissue removed from an embryo by biopsy. The cfDNA can be further processed in a manner that makes it compatible with subsequent library preparation.

[0196] Methylation profiling analysis

[0197] The present disclosure provides an amplification-free method for sample preparation for methylation profiling analysis, the method comprising:

[0198] i) providing a sample containing cell-free DNA (cfDNA) from biological material;

[0199] ii) repairing cfDNA and preferably obtaining single-base overhanging end fragments; and

[0200] iii) adding directional barcoded adapters to each end of the repaired cfDNA,

[0201] This allows the generation of barcoded libraries and avoids PCR amplification, which disrupts methylation patterns by converting methyl-C to C.

[0202] In a preferred embodiment, the adapter includes a Y adapter with a unique barcode. This enables directional addition of the adapter and allows sequencing of more samples in each run. Due to the avoidance of PCR duplication, more efficient readings can be obtained for each sample, thereby significantly increasing the information of data analysis. Alternative sequence primers / adapters can be used to enable sequencing on different NGS platforms or third generation sequencing platforms.

[0203] In a preferred embodiment, barcode libraries from different samples can be combined into a single sample for preparation. This combination is cost-effective for enriching multiple methylated cfDNA fractions.

[0204] 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. Such sequencing improves the mapping of methylated fragments and maximizes the methylation data available for analysis.

[0205] In a preferred embodiment, the Y linker can be modified to add random sequences to each primer strand in the single-stranded region, thereby conferring unique, identifiable 5' and 3' portions to each strand. Such labeling would allow direct identification of all original unique targets containing a specific methylation site.

[0206] The generated library contains methylated and unmethylated cfDNA. In a preferred embodiment, the method comprises enriching the methylated portion of the combined cfDNA library, for example by specifically capturing methylated cfDNA fragments.

[0207] In a preferred embodiment, specific capture is performed using antibodies specific for double-stranded or single-stranded methylated DNA fragments, including anti-mC monoclonal or polyclonal antibodies or anti-ds antibodies from systemic lupus erythematosus (SLE) patients.

[0208] In a preferred embodiment, the antibody (such as an anti-mC antibody) is bound to or captured by an enrichment matrix (such as magnetic beads) to effectively enrich for methylated fragments, allowing unmethylated cfDNA to be washed away and the methylated cfDNA fraction to be eluted into a single tube for NGS, further prepared for nanopore sequencing or further enrichment, such as capture of selected genes or intergenic regions. For example, the cfDNA library is enriched by hybridization to a gene chip or in solution with DNA probes designed from known methylated regions or using other matrices that preferentially bind to methylated DNA (mDNA), which can then be differentially eluted and sequenced.

[0209] In a preferred embodiment, bound methylated DNA fragments are released from the enrichment matrix and sequenced directly to reveal global methylation profiles of the DNA fragment library. These profiles can be generated by simple mapping and binning to a standard chromosome reference.

[0210] In a preferred embodiment, the method further comprises a simple amplification of the enriched DNA fragments using primers homologous to the directional adapter sequence initially added prior to mDNA capture. Since the enrichment of methylated fragments has already been performed, no information is lost during subsequent amplification. An accurate methylation signature (methylated regions only) will be identified.

[0211] In a preferred embodiment, the method includes the use of a protease to improve DNA recovery. The protease can digest any protein bound to the DNA or other protein structures in the plasma. This step is intended to remove proteins, preferably all protein structures (nucleosomes and heterochromatin) bound to DNA fragments, expose hidden methylation sites and improve the efficiency of DNA library preparation and methylation detection.

[0212] In a preferred embodiment, the protease is a protease known to effectively digest and remove protein structures bound to DNA, for example selected from trypsin and proteinase K.

[0213] In a preferred embodiment, the protease is heat-labile proteinase K or membrane-bound trypsin. Such proteases can be inactivated or removed to avoid interference with sample preparation, such as downstream molecular processing of DNA, including DNA repair, A addition and ligation.

[0214] In some embodiments, the method further comprises sequencing the methylated DNA, such as by NGS.

[0215] In a preferred embodiment, a Y-adapter sequencing primer is used to obtain the complete DNA sequence of the methylated DNA fragment in NGS.

[0216] In a preferred embodiment, NGS is performed in paired-end NGS sequencing mode to obtain the full-length sequence of each enriched methylated DNA fragment, maximizing the sequencing information of the methylated regions.

[0217] In a preferred embodiment, nanopore sequencing is used to obtain the complete DNA sequence of long tandemly enriched methylated DNA fragments, including bases A, G, C, T and methylated C.

[0218] In a preferred embodiment, the method further comprises analyzing sequencing data from methylated DNA fragments using a novel algorithm. In a preferred embodiment, the method comprises analyzing NGS data to map methylated regions to chromosome bins and generate a methylation profile for analysis. In a preferred embodiment, the method comprises analyzing nanopore sequencing data to map genome-wide methylation sites for methylation analysis.

[0219] In some embodiments, the method identifies hypermethylated and hypomethylated regions of the genome. In some embodiments, the method identifies specific genomic hypermethylated and hypomethylated sites.

[0220] This method can predict disease states (such as cancer and other chronic diseases) based on changes in the methylation profile portion of the genome. The method can also be used to enrich other selected regions of the genome, such as polymorphic sites or known mutation sites. This information can be used to assess the relative contribution of different tissues to the final cfDNA profile. In addition, the method allows for multiple different stages of cfDNA enrichment.

[0221] In some embodiments, the sample comprises cfDNA extracted from cells or tissues. In some embodiments, the cfDNA is from tissue removed from an embryo by biopsy. The cfDNA can be further processed in a manner that makes it compatible with subsequent library preparation.

[0222] Examining selected genomic regions

[0223] The present disclosure provides a method for examining a selected genomic region on a chromosome associated with a gene for amplification-free, the method comprising:

[0224] i) Providing samples containing cell-free DNA (cfDNA);

[0225] ii) repairing cfDNA fragments and preferably obtaining single-base overhanging end fragments;

[0226] iii) adding directional barcode sequencing-compatible adapters to each end of the repaired cfDNA to generate a barcoded library;

[0227] iv) enriching the library for selected genomic regions; and

[0228] v) Sequencing the barcoded library.

[0229] In a preferred embodiment, the library is enriched by using hybridization capture probes to selectively bind any cfDNA homologous to a selected genomic region. The selected genomic region may include exon or intron sites of known genes or chromosomal regions associated with mutations, methylation regions, or other regions of interest to the researcher.

[0230] Preferably, the relative ratios of any polymorphic sites identified in the captured DNA fragments can be assessed. Such information is useful for assessing the contribution of different tissues, such as transplanted organs, spontaneous chromosomal changes associated with cancer, pregnancy, or similar situations where distinguishable sequences are useful for assessing the genetic characteristics of tissues or assessing the general health of different tissues.

[0231] In preferred embodiments, the cfDNA may be cell-associated but has been extracted and processed into a form that is compatible with library preparation and subsequent manipulations.

[0232] The present disclosure provides an amplification-free method for examining a selected genomic region on a chromosome associated with a polymorphic site, the method comprising:

[0233] i) Providing samples containing cell-free DNA (cfDNA);

[0234] ii) repairing cfDNA fragments and preferably obtaining single-base overhanging end fragments;

[0235] iii) adding directional barcode sequencing-compatible adapters to each end of the repaired cfDNA to generate a barcoded library;

[0236] iv) enriching the library for selected genomic regions; and

[0237] v) Sequencing the barcoded library.

[0238] In a preferred embodiment, the library is enriched by using hybridization capture probes to selectively bind any cfDNA homologous to a selected genomic region. The selected genomic region may include a polymorphic single nucleotide site or other region of polymorphic nature of interest to the researcher.

[0239] In a preferred embodiment, selected genomic regions can be sequenced and analyzed to detect variations, such as single base substitutions or small indels.

[0240] Preferably, the relative ratios of any polymorphic sites identified in the captured DNA fragments can be assessed. Such information is useful for assessing the contribution of different tissues, such as transplanted organs, spontaneous chromosomal changes associated with cancer, pregnancy, or similar situations where distinguishable sequences are useful for assessing the genetic characteristics of tissues or assessing the general health of different tissues.

[0241] In preferred embodiments, the cfDNA may be cell-associated but has been extracted and processed into a form that is compatible with library preparation and subsequent manipulations.

[0242] In some embodiments, the sample comprises cfDNA extracted from cells or tissues. In some embodiments, the cfDNA is from tissue removed from an embryo by biopsy. The cfDNA can be further processed in a manner that makes it compatible with subsequent library preparation.

[0243] Implementation

[0244] 1. An amplification-free method for preimplantation genetic testing (PGT) of in vitro fertilization (IVF), comprising

[0245] i) Providing a sample containing cell-free DNA (cfDNA) from spent culture medium from IVF embryos;

[0246] ii) repairing cfDNA fragments and preferably obtaining single-base overhanging end fragments;

[0247] iii) adding directional barcode adapters to each end of the repaired cfDNA, thereby generating a barcoded library for sequencing; and

[0248] iv) sequencing the barcoded library and optionally

[0249] v) Fractionation of reads based on size profile.

[0250] 2. The method according to Example 1, wherein repairing cfDNA includes converting 5' and / or 3' protruding ends into blunt ends, and / or repairing internal cuts.

[0251] 3. The method according to embodiment 1, wherein the single base overhang end fragments include adding 3' dA overhang ends to each end of the repaired fragments.

[0252] 4. The method according to embodiment 1, wherein the barcode library is sequenced by paired-end sequencing.

[0253] 5. The method according to embodiment 1, wherein the barcode adapter is a Y adapter.

[0254] 6. The method according to embodiment 5, wherein the Y linker comprises a barcode in the double-stranded region.

[0255] 7. The method according to Example 6, wherein the Y linker comprises a random nucleotide sequence of 1 to 10 bases in the single-stranded region of each arm.

[0256] 8. The method according to any one of Examples 1 to 7 further comprises a step of removing proteins bound to cfDNA, for example, the cfDNA is treated with a protease to remove nucleosomes or heterochromatin structures from the cfDNA fragments.

[0257] 9. The method according to any one of embodiments 1 to 8, wherein the method comprises generating a plurality of barcode libraries having different barcodes.

[0258] 10. The method according to any one of embodiments 1 to 9, further comprising a purification step.

[0259] 11. The method according to embodiment 10, comprising the step of combining multiple barcode libraries before purification.

[0260] 12. The method according to any one of Examples 1 to 11 further comprises analyzing sequencing data to obtain a map of 24 chromosomes for determining the chromosome ploidy status of the embryo.

[0261] 13. The method according to any one of embodiments 1 to 11, further comprising analyzing the sequencing data to obtain X and Y chromosome maps for determining sex chromosome balance.

[0262] 14. The method of embodiment 13, further comprising calculating the copy number of chromosome X by comparing with the autosomal region.

[0263] 15. The method according to any one of embodiments 1-14, wherein the spent culture medium is collected from the blastocyst culture at day 5 to day 7.

[0264] 16. An amplification-free method for library generation for in vitro fertilization (IVF) preimplantation genetic testing (PGT), comprising

[0265] i) Providing a sample containing cell-free DNA (cfDNA) from spent culture medium from IVF embryos;

[0266] ii) repairing cfDNA and preferably obtaining single-base overhanging end fragments; and

[0267] iii) Adding directional barcode adapters to each end of the repaired cfDNA to generate a barcoded library for sequencing.

[0268] 17. A method according to Example 16, wherein repairing cfDNA includes converting 5' and / or 3' protruding ends into blunt ends, and / or repairing internal cuts.

[0269] 18. The method of embodiment 16, wherein the single base overhang end fragments include adding 3' dA overhang ends to each end of the repaired fragments.

[0270] 19. The method of embodiment 16, wherein the barcode linker is a Y linker.

[0271] 20. The method of embodiment 19, wherein the Y linker comprises a barcode in the double-stranded region.

[0272] 21. The method according to Example 20, wherein the Y linker comprises a random nucleotide sequence of 1 to 10 bases in the single-stranded region of each arm.

[0273] 22. The method according to any one of embodiments 15 to 21, further comprising the step of removing proteins bound to cfDNA, for example, treating cfDNA with a protease to separate the cfDNA fragments from the cfDNA fragments.

[0274] 23. The method according to any one of embodiments 15 to 22, wherein the method comprises generating a plurality of barcode libraries having different barcodes.

[0275] 24. The method according to any one of embodiments 15 to 23, further comprising a purification step.

[0276] 25. The method of embodiment 24, comprising the step of combining multiple barcode libraries prior to purification.

[0277] 26. The method of any one of embodiments 15-25, wherein the spent culture medium is collected from day 5 to day 7 blastocyst cultures.

[0278] 27. A method for identifying the genetic background of an in vitro fertilization (IVF) embryo, comprising:

[0279] i) Providing a sample containing cell-free DNA (cfDNA) from spent culture medium from IVF embryos;

[0280] ii) repairing cfDNA and preferably obtaining single-base overhanging end fragments; and

[0281] iii) adding directional barcode adapters to each end of the repaired cfDNA, thereby generating a barcoded library; and

[0282] iv) Determine the whole-genome heterozygous SNP profile or mitochondrial sequence by sequencing the barcoded library.

[0283] 28. A method according to Example 27, wherein repairing cfDNA includes converting 5' and / or 3' protruding ends into blunt ends, and / or repairing internal cuts.

[0284] 29. The method of embodiment 27, wherein the single base overhang end fragments include adding 3' dA overhang ends to each end of the repaired fragments.

[0285] 30. The method of embodiment 27, wherein the barcode library is sequenced by paired-end sequencing.

[0286] 31. The method of embodiment 27, wherein the barcode linker is a Y linker.

[0287] 32. The method of embodiment 31, wherein the Y linker comprises a barcode in the double-stranded region.

[0288] 33. The method according to embodiment 32, wherein the Y linker comprises a random nucleotide sequence of 1-10 bases in the single-stranded region of each arm.

[0289] 34. The method according to any one of Examples 27 to 33 further comprises a step of removing proteins bound to cfDNA, such as treating cfDNA with a protease to remove nucleosomes or heterochromatin structures from cfDNA fragments.

[0290] 35. The method of any one of embodiments 28 to 34, wherein the method comprises generating a plurality of barcode libraries having different barcodes.

[0291] 36. The method according to any one of embodiments 28 to 34, further comprising a purification step.

[0292] 37. The method of embodiment 36, comprising the step of combining multiple barcode libraries prior to purification.

[0293] 38. The method of any one of embodiments 28-38, wherein the spent culture medium is collected from day 5 to day 7 blastocyst cultures.

[0294] 39. An amplification-free method for determining the extent of non-embryonic DNA contamination in spent culture medium from IVF embryo culture, comprising:

[0295] i) generating a library by the method of any one of Examples 17-27;

[0296] ii) sequencing the library; and

[0297] iii) Calculate the ratio of mitochondrial DNA (mtDNA) to chromosomes,

[0298] A higher mtDNA to chromosome ratio indicates a lower level of non-embryonic DNA contamination, whereas a lower mtDNA to chromosome ratio indicates a higher level of non-embryonic DNA contamination.

[0299] and optionally

[0300] iv) assessing the X chromosome copy number when the embryo is a human 46,XY embryo.

[0301] 40. The method of embodiment 39, wherein the mtDNA to chromosome ratio is the ratio of mtDNA to autosomes.

[0302] 41. An amplification-free method for generating a cell-free DNA (cfDNA) library from biological material, the method comprising:

[0303] i) providing a sample containing cell-free DNA (cfDNA) from biological material;

[0304] ii) repairing cfDNA and preferably obtaining single-base overhanging end fragments; and

[0305] iii) Adding barcode adapters to each end of the repaired cfDNA to generate a barcoded library for sequencing.

[0306] 42. A method according to Example 41, wherein repairing cfDNA includes converting 5' and / or 3' protruding ends into blunt ends, and / or repairing internal cuts.

[0307] 43. The method of embodiment 41, wherein the single base overhang end fragments include adding 3' dA overhang ends to each end of the repaired fragments.

[0308] 44. The method of embodiment 41, wherein the barcode linker is a Y linker.

[0309] 45. The method of embodiment 44, wherein the Y linker comprises a barcode in the double-stranded region.

[0310] 46. ​​A method according to embodiment 44, wherein the Y linker comprises a random nucleotide sequence of 1-10 bases in the single-site region of each arm.

[0311] 47. The method according to any one of Examples 41 to 46 further comprises a step of removing proteins bound to cfDNA, for example, the cfDNA is treated with a protease to remove nucleosomes or heterochromatin structures from the cfDNA fragments.

[0312] 48. The method of any one of embodiments 41-46, wherein the method comprises generating a plurality of barcode libraries having different barcodes.

[0313] 49. The method according to any one of embodiments 41 to 47, further comprising a purification step.

[0314] 50. The method of embodiment 48, comprising the step of combining multiple barcode libraries prior to purification.

[0315] 51. A sequencing library generated by the method of any one of Examples 16-26 and 41-50.

[0316] 52. A method for sample gene detection, comprising sequencing the library of Example 51, and analyzing the sequencing data to determine a whole-genome heterozygous SNP map or mitochondrial sequence.

[0317] 53. A method for sample preparation without amplification for methylation profiling, the method comprising:

[0318] i) providing a sample containing cell-free DNA (cfDNA) from biological material;

[0319] ii) repairing cfDNA and preferably obtaining single-base overhanging end fragments; and

[0320] iii) Adding directional barcode adapters to each end of the repaired cfDNA to generate a barcoded library and avoid PCR amplification, which can disrupt methylation patterns by converting methyl-C to C.

[0321] 54. The method of embodiment 53, wherein the linker comprises a unique Y linker and carries a barcode.

[0322] 55. The method of embodiment 53 or 54, wherein barcode libraries from different samples can be combined into a single sample for preparation.

[0323] 56. A method according to any one of embodiments 53 to 55, wherein the method comprises performing paired-end sequencing on the Y-linker library fragments to provide the full-length sequence of each fragment.

[0324] 57. The method of any one of embodiments 53 to 56, wherein the Y linker can be modified to add a random sequence to each primer strand of the single-stranded region.

[0325] 58. The method of any one of Examples 53 to 57, comprising enriching the methylated portion of the combined cfDNA library, for example by specifically capturing methylated cfDNA fragments.

[0326] 59. The method according to embodiment 58, wherein specific capture is performed using antibodies specific 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.

[0327] 60. The method according to embodiment 59, wherein the antibody is selected from anti-mC monoclonal or polyclonal antibodies or anti-ds antibodies from systemic lupus erythematosus (SLE) patients.

[0328] 61. The method according to Example 59, wherein the antibody is bound to or captured by an enrichment matrix (such as magnetic beads) to effectively enrich the methylated fragments.

[0329] 62. The method of any one of embodiments 58 to 61, wherein the captured methylated DNA fragments are released from the enrichment matrix and directly sequenced.

[0330] 63. The method according to any one of embodiments 58 to 62, further comprising simply amplifying the enriched DNA fragments using primers homologous to the directional adapter sequence added prior to mDNA capture.

[0331] 64. The method of any one of embodiments 53 to 63, further comprising adding a protease, such as trypsin and proteinase K.

[0332] 65. The method of embodiment 64, wherein the protease is heat-labile proteinase K or membrane-bound trypsin.

[0333] 66. A method according to any one of embodiments 53 to 65, wherein the method further comprises sequencing the methylated DNA, for example by NGS.

[0334] 67. A method according to Example 66, wherein a sequencing primer with a Y linker is used to obtain the complete DNA sequence of the methylated DNA fragment in NGS.

[0335] 68. The method of embodiment 66 or 67, wherein NGS is performed in paired-end NGS sequencing mode.

[0336] 69. A method according to any one of embodiments 53 to 65, wherein the method further comprises sequencing the methylated DNA by nanopore sequencing, for example to obtain the complete DNA sequence of long tandemly enriched methylated DNA fragments, including bases A, G, C, T and methylated C.

[0337] 70. The method of any one of embodiments 53 to 69, wherein the method further comprises analyzing sequencing data from methylated DNA fragments.

[0338] 71. A method according to embodiment 70, wherein the method comprises analyzing NGS data to map methylation regions to chromosome bins and generate a methylation profile for analysis.

[0339] 72. A method according to embodiment 70, wherein the method comprises analyzing nanopore sequencing data to map genome-wide methylation sites for methylation analysis.

[0340] 73. An amplification-free method for examining a selected genomic region on a chromosome associated with a gene or polymorphic site, the method comprising:

[0341] i) Providing samples containing cell-free DNA (cfDNA);

[0342] ii) repairing cfDNA fragments and preferably obtaining single-base overhanging end fragments;

[0343] iii) adding directional barcode sequencing-compatible adapters to each end of the repaired cfDNA to generate a barcoded library;

[0344] iv) enriching the library for selected genomic regions; and

[0345] v) Sequencing the barcoded library.

[0346] 74. A method according to Example 73, wherein the library is enriched by selectively binding any cfDNA homologous to the selected genomic region using a hybridization capture probe.

[0347] 75. The method of embodiment 73 or 74, wherein the relative ratio of any polymorphic sites identified in the captured DNA fragments can be assessed.

[0348] 76. A method according to any one of Examples 73 to 75, wherein the cfDNA can be cell-associated but has been extracted and processed into a form compatible with library preparation and subsequent operations.

[0349] 77. A method according to any one of embodiments 73 to 76, wherein selected genomic regions can be sequenced and analyzed to detect variations, such as single base substitutions or small insertions and deletions.

[0350] 78. A method according to any one of embodiments 1 to 77, wherein the sample comprises cfDNA extracted from cells or tissues.

[0351] 79. A method according to any one of embodiments 1 to 78, wherein the cfDNA is from tissue removed from the embryo by biopsy and, optionally, processed in a manner that makes it compatible with subsequent library preparation.

[0352] Advantages of the Invention

[0353] The present disclosure provides a method in which sample preparation (eg, library generation) is simple, rapid, reliable, and accurate. In the method of the present invention, whole genome amplification is not required at least during library generation.

[0354] Example

[0355] Example 1. Analysis of embryo waste culture medium

[0356] 1.1 Sample

[0357] The spent media from blastocysts cultured to day 5 or later was collected for analysis; this media is usually discarded after embryos have completed development to the desired blastocyst stage.

[0358] 1.2 Library preparation

[0359] 10 μL of embryo waste medium was mixed with 10 μL of repair mixture: {0.8 μL T4 DNA polymerase (Hunan Yearthbio) (3U / μL), 0.4 μL rTaq (Hunan Yearthbio) (5U / μL), 2 μL 10xT4 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}, with a total volume of 20 μL. The mixture was incubated at 37°C for 20 minutes, and then incubated at 72°C for 30 minutes for end repair and "A addition".

[0360] The product was mixed with 10 μL of adapter mixture: {0.6 μL ADT-FL (1 μM Hunan Yearthbio), 1 μL T4 ligase (600U / μL), 1 μL 10xT4 ligase buffer (500mM Tris-HCl, 100mM MgCl2, 10mM ATP, 100mM DTT), PEG6000 (Sigma) (final concentration of 25-27%), water to 10 μL}, the total volume was about 30 μL. The barcode Y adapter ligation mixture was incubated at 20°C for 15 minutes to obtain the barcode library, and then heated at 65°C for 10 minutes to inactivate the ligase.

[0361] The libraries with different barcodes were then pooled and the modified cfDNA libraries were purified using 0.8X Agencourt AMPure magnetic beads following the manufacturer’s instructions. After washing with 80% fresh ethanol to remove unused reagents and the Y linker, the modified cfDNA was eluted from the beads using 20-40 μL DB pH 8.

[0362] 1.3 Sequencing

[0363] The combination of barcoded libraries was sequenced on an NGS platform (Illumina NovaSeq, Applied Biosystems S5, BGI MGI-T7).

[0364] To obtain relative sequencing reads for each library, the libraries were pooled and purified. The pooled libraries were adjusted to 10 pmol / μL in resuspension buffer (RSB) and injected into the Illumina NovaSeq flow cell. Sequencing was performed in 2x150 paired-end mode.

[0365] 1.4 Reading Processing

[0366] Reads were processed using Redis (for data storage), Burrow-Wheeler-Aligner (for mapping), fastq count (for raw data QC), GCcorrect (for GC correction), CNVcalling.R (for computational mapping), or their derivatives.

[0367] Procedure 2. Size Analysis of Native cfDNA in Embryo Waste Medium

[0368] The size of native cfDNA in embryonic waste media was analyzed by paired-end sequencing of the libraries followed by alignment to a reference genome and estimation of fragment size.

[0369] like Figure 2 As shown, a major peak of approximately 170 bp fragment was consistently observed in all four cfDNA samples from embryo culture media, while minor obvious multimer peaks of approximately 340 bp and approximately 510 bp were also observed.

[0370] It should be noted that the major population of cfDNA molecules with a length of approximately 170 bp or less is not easily amplified by current PCR-based WGA methods.

[0371] Example 3. Copy number analysis

[0372] Paired-end sequencing reads were size-classified after alignment with the reference genome and divided into two size fractions: <200bp and >200bp. The reads were mapped to the human genome and the results are shown in the example of chromosome 21, chromosome 22, and chromosome X (46,XX sample). Figure 3 Since the p arms of chromosomes 21 and 22 consist only of repetitive DNA structures, their fragments could not be mapped.

[0373] Copy number results were similar for both size fractions.

[0374] Example 4. Determination of sex chromosome balance

[0375] Sex chromosome balance is determined by the copy number of the X chromosome.

[0376] Figure 4 A shows the characterization of a 46,XY sample, indicating that the embryo contains two copies of each autosome and one copy of the X chromosome.

[0377] Figure 4 B shows the characterization of a 46,XX sample, indicating that the embryo contains two copies of each autosome and the X chromosome.

[0378] Figure 4 C shows the Y chromosome-specific sequences identified from the 46,XY and 46,XX samples, indicating that reads corresponding to the Y chromosome are present in the data from the 46,XY sample but absent from the data from the 46,XX sample. This figure shows segments that map only to the Y chromosome. Likewise, other segments that map to other autosomes are specific to that chromosome. Sequences shared across chromosomes were removed in the initial analysis because they could not be assigned to a single location.

[0379] Example 5. Detection of euploid and aneuploid embryos

[0380] Analyze sequencing data to detect euploidy or aneuploidy of embryos. Because chromosomes exist in one copy, two copies or more copies, and Y chromosomes are 0 copies in women. Because autosomes are usually two copies, sex chromosomes can be compared with autosomes and give a relative copy number of 1 or 2 (Y chromosomes are 0 in women).

[0381] Figure 5 Maps of 24 chromosomes are shown. Figure 5 A and 5B show the results from euploid embryos 46,XY and euploid embryos, respectively, while Figure 5 C shows the results from an aneuploid embryo, where the reads corresponding to chromosome 15 show a copy number of 1, indicating that only one chromosome 15 is present in the embryo.

[0382] Example 6. Prediction of non-embryonic DNA contamination by calculating the mtDNA:autosomal ratio

[0383] Mitochondrial DNA (mtDNA) levels (48 million base pairs or less total) are typically only 1% or less of the cellular DNA content. Comparing a large number to a very small number may ultimately be insensitive. Since each chromosome varies in size, ranging from the largest at 250 million base pairs to the smallest at about 60 million base pairs, it may be more sensitive to compare mtDNA to chromosomes that are relatively closer in DNA content. Not necessarily to a single chromosome, as multiple independent comparisons may yield a more stable estimate.

[0384] Therefore, relative mtDNA reads are compared to autosomal reads (all or selected chromosomes) to predict non-embryonic DNA contamination.

[0385] Example 7. Mitochondrial DNA Analysis

[0386] The maternal mitochondrial DNA data was used as a reference.

[0387] Figure 8 A shows the mapping and read coverage of mitochondrial DNA sequences. Mitochondrial DNA reads are aligned to the mitochondrial DNA reference genome. The number of reads at any position indicates the depth and copy number of mitochondrial DNA relative to autosomal fragments.

[0388] Figure 8 B shows the differentiation of embryos from different patient cohorts by mitochondrial DNA polymorphisms. In particular, in one set of embryos, embryos 2, 5, and 9 showed the same polymorphisms as the mitochondrial DNA reads of patient A and were therefore determined to belong to the same cohort. In another set of embryos, embryos 1 and 5 showed the same polymorphisms as the mitochondrial DNA reads of patient B and were therefore determined to belong to the same cohort.

[0389] Example 8. Enrichment of methylated DNA fragments

[0390] 8.1 Sample

[0391] Samples included human genomic DNA and cfDNA isolated from the apheresis cell fraction of blood.

[0392] Genomic DNA was isolated and purified according to standard protocols. A portion of the DNA was sonicated to produce an average fragment size of approximately 200 bp. Sources included individuals with known malignancies and individuals without any known underlying disease.

[0393] cfDNA was prepared from the serum / plasma portion of the patient's blood according to standard protocols. The patients included those with known colorectal cancer and those without known malignancies.

[0394] 8.2 Library preparation

[0395] Use 160 ng of sheared genomic DNA or cfDNA purified from 1 ml of plasma (approximately 10-20 ng) to prepare the library as follows:

[0396] 10 μL of embryo waste medium was mixed with 10 μL of repair mixture: {0.8 μL T4 DNA polymerase (Hunan Yearthbio) (3U / μL), 0.4 μL rTaq (Hunan Yearthbio) (5U / μL), 2 μL 10xT4 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}, with a total volume of 20 μL. The mixture was incubated at 37°C for 20 minutes, and then incubated at 72°C for 30 minutes for end repair and "A addition". The product was mixed with 10 μL of adapter mixture: {0.6 μL ADT-FL (1 μM Hunan Yearthbio), 1 μL T4 ligase (600 U / μL), 1 μL 10xT4 ligase buffer (500 mM Tris-HCl, 100 mM MgCl2, 10 mM ATP, 100 mM DTT), PEG6000 (Sigma) (final concentration of 25-27%), water to 10 μL}, the total volume was about 30 μL. The barcode Y adapter ligation mixture was incubated at 20°C for 15 minutes to obtain the barcode library, and then heated at 65°C for 10 minutes to inactivate the ligase.

[0397] 8.3 Methylated DNA Enrichment

[0398] The enriched mDNA fraction was prepared using the Methylated-DNA IP Kit (Zymo Research, catalog number D5101) according to the manufacturer's instructions.

[0399] Basically: Mix the library preparation fragments from 8.2 with DNA denaturation buffer in a total volume of 50 μL. Heat it at 98°C for 5 minutes. Sequentially, prepare 250 μL MIP buffer by adding 15 μL ZymoMag Protein A and 0.8 μL anti-methylcytosine antibody. Add the denatured DNA and incubate the mixture at 37°C for 0.5-1 hour. Separate the beads on a magnetic stand and discard the supernatant. Wash the beads twice with 500 μL MIP buffer. Elute the DNA with 500 μL DNA elution buffer. Incubate the tube at 75°C for 5 minutes and then centrifuge in a microcentrifuge for 2 minutes. The enriched DNA is in the supernatant fraction.

[0400] 8.4 Sequencing

[0401] The combination of barcoded libraries was sequenced on an NGS platform (Illumina NovaSeq, Applied Biosystems S5, BGI MGI-T7).

[0402] 8.5 Reading Processing

[0403] Reads were processed using Redis (for data storage), Burrow-Wheeler-Aligner (for mapping), fastq count (for raw data QC), GCcorrect (for GC correction), CNVcalling.R (for computational mapping), or their derivatives.

[0404] The results are as follows Fig. 9 and 10 In particular, Fig. 9 Shown is a comparison of fragments captured in three different samples at a randomly selected region of chromosome 4. This demonstrates the overall reliability of the procedure across different samples. Fig.10 Similar and divergent regions of mDNA profiles from different samples are shown, demonstrating the usefulness of this method for examining methylation commonalities and differences between samples.

[0405] Example 9. Detection and quantification of minor alleles

[0406] 9.1 Sample

[0407] Samples include:

[0408] Plasma from women and plasma from women carrying a known male fetus.

[0409] Male plasma cfDNA was prepared from the serum / plasma fraction of blood according to standard protocols.

[0410] 9.2 Library Preparation

[0411] Purify cfDNA (approximately 10-20 ng) from 1 ml of plasma and prepare the library as follows:

[0412] 10 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 10xT4 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 volume 20 μL). The mixture was incubated at 37°C for 20 minutes and then incubated at 72°C for 30 minutes for end repair and "A addition".

[0413] The product was mixed with 0.6 μL ADT-FL (1 μU), 1 μL T4 ligase (600 U / μL), 8.7 μL Buffer 2 (1 μL 10xT4 ligase buffer (500 mM Tris-HCl, 100 mM MgCl2, 10 mM ATP, 100 mM DTT), PEG6000 (Sigma) (final concentration in the ligation mixture was 6%, add water to 8.7 μL). The mixture was incubated at 20° C. for 15 minutes to ligate the barcode Y linker to obtain a barcode library, and then incubated at 65° C. for 10 minutes to inactivate the ligase.

[0414] 9.3 Polymorphic SNP Enrichment

[0415] SNP enrichment was performed using the cfDNA Library NanoID Panel Capture Kit according to the manufacturer's instructions.

[0416] Briefly, the purified cfDNA library was dried after adding 5 μL human Cot DNA and 2 μL NadPrep Nano Blockers. The dried probes were redissolved in a hybridization mix containing 8.5 μL Hyb#1, 2.7 μL Hyb#2, and 6 μL NanoIDPanel Probe. After denaturation at 95°C for 30 seconds, the mixture was hybridized at 65°C overnight. Streptavidin magnetic beads (50 μL) were prepared according to the manufacturer's instructions and added to the hybridization mix. It was kept at 65°C for 45 minutes with intermediate mixing every 10-12 minutes. 100 μL Wash Buffer 1 was added to the library / magnetic beads mixture and the magnetic beads were separated on a magnetic stand. The beads were washed twice with Wash Buffer at 65°C for 5 minutes each, once in Wash Buffer 1 (2 minutes at room temperature), once in Wash Buffer 2 (2 minutes at room temperature), and once in Wash Buffer 3 (2 minutes at room temperature), and then resuspended in 23 μL H2O. The bead mixture was added to a PCR reaction mixture containing 25 μL HiFi HotStart ReadyMix, 2 μL P5+P7 Primer mix (25 μM), and cycled 1x98°C x 45 seconds; 15x (98°C x 15 seconds / 60°C x 30 seconds / 72°C x 30 seconds); 1x72°C x 1 minute. The library was then purified using 54 μL VAHTSDNAClean Beads according to the manufacturer's instructions.

[0417] 9.4 Sequencing

[0418] The combination of barcoded libraries was sequenced on an NGS platform (Illumina NovaSeq, Applied Biosystems S5, BGI MGI-T7).

[0419] 9.5 Reading Processing

[0420] Reads were processed using Redis (for data storage), Burrow-Wheeler-Aligner (for mapping), fastq count (for raw data QC), GCcorrect (for GC correction), CNVcalling.R (for computational mapping), or their derivatives.

[0421] The results are as follows Fig.11 In particular, Fig.11 The SNP profile of the minor allele in a mixed genetic sample is shown, demonstrating its utility in implantation monitoring, pregnancy monitoring, or any other application where different genetic contributions are present and relative amount estimates may be a useful analysis.

Claims

1. A method for analyzing cell-free DNA (cfDNA) in a biological sample without amplification, comprising: i) Providing samples containing cell-free DNA (cfDNA); ii) repairing cfDNA fragments to obtain single-base overhanging end fragments; iii) adding barcode adapters to each end of the repaired cfDNA to generate a barcoded library for sequencing; and iv) The barcoded library is sequenced or processed through further enrichment steps prior to sequencing.

2. An amplification-free method for preimplantation genetic testing (PGT) of in vitro fertilization (IVF) embryos, comprising: i) Providing a sample containing cell-free DNA (cfDNA) from spent culture medium from IVF embryos; ii) repairing cfDNA fragments to obtain blunt-ended fragments; iii) adding barcode adapters to each end of the repaired cfDNA to generate a barcoded library for sequencing; and iv) Sequencing the barcoded library.

3. The method according to claim 1 or 2, wherein repairing cfDNA comprises converting 5' and / or 3' protruding ends into blunt ends, and / or repairing internal cuts.

4. The method according to claim 1 or 2, wherein the single base overhanging end fragments comprises adding 3' dA overhanging ends to each end of the repaired fragments.

5. The method according to claim 1 or 2, wherein the barcode library is sequenced by paired-end sequencing.

6. The method according to claim 1 or 2, wherein the barcode adapter is a Y adapter.

7. The method according to claim 6, wherein the Y linker comprises a barcode in the double-stranded region.

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

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

10. The method according to any one of claims 1 to 9, wherein the method comprises generating a plurality of barcode libraries having different barcodes.

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

12. The method according to claim 11, further comprising the step of combining multiple barcode libraries prior to purification.

13. The method according to any one of claims 1 to 12 further comprises analyzing sequencing data to obtain a 24 chromosome map for determining the chromosome ploidy state of the embryo.

14. The method according to any one of claims 1 to 12, further comprising analyzing the sequencing data to obtain X and Y chromosome maps for determining sex chromosome balance.

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

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

17. An amplification-free method for library generation for in vitro fertilization (IVF) preimplantation genetic testing (PGT), comprising: i) Providing a sample containing cell-free DNA (cfDNA) from spent culture medium from IVF embryos; ii) repairing cfDNA to obtain blunt-ended fragments; and iii) Adding barcode adapters to each end of the repaired cfDNA to generate a barcoded library for sequencing.

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

19. The method of claim 15, wherein the single base overhanging end fragments comprises adding 3' dA overhangs to each end of the repaired fragments.

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

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

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

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

24. The method according to any one of claims 17 to 23, wherein the method comprises generating a plurality of barcode libraries having different barcodes.

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

26. A method according to claim 25, comprising the step of combining multiple barcode libraries prior to purification.

27. The method according to any one of claims 17 to 26, wherein the spent culture 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: i) Providing a sample containing cell-free DNA (cfDNA) from spent culture medium from IVF embryos; ii) repairing cfDNA to obtain blunt-ended fragments; iii) adding barcode adapters to each end of the repaired cfDNA to generate a barcoded library; and iv) Determine the whole-genome heterozygous SNP profile or mitochondrial sequence by sequencing the barcoded library.

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

30. The method of claim 28, wherein the single base overhanging end fragments comprises adding 3' dA overhangs to each end of the repaired fragments.

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

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

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

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

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

36. A method according to any one of claims 28 to 34, wherein the method comprises generating a plurality of barcode libraries having different barcodes.

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

38. A method according to claim 36, comprising the step of combining multiple barcode libraries prior to purification.

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

40. An amplification-free method for determining the extent of non-embryonic DNA contamination in spent culture medium from IVF embryo culture, comprising: i) generating a library by the method of any one of claims 17 to 27; ii) sequencing the library; and iii) Calculate the ratio of mitochondrial DNA (mtDNA) to chromosomes; Among them, a higher mtDNA to chromosome ratio indicates a lower level of non-embryonic DNA contamination, while a lower mtDNA to chromosome ratio indicates a higher level of non-embryonic DNA contamination.

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

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

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

44. The method of claim 42, wherein the single base overhanging end fragments comprises adding 3' dA overhangs to each end of the repaired fragments.

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

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

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

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

49. The method according to any one of claims 42 to 48, wherein the method comprises generating a plurality of barcode libraries having different barcodes.

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

51. A method according to claim 50, comprising the step of combining multiple barcode libraries prior to purification.

52. An amplification-free method for sample preparation for methylation profiling, the method comprising: i) providing a sample containing cell-free DNA (cfDNA) from biological material; ii) repairing cfDNA to obtain single-base overhanging end fragments; and iii) Adding directional barcode adapters to each end of the repaired cfDNA to generate barcoded libraries and avoid PCR amplification, which disrupts methylation patterns by converting methyl-C to C.

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

54. The method according to claim 52 or 53, further comprising enriching the methylated portion of the library.

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

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

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