Genetic integrated single molecule sequencing detection kit and system before embryo implantation

Abstract

The invention belongs to the field of genetics detection, and particularly relates to a genetics-integrated single-molecule sequencing detection kit and system before embryo implantation. Specifically, the kit and the system provided by the invention can realize synchronous detection of human pre-implantation embryo aneuploid, copy number variation, chromosome structure abnormality and monogenic diseases. According to the method, the parent sample and the embryo sample are subjected to single molecule length reading sequencing, family members except a certificate and parents are not needed, direct detection of aneuploidy, copy number variation, chromosome structure abnormality and the like and indirect detection based on haplotype inference are achieved, the detection result is accurate, sensitive and visual, and information is comprehensive. According to the kit and the system disclosed by the invention, only the same experimental system and the same single-molecule long-fragment sequencing platform are needed, so that PGT-A, PGT-M, PGT-SR and haplotype genetic condition detection can be completely supported, the process is simple and convenient, the application range is wide, and therefore, the kit and the system have a good practical application value.

Description

Technical Field

[0001] This invention belongs to the field of genetic testing, specifically relating to a single-molecule sequencing test kit and system for preimplantation genetic testing. Background Technology

[0002] Preimplantation genetic testing (PGT) is a type of genetic testing performed on embryos fertilized in vitro during assisted reproductive technology (ART). Its purpose is to select embryos for implantation into the mother's uterus from a genetic perspective, avoiding those carrying genetic variations that could lead to implantation failure, miscarriage, or birth defects. PGT is generally divided into three categories: First, preimplantation genetic testing for aneuploidy (PGT-A), which detects both aneuploidy and copy number variants (CNVs) in the embryo. Second, preimplantation genetic testing for monogenic diseases (PGT-M), primarily targeting cases where the parents carry specific gene mutations. Third, preimplantation genetic testing for structural rearrangements (PGT-SR), which detects chromosomal rearrangements in the parents, such as inversions, balanced translocations, Robertsonian translocations, and insertional translocations.

[0003] PGT technology first requires obtaining detectable genetic material through biopsy. Since blastocyst biopsies only yield a few cells with limited genetic material, whole genome amplification (WGA) is generally performed before PGT. Several WGA methods exist, among which MDA (multiple displacement amplification) is widely used due to its simplicity, high yield, good coverage, and high fidelity. MDA-based WGA products are relatively long, possessing the potential to be adapted to single-molecule long-read sequencing platforms. However, due to the complex branched structure of MDA products, the sequencing yield is extremely low when constructing single-molecule long-read libraries using standard procedures, limiting the application of single-molecule long-read sequencing technology in embryo biopsy samples.

[0004] Haplotype linkage analysis is required when performing PGT-M and PGT-SR. There are two main reasons for this: First, WGA inevitably contains amplification bias, allele dropout (ADO), and the possibility of introducing amplification errors, thus direct detection of embryos carries the risk of false positives and false negatives. Second, for certain genetic variations, such as those occurring in highly homologous regions or structural variations like balanced translocations, current techniques are insufficient for direct detection based on embryonic WGA DNA; indirect inference based on linkage relationships is necessary. Currently, there are two main types of linkage detection methods: short tandem repeat (STR) analysis and SNP (single-nucleotide polymorphism) analysis based on microarrays and next-generation sequencing (NGS) technologies. These methods are limited to detecting Indels, SNPs, etc., and do not directly provide haplotype information. When constructing linkage relationships, they rely on genetic pedigree analysis, which requires testing the proband or two generations of family members in addition to the embryo and parents. They are also difficult to detect new variants in parents or homologous recombination near the target region of the embryo.

[0005] To overcome the aforementioned limitations, a series of PGT (Programmable Genetic Testing) protocols based on single-molecule long-read sequencing (also known as "third-generation sequencing") have been developed in recent years. Their main characteristics are: high-throughput single-molecule long-read sequencing is performed on carriers of single-gene diseases or structural variations in the parents, simultaneously obtaining the genotype and haplotype of the target detection region; for embryos, NGS or microarrays are used to provide SNP information during PGT-A for linkage analysis; and the genetic information of the embryo is derived from linkage analysis. This type of method overcomes the dependence on probands and maternal family members and also has the ability to detect novel variants. However, embryo detection still remains at the NGS or microarray level, meaning it is still largely dependent on indirect inference based on linkage relationships and cannot comprehensively achieve direct detection of embryonic genotypes and haplotypes. Furthermore, these methods require the integration of multiple technology platforms, such as "third-generation sequencing + second-generation sequencing" or "third-generation sequencing + microarray," involving platform switching, numerous procedures, and high requirements for specialized equipment, thus limiting application scenarios.

[0006] In summary, existing PGT solutions have the following limitations: 1. Limited direct detection capability: Existing methods have weak direct detection capability for embryos carrying variants (especially complex genes PGT-M and PGT-SR), and sometimes personalized detection methods are required, which limits the detection accuracy and applicability. 2. Linkage analysis based on independent loci: The PGT protocol, which indirectly infers linkage relationships using Indel and SNP, does not directly obtain embryo haplotype information. Its detection capability decreases when there is no proband, incomplete family lineage, or a small number of embryos. Furthermore, some methods are unable to detect embryos with new variants in parents and homologous recombination near the target region, thus limiting its application scope. 3. Complex process: The existing process for PGT-A, PGT-M, PGT-SR and linkage analysis often requires separate independent testing, involving different technology platform combinations, which is cumbersome, requires a lot of specialized equipment, and has a long cycle, thus limiting clinical use. Summary of the Invention

[0007] To overcome the shortcomings of the existing technologies, this invention provides a kit and system for integrated preimplantation genetic testing based on long-read single-molecule sequencing, enabling simultaneous detection of aneuploidy, copy number variations, chromosomal structural abnormalities, and single-gene diseases in human preimplantation embryos. This invention performs single-molecule long-read sequencing on both parental and embryonic samples, eliminating the need for probands and family members other than parents. It achieves direct detection of aneuploidy, copy number variations, and chromosomal structural abnormalities, as well as indirect detection based on haplotype inference. The results are accurate, sensitive, intuitive, and comprehensive. According to the kit and system of this invention, only the same experimental system and the same single-molecule long-fragment sequencing platform are required to fully support the detection of PGT-A, PGT-M, PGT-SR, and haplotype genetic status, simplifying the process and broadening its applicability.

[0008] In particular, as mentioned above, the WGA products generated by the MDA method have a complex branched structure, making them unsuitable for constructing single-molecule long-read libraries and resulting in extremely low sequencing yields. However, the kit and system provided by this invention enable the WGA products generated by the MDA method to be used for the construction of single-molecule long-fragment sequencing libraries and sequencing output, which is beneficial for the application of long-fragment single-molecule sequencing technology in clinical testing.

[0009] Therefore, in a first aspect, the present invention provides a kit for integrated preimplantation genetic testing of embryos based on long-read single-molecule sequencing, the kit comprising the following reagents: (1) Reagents used for whole genome amplification of embryo samples; (2) Reagents used to construct long-fragment sequencing libraries of parental genome samples; (3) Reagents used to construct long-fragment sequencing libraries of embryonic samples; The reagents used to construct long-fragment sequencing libraries of embryonic samples contain at least an endonuclease.

[0010] In some embodiments, the endonuclease is T7 endonuclease I (abbreviated as T7 endonuclease I) and / or FEN1.

[0011] In some embodiments, the reagents for whole-genome amplification of embryonic samples include whole-genome amplification buffer, random primers, and whole-genome amplification DNA polymerase.

[0012] In some implementations, the reagents used to construct long-fragment sequencing libraries of parental genomes include DNA fragmentation tubes, repair buffers, DNA repair enzymes, DNA end repair enzymes, ligation buffers, sequencing adapters, ligases, exonucleases, and DNA purification magnetic beads.

[0013] In some implementations, the reagents used to construct long-fragment sequencing libraries of embryonic sample genomes further include amplification adapters, amplification primers, DNA polymerase, amplification buffer, repair buffer, DNA repair enzyme, DNA end repair enzyme, ligation buffer, sequencing adapters, ligase, exonuclease, and DNA purification magnetic beads.

[0014] In some implementations, the parental sample comprises peripheral blood or tissue of parental origin.

[0015] In some embodiments, the embryo sample is a biopsy sample obtained from an embryo at the blastocyst or blastocyst stage.

[0016] PGT technology, as is known in the art, first requires obtaining the genetic material of the embryo, commonly through embryo biopsy at the blastocyst or blastocyst stage, such as blastocyst biopsy or trophoblast cells of the blastocyst. Specifically, for example, the 5th embryo after in vitro fertilization... After 6 days, when the embryo develops to the blastocyst stage, there are approximately 100 embryos. 150 cells can yield 5-10 blastocyst trophoblast cells for genetic testing.

[0017] In some implementations, the single-molecule sequencing is based on a single-molecule real-time sequencing or nanopore technology platform.

[0018] In some implementations, the kit is used for integrated preimplantation genetic testing based on long-read single-molecule sequencing of parental and embryo samples.

[0019] In some implementations, the integrated preimplantation genetic testing includes simultaneous testing of PGT-A, PGT-M, and PGT-SR.

[0020] In some implementations, the integrated preimplantation genetic testing includes haplotype construction and linkage analysis, as well as simultaneous detection of aneuploidy, copy number variation, single nucleotide variation, deletion, insertion, and chromosomal structural variation.

[0021] On the other hand, the present invention provides a system for integrated preimplantation genetic testing of embryos based on long-read single-molecule sequencing, which includes the following modules: (1) Whole genome amplification module: used to obtain the whole genome amplification products of embryo samples; (2) Preprocessing module: used to convert the whole genome amplification product into a preprocessing product suitable for the amplification module; (3) Amplification module: used to amplify the preprocessed product and convert it into an amplified product suitable for constructing a long fragment single molecule sequencing library; (4) Fragmentation module: used to obtain the genome fragmentation products of the parental sample; (5) Library construction module: used to construct long-fragment single-molecule sequencing libraries; (6) Sequencing module: used for high-throughput long-read single-molecule sequencing; The preprocessing module uses an endonuclease to convert the whole genome amplification product into a preprocessing product suitable for the amplification module.

[0022] In some embodiments, the endonuclease is T7 endonuclease I and / or FEN1.

[0023] In some implementations, the whole genome amplification module uses a reagent comprising a reaction buffer, random primers, and DNA polymerase to obtain the whole genome amplification product of the embryo sample.

[0024] In some implementations, the amplification module uses reagents comprising amplification adapters, amplification primers, and DNA polymerase to amplify the pretreated product and convert it into an amplified product suitable for constructing long-fragment single-molecule sequencing libraries.

[0025] In some implementations, the fragmentation module uses a reagent containing a DNA breaking tube to obtain a genome fragmentation product from the parent sample.

[0026] In some implementations, the library construction module uses reagents comprising reaction buffer, DNA repair enzyme, DNA end repair enzyme, sequencing adapter, ligase, exonuclease and DNA purification magnetic beads to construct long-fragment single-molecule sequencing libraries.

[0027] In some implementations, the sequencing module uses a single-molecule real-time sequencing or nanopore technology platform to perform high-throughput long-read single-molecule sequencing.

[0028] In some implementations, the parental sample comprises peripheral blood or tissue of parental origin.

[0029] In some embodiments, the embryo sample is a biopsy sample obtained from an embryo at the blastocyst or blastocyst stage.

[0030] In some implementations, the system is used for integrated preimplantation genetic testing based on long-read single-molecule sequencing of parental and embryo samples.

[0031] In some implementations, the integrated preimplantation genetic testing includes simultaneous testing of PGT-A, PGT-M, and PGT-SR.

[0032] In some implementations, the integrated preimplantation genetic testing includes haplotype construction and linkage analysis, as well as simultaneous detection of aneuploidy, copy number variation, single nucleotide variation, deletion, insertion, and chromosomal structural variation.

[0033] In some embodiments, the kits and systems according to the present invention can simultaneously support at least the following detections: chromosomal aneuploidy and copy number variations >4 Mb in size across the entire genome of the parent and embryo; single nucleotide variants, deletions, and insertions across the entire genome of the parent and embryo; inversions, unbalanced or balanced translocations (including balanced translocation-carrying embryos), and insertional translocations across the entire genome of the parent and embryo; and haplotype linkages across the entire genome of the parent and embryo.

[0034] In some embodiments, the DNA polymerase for the whole-genome amplification module is selected from Phi 29 DNA polymerase. In some embodiments, the DNA repair enzyme is selected from PreCR DNA Repair mix (NEB, M0309), NEBNext FFPE DNA Repair Mix (NEB, M6630), NEBNext FFPE DNA Repair v2 Module (NEB, E7360), and combinations thereof. In some embodiments, the DNA end repair enzyme is selected from T4 DNA polymerase, Klenow Fragment, ER / A-TailingEnzyme Mix (Qiagen, Y9420), NEBNext Ultra II End Repair / dA-Tailing Module (NEB, E7546), and combinations thereof. In some embodiments, the ligase is selected from T4 DNA ligase or NEBNext Ultra II Ligation Module (NEB, E7595). In some embodiments, the DNA polymerase used in the amplification module is selected from repliQa HiFi ToughMix (Quantabio, 95200), KOD Xtreme hot-start polymerase (Merck, 71975), KOD FX (TOYOBO, KFX-101), or KOD-Multi&Epi- (TOYOBO, KME-101). In some embodiments, the exonuclease is selected from exonuclease I, exonuclease III, exonuclease VII, T7 exonuclease, and combinations thereof. In some embodiments, the DNA purification magnetic beads are selected from AMPure XP beads (Beckman Coulter, A63882), AMPure PB Beads (PacBio, 100-265-900), SMRTbell cleanup beads (PacBio, 102-158-300), and combinations thereof. The specific configuration of the reaction system and reaction conditions depends on the specific reagents used.

[0035] In some implementations, the ligation of amplification adapters in the amplification module and sequencing adapters in the library construction module can be achieved using blunt-end ligation or TA ligation. For TA ligation, T4 polynucleotide kinase, Klenow fragment (3'-5' exo-), or combinations thereof, can be used for A-tail addition. The specific configuration of the reaction system and reaction conditions depends on the specific reagents used.

[0036] In one specific implementation, the pair of oligonucleotide sequences involved in the blunt-end amplification adapter are as follows, and the adapter is formed by annealing: 5'-pAATGATACGGCGACCACCGAGCTCTTCCGATC-3' (SEQ ID NO: 1); 5'-pGATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG-3' (SEQ ID NO: 2).

[0037] In one specific implementation, the pair of oligonucleotide sequences involved in the TA-ligation amplification adapter are as follows, and the adapter is formed by annealing: 5'-pAATGATACGGCGACCACCGAGCTCTTCCGATCT-3' (SEQ ID NO: 3); 5'-pGATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG-3' (SEQ ID NO: 2).

[0038] In one specific implementation, the pair of oligonucleotide sequences involved in the amplification primers in the amplification module are as follows: 5'-pAATGATACGGCGACCACCGA-3' (SEQ ID NO: 4); 5'-pCAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 5).

[0039] In some implementations, single-molecule long fragment sequencing is selected from Pacific Biosciences' (PacBio) single-molecule real-time (SMRT) sequencing platform or Oxford Nanopore Technologies' (ONT) nanopore sequencing platform.

[0040] In some implementations, the library construction module can be selected according to the construction method corresponding to the SMRT sequencing platform or the nanopore sequencing platform, and the resulting SMRT sequencing library or nanopore sequencing library is matched with the SMRT sequencing platform provided by PacBio or the nanopore sequencing platform provided by ONT.

[0041] In some implementations, sequencing library adapter ligation can be performed using blunt-end ligation or TA ligation.

[0042] In one specific implementation, the universal blunt-ended adapter sequence for SMRT is as follows: 5'-pATCTCTCTCTTTTCCTCCTCCTCCGTTGTTGTTGTTGAGAGAGAT-3' (SEQ ID NO: 6), which, through annealing, forms a blunt-ended stem-loop adapter aptamer. Different DNA sequences of 5-50 nt can be added to the stem to form different barcode-bearing adapter aptamers. Libraries with different barcodes can be mixed for sequencing.

[0043] In one specific implementation, the universal TA adapter sequence for SMRT is as follows: 5'-pATCTCTCTCTTTTCCTCCTCCTCCGTTGTTGTTGTTGAGAGAGATT-3' (SEQ ID NO: 7), which forms a suspended T-terminal stem-loop adapter aptamer through annealing. Different DNA sequences of 5-50 nt can be added to the stem to form different barcode-bearing adapter aptamers. Libraries with different barcodes can be mixed for sequencing.

[0044] In one specific implementation, the sequencing adapter may or may not carry a barcode. Preferably, the sequencing adapter may carry a barcode designed by PacBio (or ONT) or a custom-designed barcode, which may be selected by those skilled in the art as needed.

[0045] The superior technical effects of the reagent kit and method described in this invention are mainly in the following aspects: (1) The same platform meets multiple PGT requirements. The present invention can simultaneously support PGT-A, PGT-M, PGT-SR and related chain analyses within one system, reducing the time and equipment costs caused by the need to perform multiple independent tests.

[0046] (2) Single-molecule long-read sequencing of embryos provides strong direct detection capabilities. This invention overcomes the problem of low sequencing output when constructing single-molecule long-read libraries using embryo MDA DNA products according to the general procedure, and realizes single-molecule whole-genome sequencing of embryos, enabling direct and comprehensive detection of genetic variations in embryos. In terms of PGT-M, it has excellent detection capabilities for conventional and even single-gene diseases containing pseudogenes and homologous regions. In terms of PGT-SR, it can directly detect chromosomal structural variation break sites in embryos and can distinguish between normal embryos and balanced translocation carrier embryos.

[0047] (3) High efficiency in genetic analysis of variant sites. It can directly detect haplotypes of parents and embryos, and intuitively and accurately determine the genetic status of variants through haplotype analysis. Attached Figure Description

[0048] Figure 1It is an integrated preimplantation genetic testing process based on single-molecule sequencing.

[0049] Figure 2 This is the fragment size distribution of MDA DNA after being processed in five different ways.

[0050] Figure 3 These are schematic diagrams illustrating the mechanisms of action of different treatment methods; among them, (A) illustrates the mechanism of action of MDA DNA by mechanical fragmentation or transposase fragmentation, (B) illustrates the mechanism of action of MDA DNA by T7 endonuclease I treatment, and (C) illustrates the mechanism of action of MDA DNA by endonuclease FEN1 treatment.

[0051] Figure 4 yes FBN1 The maternal MLPA test results of the E1-2 missing family PGT-M control results.

[0052] Figure 5 yes FBN1 Linkage analysis of parental and embryonic SNPs in E1-2 deletion families with PGT-M control results, M1 being... FBN1 E1-2 is missing linked SNP.

[0053] Figure 6 yes FBN1 E1-2 missing family pedigree utilizes the PGT-A results of the present invention; wherein, (A) is FBN1 The results of PGT-A using the present invention for embryos from the E1-2 missing family P001E1 are (B) FBN1 The results of the PGT-A experiment using the E1-2 deletion family P001E2 embryos of the present invention, (C) are: FBN1 The results of PGT-A of the present invention were obtained from the E1-2 missing family P001E3 embryo.

[0054] Figure 7 yes FBN1 The results of PGT-A comparison in E1-2 missing families using NGS; where (A) is FBN1 The result of PGT-A using NGS on P001E1 embryos from the E1-2 deletion family is (B). FBN1 The result of PGT-A using NGS on P001E2 embryos from the E1-2 deletion family, (C) is... FBN1 Results of PGT-A analysis using NGS on P001E3 embryos from the E1-2 deletion family.

[0055] Figure 8 The maternal Ribbon diagram t(x;19)(p22.31;p13.1) represents the equilibrium translocation breakpoint in the representative PGT-SR family pedigree test results.

[0056] Figure 9 The results are PGT-SR control results for the t(x;19)(p22.31;p13.1) family; where (A) is the result of chromosome 19 in the PGT-SR control results for the t(x;19)(p22.31;p13.1) family, and (B) is the result of chromosome X in the PGT-SR control results for the t(x;19)(p22.31;p13.1) family.

[0057] Figure 10 The results are as follows: (A) is the PGT-A result of the t(x;19)(p22.31;p13.1) family embryo P002E1 using the present invention; (B) is the copy number of chromosome 19 in the t(x;19)(p22.31;p13.1) family embryo P002E1 using the present invention; and (C) is the copy number of chromosome X in the t(x;19)(p22.31;p13.1) family embryo P002E1 using the present invention.

[0058] Figure 11 The results are embryo testing results for the t(x;19)(p22.31;p13.1) family; among which, (A) is the result of embryo P002E2 of the t(x;19)(p22.31;p13.1) family using the PGT-A of the present invention, and (B) is the result of embryo P002E3 of the t(x;19)(p22.31;p13.1) family using the PGT-A of the present invention.

[0059] Figure 12 The results are the PGT-A control results for the t(x;19)(p22.31;p13.1) family; among them, (A) is the result of PGT-A performed on the P002E1 embryo of the t(x;19)(p22.31;p13.1) family using NGS, (B) is the result of PGT-A performed on the P002E2 embryo of the t(x;19)(p22.31;p13.1) family using NGS, and (C) is the result of PGT-A performed on the P002E3 embryo of the t(x;19)(p22.31;p13.1) family using NGS. Detailed Implementation

[0060] The exemplary embodiments of the present invention described in detail are not intended to limit the invention, but rather to provide a more detailed description of certain aspects, features, and implementations of the invention.

[0061] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included within the invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range. While any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this invention, preferred materials and methods are described below.

[0062] Example 1: Construction of a single-molecule long-read SMRT sequencing library using the present invention Step 1: Embryo whole genome amplification Embryonic whole genome amplification involves the whole genome amplification module of this invention, which uses MDA-type methods to amplify the whole genome.

[0063] The blastocyst trophoblast cell biopsy sample to be amplified was placed in 4 μL of phosphate buffer, and 3 μL of lysis buffer (400 mM KOH, 100 mM DTT, 10 mM...) was added. EDTA (dissolved in ddH2O) was vortexed and incubated at room temperature for 10 min. 9 μL of ddH2O, 27 μL of whole genome amplification buffer [80 mM Tris-HCl, 160 mM (NH4)2SO4, 16 mM MgCl2, 1.5 mM dNTPs, pH 7.5], 0.04 nmol of random primers (ThermoFisher, N8080127), and 2 μL of Phi29 DNA polymerase (NEB, M0269S) were mixed and added to the previous lysis mixture. The reaction was carried out in a thermal cycler at 30°C for 20 min, 65°C for 3 min, and then at 4°C. The resulting product was the whole genome amplification (WGA) product of the embryonic sample, i.e., MDA DNA.

[0064] Step 2: Constructing SMRT sequencing libraries of embryonic samples The construction of long-fragment sequencing libraries from embryonic samples involves the preprocessing module, amplification module, and library construction module of this invention.

[0065] WGA products from embryonic samples were analyzed for DNA concentration using Qubit dsDNA HS reagent (ThermoFisher, Q32851) on a Qubit 3 Fluoromter (ThermoFisher, Q33216). DNA was purified using 1X AMPure PB magnetic beads (PacBio, 100-265-900) according to the manufacturer's instructions.

[0066] Take purified WGA DNA and pretreat it with the restriction enzyme FEN1 in the pretreatment module. Prepare the reaction system according to Table 1 below: Table 1. Reaction system pretreated with endonuclease FEN1

[0067] Place in a thermal cycler and incubate at 65°C for 1 hour, then cool to 4°C.

[0068] Optionally, pretreatment with T7 endonuclease I is performed. The reaction system is prepared according to Table 2 below, placed in a thermal cycler, and incubated at 37°C for 1 h, then cooled to 4°C. The product needs to be purified using 1X AMPure PB magnetic beads.

[0069] Table 2. Reaction system pretreated with T7 endonuclease I

[0070] Optionally, pretreatment can be performed using both FEN1 and T7 endonuclease I. In this case, the reaction system should be prepared according to Table 3, placed in a thermal cycler, and incubated at 37°C for 30 min, then at 65°C for 30 min, and cooled to 4°C. The product needs to be purified using 1X AMPure PB magnetic beads.

[0071] Table 3. Reaction system pretreated with T7 endonuclease I and FEN1

[0072] Then proceed to the amplification module, take the product from the previous step, and prepare the reaction system according to Table 4: Table 4. Reaction system for amplification adapter ligation of pretreated products.

[0073] Incubate at room temperature for 30 min, then purify the product with 1X AMPure PB magnetic beads, eluting with 25 μL. The reaction system was then prepared according to Table 5: Table 5. Reaction system for amplification of pretreated products

[0074] Place the sample into a thermal circulator and react according to Table 6: Table 6. Reaction temperature and cycle settings for amplification of pretreated products.

[0075] The amplified product was purified with 0.6X AMPure PB magnetic beads, eluted with 23 μL H2O, and then entered the library construction module. The reaction system was prepared according to Table 7. Table 7 Reaction system for amplification product repair

[0076] Place the mixture in a thermal cycler and react as follows: 37°C for 30 min, 65°C for 5 min, and then hold at 4°C. Prepare the reaction mixture according to Table 8 and perform sequencing adapter ligation. Table 8 Sequencing adapter ligation reaction system for amplification products

[0077] Incubate in a thermal cycler at 20°C for 30 min. Prepare the reaction system according to Table 9 below for exonuclease digestion: Table 9. Exonuclease digestion reaction system for embryo libraries

[0078] Place the sample in a thermal cycler and incubate at 37°C for 30 min. Purify the product with 1X AMPure PB magnetic beads to obtain the SMRT library. Fragment screening can be performed using BluePippin, PippinHT (Sage Science), or LightBench (Yourgene Health) to enrich libraries >4 kb.

[0079] Step 3: Constructing long-fragment sequencing libraries of parental genome samples The construction of long-fragment sequencing libraries from parental samples involves the fragmentation module and library construction module of this invention.

[0080] Genomic DNA (gDNA) from parental sources (peripheral blood, tissue) was analyzed for concentration using Qubit dsDNA HS reagent on a Qubit 3 Fluorometer. Fragment size was determined by pulsed-field agarose gel electrophoresis, requiring no significant degradation and major bands located in the region above 60 kb. DNA purity was assessed using Nanodrop, requiring 1.8 ≤ OD 260 / 280 ≤ 2.0 and OD 260 / 230 ≥ 2.0. After passing quality control, the DNA was purified using 1X AMPure PB magnetic beads.

[0081] Take the purified gDNA and fragment it using a DNA fragmentation tube (Covaris, 520079) in the fragmentation module. After purification with magnetic beads, the fragmented product is transferred to the library construction module, and the reaction system is prepared according to Table 10 below: Table 10 Reaction systems for fragmentation product repair

[0082] Place the container in a thermal cycler and react as follows: 37°C for 30 min, 65°C for 5 min, and then hold at 4°C. Prepare the reaction mixture according to Table 11 and perform sequencing adapter ligation. Table 11 Fragmentation product sequencing adapter ligation system

[0083] Place the container in a thermal cycler and incubate at 20°C for 30 min. Prepare the reaction system according to Table 12 below and perform exonuclease digestion: Table 12 Exonuclease digestion reaction system of parent library

[0084] Incubate in a thermal cycler at 37°C for 45 min. Purify the product with 1X AMPure PB magnetic beads to obtain the SMRT library. Fragment screening can be performed using BluePippin, PippinHT (Sage Science), or LightBench (Yourgene Health) to enrich libraries >10 kb.

[0085] Step 4: PacBio sequencing and analysis The DNA concentration of the library was determined using the Qubit dsDNA HS reagent on a Qubit 3 Fluoromter. Based on the library concentration, the library was prepared into a sequenceable library using the PacBio kit (Vega polymerase kit, 103-517-600; or RevioSPRQ polymerase kit, 103-520-100) according to the relevant product instructions. Then, it was sequenced in CCS (circular consensus sequencing) mode using the corresponding reagent type (Vegasequencing plate, 103-274-300; or Revio SPRQ sequencing plate, 103-504-900) and the chip (Vega SMRT Cell tray, 103-406-700; or Revio SMRT Cell tray, 102-202-200). The sequencing times were 24 h (Vega) and 30 h (Revio).

[0086] Example 2: The necessity of preprocessing and amplification modules for single-molecule long-read whole-genome sequencing of MDA-based DNA products from embryonic samples. After treatment with NGS fragmentation enzyme, embryonic sample MDA DNA could be successfully used for short-read NGS sequencing. To verify whether MDA DNA treated with NGS fragmentation enzyme is suitable for single-molecule long-read sequencing, the following treatments were performed: (Table 13 below) Table 13 MDA DNA treatment conditions

[0087] When treating with F2, prepare the reaction system according to Table 14 below, and incubate it in a thermal cycler: 4°C for 1 min, 32°C for 10 min, 65°C for 30 min, and then keep at 4°C.

[0088] Table 14 Reaction system for NGS fragmentation enzyme treatment of MDA DNA

[0089] For F3 and F4 treatments, the pretreatment module in step 2 of Example 1 was followed. For F5 treatment, 1 μg of MDA DNA was diluted in 50 μL TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0) and fragmented in a DNA fragmentation tube g-TUBE (Covaris, 520079) (5000 rpm, membrane pass twice). 20 ng of each of the F1–F5 products were analyzed for fragment size using GenomicDNA ScreenTape (Agilent, 5067-5365). The results showed that the MDA DNA fragments after NGS fragmentation enzyme treatment were too short (concentrated around 250 bp) and unsuitable for long-read sequencing; other treatment methods preserved longer DNA fragments. Figure 2 ).

[0090] Figure 3 Tables (A) to (C) compare the effects of random fragmentation, T7 endonuclease I, and FEN1 on MDA DNA processing from a fundamental perspective: Random fragmentation (such as mechanical methods using DNA breaking tubes or enzymatic digestion using transposases) is a common processing method for single-molecule long-read sequencing, randomly introducing breaks or notches into the DNA. This method can effectively improve the output of single-molecule long-read sequencing when used to process long straight-chain DNA (such as high molecular weight genomic DNA). However, when applied to MDA DNA, it cannot effectively remove branched structures. T7 endonuclease I specifically recognizes the incompletely complementary parts within the DNA double strand and digests both strands simultaneously, thereby causing MDA DNA to specifically break at the branching points, producing a normal double-stranded structure. FEN1 specifically recognizes the 5' flanking points of DNA branches, introducing notches, which can also cause MDA DNA to specifically break at the branching points, producing a double-stranded structure with gaps. After repair, a complete double-stranded structure can be generated. In principle, treatment with T7 endonuclease I or FEN1 can improve the ability of MDA DNA to produce single-molecule long reads while preserving long DNA fragment information.

[0091] Further experimental verification of the above theoretical analysis was conducted. Take 1 μg of the same MDA DNA and treat it under 9 conditions: Under T01, no pretreatment or amplification is performed; Under T02, pretreatment is performed only as per step 2 of Example 1, without amplification, wherein T02-1 is pretreated only with T7 restriction enzyme I, T02-2 is pretreated only with FEN1, and T02-3 is treated with both T7 restriction enzyme I and FEN1; Under T03, pretreatment and amplification are performed simultaneously as per step 2 of Example 1, wherein T03-1 is pretreated only with T7 restriction enzyme I, T03-2 is pretreated only with FEN1, and T03-3 is treated with both T7 restriction enzyme I and FEN1; Under T04, MDA DNA is treated with a DNA fragmentation tube gTUBE without T7 restriction enzyme I or FEN1 treatment; Under T05, MDA DNA is treated with transposase (using the LongPlex Multiplexing Kit, seqWell; refer to the product instructions), without T7 restriction enzyme I or FEN1 treatment. All the above products were used for SMRT library construction according to step 2 of Example 1. After preparing the libraries into sequenceable libraries using the Sequel II binding kit 2.0 (PacBio, 101-789-500), the same mass was used for CCS sequencing on the Sequel II sequencing platform using Sequel II sequencing kit 2.0 (PacBio, 101-820-200) reagents and an SMRT Cell 8M single-use tray (PacBio, 102-281-700) chip. The sequencing time was 30 h. The sequencing results are shown in Table 15 below.

[0092] Table 15 Sequencing results of libraries constructed from MDA DNA after different treatments

[0093] The results showed that when MDA DNA without pretreatment and amplification was directly used for SMRT library construction (T01), the single-chip sequencing yield was 0.06 Gb of bases and ~0.01 M of fragments. After pretreatment with T7 restriction enzyme I or FEN1 (T02), the total base yield was 8 times that of T01; the fragment yield was approximately 10 times. Meanwhile, after pretreatment and amplification with T7 restriction enzyme I or FEN1 (T03), the base yield was 4–8 Gb, a 60–120-fold increase compared to T01; the fragment yield was 2.1 M, an approximately 150-fold increase compared to T01, reaching the typical yield level (2–3 M) of a single Sequel II 8M tray chip. Using DNA fragmentation tubes (T04) or transposase treatment (T05) without pretreatment with FEN1 or T7 restriction enzyme I resulted in yields similar to those under T01 conditions, indicating no effective improvement in yield.

[0094] In summary, T7 endonuclease I or FEN1 pretreatment and amplification modules are necessary for the normal output of sequencing data from MDA DNA on long-read single-molecule sequencing platforms.

[0095] Example 3: The present invention applies to FBN1 PGT-M testing for E1-2 missing family lineage Family history: Maternal (P001M) genes FBN1 A single-gene variant with exon E1-2 deletion was present; the paternal side (P001F) was negative. SMRT libraries were constructed and whole-genome single-read sequencing was performed on both parents and three embryos (P001E1-3) according to Example 1, with the preprocessing module using only the FEN1 restriction enzyme. Sequencing data are shown in Table 16. Table 16 FBN1 E1-2 missing family sequencing data

[0096] The results of relevant variant detection are shown in Table 17. Samples P001M and P001E3 showed heterozygous deletions, with a deletion range of chr15: 48,625,733-48,708,356, including... FBN1 Gene exons E1-2. In haplotype, P001E3 inherits from the maternal side. FBN1 E1-2 lacks the haplotype; P001E1-2 inherits the normal haplotype from the mother. The control results were obtained by detecting the maternal haplotype using multiplex ligation-dependent probe amplification (MLPA). Figure 4 ), using NGS for familial linkage analysis ( Figure 5 The results are consistent with those of the present invention.

[0097] Table 17 FBN1 E1-2 Missing Family PGT-M Test Results

[0098] The results of aneuploidy and copy number variation detection are as follows: Figure 6 As shown in (A) to (C) and Table 18, the three embryos do not carry aneuploidy or CNVs larger than 4 Mb. The control results were obtained using NGS, and the results are as follows: Figure 7 As shown in (A) to (C), they are consistent with the present invention.

[0099] Table 18 FBN1 E1-2 Missing Family PGT-A Test Results

[0100] Example 4: t(x;19)(p22.31;p13.1) PGT-SR family pedigree testing Family history: The mother (P002M) exhibited a balanced t(x;19)(p22.31;p13.1) translocation; the father (P002F) was negative. SMRT libraries were constructed and whole-genome single-read sequencing was performed on both parents and three embryos (P002E1-3) according to Example 1, with the preprocessing module using only the FEN1 restriction enzyme. Sequencing data are shown in Table 19 below: Table 19. Family sequencing data for t(x;19)(p22.31;p13.1)

[0101] The results of the mutation detection are as follows Figure 8 As shown in Table 20 below. A balanced translocation of t(x;19) was detected in the maternal embryo, with breakpoints at chr19: 7,692,310-7,692,363 and chrX: 2,530,124-2,530,128; translocated chromosomes were present in samples P002M, P002E1, P002E2, and P002E3; among them, P002E1 was an unbalanced embryo, inheriting chromosome 19 derived from the maternal translocation [der(19)Xpter→Xp22.23::19p13.2→19qter] and a normal X chromosome in its haplotype; P002E2 and P002E3 inherited chromosome 19 derived from the maternal translocation [der(19)Xpter→Xp22.23::19p13.2→19qter] and [der(X)19pter→19p13.2:: The Xp22.3→Xqter] translocation-derived chromosome is a balanced translocation carrier. The control results included maternal karyotyping and family linkage analysis using NGS. Figure 9As shown in (A), embryos P005E1, P005E2, and P005E3 carry translocation-derived chromosome 19, as... Figure 9 As shown in (B), P005E2 and P005E3 carry translocation-derived chromosome X, and the conclusion is consistent with the results shown in this invention.

[0102] Table 20. Family PGT-SR Results (t(x;19)(p22.31;p13.1))

[0103] The results of aneuploidy and copy number variation detection are as follows: Figure 10 , 11 And as shown in Table 21 below. Figure 10 As shown in (A) to (C), embryo P002E1 exhibits copy number variations of del(19)(p13.3p13.2), dupX(p22.33), consistent with the PGT-SR results of chromosome 19 derived from the maternal translocation [der(19)Xpter→Xp22.23::19p13.2→19qter] and the normal maternal X chromosome; Figure 11 As shown in (A) to (B), embryos P002E2 and P002E3 do not carry CNVs greater than 4 Mb. Figure 12 As shown in (A) to (C), the control group used NGS for PGT-A, and the results were consistent with those shown in this invention.

[0104] Table 21. Family PGT-A Results (t(x;19)(p22.31;p13.1))

[0105] Example 5: PGT detection of 16 families according to the present invention For 16 families undergoing assisted reproduction, PGT was detected according to Example 1 (where the pretreatment module used only the endonuclease FEN1), and the results were consistent with the control results (Table 22).

[0106] Table 22 PGT results of 16 assisted reproductive families

[0107] It should be noted that although the above-described embodiments have demonstrated a series of features of the present invention, researchers and those skilled in the art can utilize the ideas of the present invention to make corresponding adjustments and changes to the reaction reagents, reaction conditions, etc., involved in library construction. Therefore, those skilled in the art can make several simple substitutions without departing from the concept and principles of the present invention, and these should all be included within the scope of protection of the present invention.

Claims

1. A reagent kit, characterized in that, It is used for integrated preimplantation genetic testing of embryos based on long-read single-molecule sequencing, and the kit contains the following reagents: (1) Reagents used for whole genome amplification of embryo samples; (2) Reagents used to construct long-fragment sequencing libraries of parental genome samples; (3) Reagents used to construct long-fragment sequencing libraries of embryonic samples; The reagents used to construct long-fragment sequencing libraries of embryonic samples contain at least an endonuclease; the endonuclease is T7 endonuclease I and / or FEN1.

2. The reagent kit according to claim 1, characterized in that, The reagents used for whole genome amplification of embryo samples include whole genome amplification buffer, random primers, and whole genome amplification DNA polymerase.

3. The reagent kit according to claim 1, characterized in that, The reagents used to construct long-fragment sequencing libraries of parental genome samples include DNA fragmentation tubes, repair buffer, DNA repair enzyme, DNA end repair enzyme, ligation buffer, sequencing adapter, ligase, exonuclease, and DNA purification magnetic beads.

4. The reagent kit according to claim 1, characterized in that, The reagents used to construct long-fragment sequencing libraries from embryonic samples include restriction enzyme buffers, amplification adapters, amplification primers, PCR DNA polymerase, repair buffers, DNA repair enzymes, DNA end repair enzymes, ligation buffers, sequencing adapters, ligases, exonucleases, and DNA purification magnetic beads.

5. The reagent kit according to claim 1, characterized in that, The parental sample includes peripheral blood or tissue from the parent.

6. The reagent kit according to claim 1, characterized in that, The embryo samples are biopsies of embryos in the blastocyst or blastocyst stage.

7. The kit according to claim 1, characterized in that, The single-molecule sequencing is based on a single-molecule real-time sequencing or nanopore technology platform.

8. The kit according to any one of claims 1-7, characterized in that, The kit is used for integrated preimplantation genetic testing based on long-read single-molecule sequencing of parental and embryo samples.

9. The reagent kit according to claim 8, characterized in that, The integrated preimplantation genetic testing includes simultaneous detection of PGT-A, PGT-M, and PGT-SR.

10. The reagent kit according to claim 9, characterized in that, The integrated preimplantation genetic testing includes haplotype construction and linkage analysis, as well as simultaneous detection of aneuploidy, copy number variation, single nucleotide variation, deletion, insertion and chromosomal structural variation.

11. A system for integrated preimplantation genetic testing of embryos based on long-read single-molecule sequencing, characterized in that, It includes the following modules: (1) Whole genome amplification module: used to obtain the whole genome amplification products of embryo samples; (2) Preprocessing module: used to convert the whole genome amplification product into a preprocessing product suitable for the amplification module; (3) Amplification module: used to amplify the preprocessed product and convert it into an amplified product suitable for constructing a long fragment single molecule sequencing library; (4) Fragmentation module: used to obtain the genome fragmentation products of the parental sample; (5) Library construction module: used to construct long-fragment single-molecule sequencing libraries; (6) Sequencing module: used for high-throughput long-read single-molecule sequencing; The preprocessing module uses an endonuclease to convert the whole genome amplification product into a preprocessing product suitable for the amplification module; the endonuclease is T7 endonuclease I and / or FEN1.

12. The system according to claim 11, characterized in that, The whole genome amplification module uses reagents containing whole genome amplification buffer, random primers, and whole genome amplification DNA polymerase to obtain whole genome amplification products from embryo samples.

13. The system according to claim 11, characterized in that, The amplification module uses reagents containing amplification adapters, amplification primers, and PCR DNA polymerase to amplify the pretreated product and convert it into an amplified product suitable for constructing long-fragment single-molecule sequencing libraries.

14. The system according to claim 11, characterized in that, The fragmentation module uses reagents containing DNA-breaking tubes to obtain genomic fragmentation products from parental samples.

15. The system according to claim 11, characterized in that, The library construction module uses reagents containing repair buffer, DNA repair enzyme, DNA end repair enzyme, ligation buffer, sequencing adapter, ligase, exonuclease and DNA purification magnetic beads to construct long fragment single-molecule sequencing libraries.

16. The system according to claim 11, characterized in that, The sequencing module uses a single-molecule real-time sequencing or nanopore technology platform to perform high-throughput long-read single-molecule sequencing.

17. The system according to claim 11, characterized in that, The parental sample includes peripheral blood or tissue from the parent.

18. The system according to claim 11, characterized in that, The embryo samples are biopsies of embryos in the blastocyst or blastocyst stage.

19. The system according to any one of claims 11-18, characterized in that, The system is used for integrated preimplantation genetic testing based on long-read single-molecule sequencing of parental and embryo samples.

20. The system according to claim 19, characterized in that, The integrated preimplantation genetic testing includes simultaneous detection of PGT-A, PGT-M, and PGT-SR.

21. The system according to claim 20, characterized in that, The integrated preimplantation genetic testing includes haplotype construction and linkage analysis, as well as simultaneous detection of aneuploidy, copy number variation, single nucleotide variation, deletion, insertion and chromosomal structural variation.

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