Kit for targeted amplification of wga product of embryo based on third-generation sequencing and application

Abstract

The application relates to the technical field of detection, and discloses a reagent kit for a targeted amplification primer group of embryo WGA products based on third-generation sequencing and application, which comprises a targeted capture probe group; the targeted capture probe group comprises specific probes for targeting a full-length region of a single-gene genetic disease pathogenic gene, a copy number variation region, and a single nucleotide polymorphism site within a range of 0-5 Mb upstream and downstream of the pathogenic gene; the length of the specific probes is 100 nt; and the paving interval of the specific probes in the targeted region is one probe per 450 bp. The application is characterized in that specific probes are designed and targeted to enrich target gene regions, pathogenic variation regions, large-fragment variation related regions and key upstream and downstream linkage regions, the interference of inherent defects of WGA products on third-generation sequencing is reduced, and the effective sequencing utilization rate and detection accuracy of embryo WGA products are improved.

Description

Technical Field

[0001] This invention relates to the field of detection technology, and in particular to a kit and its application based on a primer set for targeted amplification of WGA products from third-generation sequencing embryos. Background Technology

[0002] Single-gene genetic diseases are caused by mutations in a single gene, and these mutations are complex and diverse, including point mutations, small insertions / deletions, large structural variations, and dynamic mutations. Traditional short-read sequencing technologies have inherent limitations in detecting large structural variations, highly homologous regions, and complex rearrangements, often requiring the combined use of multiple technologies such as MLPA and LD-PCR. This multi-technology platform approach is not only cumbersome, time-consuming, and costly, but also struggles to achieve comprehensive coverage of all potential variant types.

[0003] In preimplantation genetic testing for single-gene disorders, embryonic biopsy cells are typically used. Due to the extremely small number of embryonic biopsy cells, whole-genome amplification (WGA) is usually required to obtain sufficient DNA template. However, WGA amplification products often suffer from amplification bias, uneven region coverage, fragment discontinuities, non-specific amplification, and chimeric fragments, which can easily affect subsequent sequencing and interpretation of genetic results. Currently, the main approach is to indirectly diagnose variant sites using pedigree haplotype linkage analysis based on next-generation sequencing (NGS). This requires first constructing a family pedigree, then performing the sequencing, and finally conducting linkage analysis on the embryos. While existing NGS short-read sequencing technologies offer high throughput and relatively low cost, their short read lengths make it difficult to directly cross pathogenic sites and multiple surrounding linked SNPs, and also hinder accurate resolution of repetitive regions, complex structural variations, and long-distance haplotypes. Therefore, in families without probands, with incomplete kinship samples, or with de novo mutations or complex variations, NGS-based PGT-M testing still faces challenges such as difficulty in haplotype construction, insufficient interpretation of complex regions, and high uncertainty in results.

[0004] Third-generation long-read sequencing technology can obtain longer sequence reads, which has advantages in repetitive region resolution, complex variant identification, structural variant detection, and haplotype construction. However, embryonic WGA product template sequences are relatively short and contain many aberrant amplification products, which does not match the requirements of conventional third-generation sequencing for template integrity and continuity. If whole-genome third-generation sequencing is performed directly, there are problems such as analysis of chimeric fragment molecular interference, high sequencing costs, and heavy analytical burden, which make it difficult to meet the clinical application needs of simple, accurate, and economical preimplantation genetic testing.

[0005] Currently, the mainstream strategies for improving PGT-M accuracy are based on next-generation sequencing and probe-targeted capture detection technologies or microarray technologies. However, existing technologies have the following drawbacks: 1. Short-read sequences typically struggle to cross pathogenic sites and multiple upstream and downstream SNP linkage markers, and also find it difficult to directly determine whether multiple variants or SNPs reside on the same parental chromosome. Therefore, in PGT-M testing, next-generation sequencing often relies on indirect linkage analysis using family members, proband samples, or embryo reference information. For cases without probands, with incomplete relative samples, novel mutations, or complex families, short-read sequences present significant limitations in constructing disease-related haplotypes.

[0006] 2. Regardless of whether haplotype analysis is based on STR, SNP microarrays, or NGS, determining whether an embryo carries a pathogenic haplotype typically requires combining the pedigree pathogenic locus with multiple surrounding SNP loci, including preliminary haplotype construction and embryo haplotype construction, a cumbersome and time-consuming process. Furthermore, a single short-read sequence cannot cover a sufficient number of linked SNPs, and it is difficult to directly observe recombination events near the pathogenic locus. Therefore, when recombination occurs near the pathogenic locus, or when allele deletions occur at key SNP loci, next-generation sequencing results may show uncertain or misclassified haplotype interpretations.

[0007] 3. Short-read sequencing relies primarily on short fragment alignment and sequence depth variations to infer structural variations. It is significantly affected by WGA amplification bias and uneven target region coverage, making it difficult to accurately determine breakpoint locations, variation directions, and parental origins. For regions containing homologous sequences, pseudogenes, repetitive sequences, or complex gene clusters, the alignment and interpretation difficulties of short-read sequencing further increase.

[0008] 4. Theoretically, direct sequencing of embryonic WGA products is the ideal solution for PGT-M. However, due to amplification bias in embryonic WGA products, such as the presence of branches, gaps, or chimeric fragments in the amplified sequences, the risk of analysis failure is high when directly detecting embryonic amplified products. While third-generation long-read sequencing provides the possibility of directly detecting embryonic variations, it faces the following problems: the template integrity requirements of embryonic WGA products and conventional third-generation sequencing are incompatible, resulting in low efficiency of SMRTbell library construction, low effective insertion ratio, and low sequencing read quality; chimeric fragments and rearrangement signals introduced by WGA can interfere with long-read analysis, easily misclassifying artificially amplified products as real structural variations; if whole-genome sequencing is performed directly, the sequencing cost is high and the utilization rate of non-target region data is low; in addition, existing solutions lack a standardized targeted enrichment and analysis system for embryonic WGA products, making it difficult to simultaneously meet the integrated needs of enriching multiple complex gene target regions, long-read sequencing, variation detection, haplotype construction, WGA artificial product filtering, and embryonic genetic status interpretation. Summary of the Invention

[0009] To address the aforementioned technical problems, this invention provides a primer kit based on targeted amplification of WGA products from third-generation sequencing embryos, comprising: The targeted capture probe set includes specific probes targeting the full-length region, copy number variation region, and single nucleotide polymorphism sites within the 0-5 Mb range upstream and downstream of the pathogenic gene of a single-gene genetic disease; the specific probes are 100 nt in length and the tiling spacing of the specific probes within the target region is one probe every 450 bp. Library amplification primer set; Library construction reagents include fragmentation reagents containing Tn5 transposase, long-fragment PCR amplification enzymes, and fragment screening reagents for removing nucleic acid fragments shorter than 5kb.

[0010] The targeted capture probe set has probe dose compensation in the high GC region of the target area, and the error tolerance of the sequence of the specific probe and the sequence of the target area is less than 5%.

[0011] The pathogenic gene for the single-gene hereditary disease is selected from at least one of ASS1, ATP7B, CYP21A2, DMD, F8, F9, FMR1, FXN, GAA, GBA1, GJB2, GLA, HBA1 / HBA2, HBB, IDS, MMACHC, MMUT, PAH, SLC22A5, SLC25A13, SLC26A4, and STS.

[0012] The fragment screening reagent is AMPure PB screening magnetic beads diluted to a volume concentration of 35%; the long fragment PCR amplification enzyme is PK512 DNA polymerase.

[0013] The kit also includes a hybridization elution reagent comprising a hybridization reaction solution, streptavidin magnetic beads, and a heat washing buffer.

[0014] Secondly, the present invention also provides an in vitro method for constructing a third-generation targeted sequencing library of embryonic WGA products, using a primer kit based on third-generation sequencing embryonic WGA products targeted amplification, the method comprising the following steps: Fragmentation and adapter introduction: The purified embryonic WGA product was fragmented and sequenced using the fragmentation reagent to obtain fragmented products; Pre-capture library amplification: The fragmented product was amplified by PCR using the library amplification primer set and the long fragment PCR amplification enzyme to obtain the original pre-capture library; Fragment screening: The original pre-capture library is screened using the fragment screening reagent to remove nucleic acid fragments with a length of less than 5kb, thereby obtaining the screened pre-capture library; Hybridization capture and elution: The screened pre-capture library and the target capture probe group were added to the hybridization reaction solution for liquid-phase hybridization incubation, streptavidin magnetic beads were added to bind the target fragment, and a washing solution was used for washing. Third-generation sequencing library construction: The target fragment after washing and elution was amplified using PCR primers to obtain a captured library, and the captured library was subjected to damage repair, end repair, A addition reaction and third-generation sequencing adapter ligation reaction in sequence.

[0015] In the fragmentation and connector introduction steps: The total volume of the reaction system for a single interruption reaction is 50 μL, the amount of Tn5 transposase in the reaction system is 3 μL, and the reaction time of the interruption reaction is 10 minutes; the main peak length of the nucleic acid fragment of the obtained interruption product is distributed between 6kb and 7kb.

[0016] In the hybridization capture and elution step: The cleaning operation is as follows: add a preheated hot cleaning buffer to the streptavidin magnetic beads, incubate and clean at a constant temperature of 50°C, and repeat the constant temperature incubation and cleaning operation a total of 4 times.

[0017] Thirdly, the present invention provides a data processing method, executed by a computer system, comprising the following steps: Obtain circularly consistent sequence sequencing data from third-generation sequencing libraries prepared by in vitro construction methods; The circular consensus sequence sequencing data is aligned to the target region of the reference genome, and split reads are extracted; Assess read internal consistency and filter out chimeric molecular fragments and aberrantly connected fragments introduced by embryonic whole-genome amplification in the sequencing data; Based on the filtered sequencing data, local genome assembly and variant detection are performed, and local haplotypes are constructed based on the single nucleotide polymorphism sites to output genetic variation status data of the target embryo region.

[0018] Fourthly, the present invention also includes the application of a kit for targeted amplification primer sets based on third-generation sequencing embryo WGA products in the preparation of in vitro detection products for preimplantation genetic diseases.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention establishes a targeted long-read sequencing strategy adapted to the characteristics of embryonic WGA products. After embryonic biopsy cells are amplified by WGA, the products often contain numerous branching structures, gaps, discontinuous fragments, non-specific amplified fragments, and amplification-biased regions. Third-generation sequencing (NGS) requires high integrity and continuity of template molecules. To address this contradiction, this invention employs specific probe design and targeted enrichment for target gene regions, pathogenic variant regions, large-fragment variant-related regions, and key upstream and downstream linkage regions. Simultaneously, it optimizes key library construction steps, such as long-fragment amplification enzyme selection, fragmentation conditions, and the number of hot washes. This effectively avoids non-target invalid fragments and amplification-biased regions in WGA products, reduces the interference of inherent defects in WGA products on NGS, and improves the effective sequencing utilization and detection accuracy of embryonic WGA products.

[0020] This invention establishes a method for quality control and interference removal of long-read artificial products amplified by embryonic WGA. Embryonic WGA products may contain a large number of chimeric molecules, artificially rearranged fragments, branched structures, and discontinuous template molecules introduced during the amplification process. Directly entering third-generation sequencing analysis may lead to incorrect alignments, false structural variations, or misjudgments of variations. This invention utilizes circular consistency sequences generated by PacBio HiFi sequencing, combined with target region alignment, split read identification, read internal consistency assessment, local genome assembly, and abnormal read filtering, to identify and exclude chimeric molecules and artificially rearranged signals introduced by WGA, thereby obtaining more reliable results for detecting variations in target regions.

[0021] This invention proposes a probe set design method for PGT-M applications. The probe set of this invention not only covers the exons, introns, and key regulatory regions of target genes associated with complex monogenic diseases, but also covers known pathogenic variant sites, common large-fragment deletion / duplication regions, regions near structural variation breakpoints, and effective SNP sites within the 0-5Mb range upstream and downstream of the target gene. By combining the direct pathogenic variant detection region with the local haplotype linkage analysis region, this invention can simultaneously support comprehensive analysis of SNVs, Indels, CNVs, large-fragment deletions / duplications, structural variations, and disease-related haplotypes, constructing local haplotypes, identifying allele deletion risks, and improving the coverage, flexibility, and interpretation accuracy of embryonic monogenic disease detection. In the probe set of this invention, the selection of target genes comprehensively considers the clinical incidence of the disease, the needs of PGT-M applications, the complexity of gene structure, and the limitations of existing short-read detection methods, prioritizing the inclusion of monogenic disease-related genes with clear clinical needs, complex variant types, and deficiencies in existing detection methods.

[0022] This invention enables parallel targeted detection of multiple complex monogenic genetic disease-related genes in embryonic WGA products. Conventional PGT-M testing often relies on family pedigrees and probands to construct family haplotypes, limiting clinical applicability, and involving complex, lengthy, and costly procedures. Furthermore, alignment and interpretation are challenging in regions containing homologous sequences, pseudogenes, repetitive sequences, or complex gene clusters. This invention constructs a targeted detection system covering multiple complex monogenic disease-related target genes, pathogenic loci, and their surrounding linked SNP loci. This allows the same detection process to be adapted to multiple common complex disease genes, enabling direct detection of embryonic pathogenic genes, reducing testing costs, and improving the throughput and clinical applicability of PGT-M testing.

[0023] This invention provides an integrated testing kit and analytical workflow adapted to the needs of preimplantation genetic testing (PGD). This invention integrates WGA product quality control, target region enrichment, PacBio HiFi library construction, sequencing, data quality control, variant detection, and embryo genetic status interpretation into a standardized workflow. It can further be developed into an integrated kit containing capture probe sets, library construction reagents, quality control standards, and analytical software or parameters. This design simplifies experimental procedures, lowers the technical threshold, and shortens the testing cycle, making this method more suitable for the clinical needs of rapid, accurate, low-cost, and parallel detection of multiple complex genes in PPD. Attached Figure Description

[0024] Figure 1 The image shows the analysis results of three enzymes (taking the F9 region as an example). Detailed Implementation

[0025] The terms “first,” “second,” “third,” “fourth,” etc. (if present) in the specification, claims, and accompanying drawings of this invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms “comprising” or “having,” and any variations thereof, are intended to cover a non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0026] Definition of the addition of A / A reaction: Adding a single adenine deoxynucleotide (dA) to the 3' end of a DNA fragment via an enzymatic reaction. This reaction is typically catalyzed by enzymes with terminal transferase activity (such as Taq DNA polymerase, Klenow Fragment, etc.), transforming a blunt-ended DNA fragment into a sticky end with an A protruding at the 3' end. This allows for complementary pairing with sequencing adapters that have a T protruding at the 3' end, achieving efficient and specific adapter ligation while reducing fragment self-ligation and background interference. In third-generation sequencing library construction, adding an A is a crucial step after end repair and before adapter ligation.

[0027] This invention uses embryonic biopsy cell WGA products as a detection template, combining target region probe capture and enrichment with PacBio HiFi long-read sequencing technology to perform targeted enrichment and sequencing analysis on target gene regions related to single-gene diseases, known pathogenic variant regions, complex repetitive regions, large fragment deletion / repetition regions, structural variant-related regions, and 0-5Mb linked SNP regions upstream and downstream of the target gene. Through specific probe capture and enrichment methods, this invention can reduce the interference of non-target fragments, low-quality amplified fragments, and amplification bias regions in WGA products on sequencing results, improving the sequencing depth of target regions and the effective utilization rate of sequencing data. Simultaneously, leveraging the advantages of PacBio HiFi long-read sequencing in repetitive sequences, complex variants, large fragment variants, and haplotype resolution, it improves the comprehensive detection capability of SNVs, Indels, CNVs, large fragment deletions / repetitions, and structural variants in embryonic WGA products. The probe set design of this invention can simultaneously cover multiple complex single-gene genetic disease-related genes and their key upstream and downstream linkage analysis regions, enabling the same detection system to adapt to the detection needs of various complex single-gene diseases that cannot be covered by conventional next-generation sequencing. By employing a single targeted capture and third-generation sequencing experiment, multiple target gene regions in embryonic WGA products can be detected and comprehensively analyzed in parallel. This reduces the need for repeatedly developing testing protocols for individual families or single genes, lowers testing costs, and increases throughput and process standardization. Furthermore, this invention establishes a long-read data analysis workflow tailored to the characteristics of WGA products. It identifies and filters artificial chimeric molecules, aberrant connection fragments, and abnormally covered regions that may arise during amplification. Combined with sequence consistency analysis, local assembly, variant detection, and haplotype analysis, it provides a comprehensive interpretation of the embryonic genetic status. Therefore, this invention improves the accuracy, stability, and interpretability of complex single-gene genetic disease detection using embryonic WGA products, providing a more efficient, economical, and easily standardized technical solution for preimplantation genetic disease detection.

[0028] This invention primarily constructs a detection method for embryo amplification products based on third-generation targeted sequencing, while optimizing key steps and selecting the best conditions. Furthermore, comparative examples verify the detection accuracy of this invention.

[0029] The specific process is as follows: The database creation process includes the following steps: Embryonic biopsy cell WGA amplification: A small number of biopsy cells were lysed and isothermally amplified, and the amplification products were purified to obtain usable WGA amplification products.

[0030] Transposase fragmentation: Transposases are used for fragmentation and introduction of sequencing adapters to complete the pre-capture library construction.

[0031] Pre-capture library amplification: PCR amplification was performed using tagged primers, resulting in library enrichment.

[0032] Pre-capture library filtering: The pre-capture library is segmented to remove libraries with a segment length of less than 5kb as much as possible, resulting in a filtered pre-capture library.

[0033] Hybridization capture and post-capture library amplification: Customized biotin-labeled probes were added to the pre-capture library, hybridized and incubated, and then the target fragment was captured using streptavidin magnetic beads. The post-capture library was then amplified and enriched using PCR primers to obtain the post-capture library.

[0034] Construction of third-generation libraries: Construct corresponding third-generation libraries according to different third-generation sequencing platforms.

[0035] Capture probe design: The screening panel of this invention incorporates 25 complex genes. Key information about the probes is as follows: The probe covers the full length of the gene, the CNV coordinate region, and SNP sites in the 0-5Mb range upstream and downstream of the gene; Different numbers of probes were designed and synthesized based on the length of different genes, and dose compensation was performed in high GC regions.

[0036] The probe laying density is 0.2268x, meaning that one probe is laid every 450bp.

[0037] The probe length is 100nt.

[0038] The probe's fault tolerance rate is <5%.

[0039] Table 1 lists 25 complex genes: Table 1

[0040] The probe sequence is shown in the sequence listing SEQ ID NO: 0001-1303. The corresponding chromosome positions are in the order of 1-1303, as detailed in the sequence listing.

[0041] Determination of long-fragment amplification enzymes: The expected size of the Chinese library fragments in this invention is 4-5k, while the performance of the PCR enzymes in the transposase library preparation kit is far from sufficient; furthermore, for certain complex regions, amplification enzymes with special properties are required to meet the requirements. Therefore, we first investigated and screened long-fragment amplification enzymes that met the criteria, tested the candidate enzymes, and compared the amplification efficiency, amplification rate, and coverage of different enzymes.

[0042] Determination of transposase fragmentation conditions: The standard reaction system of the transposase library construction kit is 10 µL of transposase, 100 µL of reaction volume, and 10 min of reaction time, resulting in a 2-3 kb library. However, the library fragment required in this invention is 6-7 kb. The optimal fragmentation conditions were determined through testing of the reaction volume, reaction time, and transposase dosage.

[0043] Determining the number of thermal cleaning cycles: Increasing the number of thermal cleaning cycles can reduce non-specific binding and improve capture efficiency. By setting a gradient of cleaning cycles and comparing the capture efficiency, the required number of cleaning cycles can be determined.

[0044] Example 1: Screening test for long fragment amplification enzymes Considering amplification performance, cost, and delivery time, several domestically produced long-fragment amplification enzymes suitable for transposase library construction were first screened. Using the physical fragmentation method g-tube (excluding fragmentation bias) combined with KOD (TOYOBO# KFX-201) amplification enzyme (i.e., g-tube+KOD) as the standard, the amplification effects of Tn5+P512 (Novizan P521), Tn5+PK512 (Novizan PK512), and Tn5+KOD (TOYOBO# KFX-201) were compared.

[0045] The results showed that: a) the concentration of Tn5+KOD was extremely low after removing the 5k fragment, and the yield was insufficient for hybridization capture. b) PK521+Tn5 showed a more pronounced preference than PK512+Tn5. c) the regional coverage difference between PK512+Tn5 and the standard condition g-tube+KOD was basically consistent, with the former having a slightly lower average coverage than the standard condition. PK512+Tn5 was determined to be the experimental condition for this project. Specific results are as follows (Table 2-4): Table 2: Library concentrations before and after capture (unscreened, screened before and after capture)

[0046] Table 3: Quality control data charts for three types of enzyme-based machines

[0047] Table 4: Results of comparing capture efficiency with different washing cycles

[0048] Example 2: Determination of transposase fragmentation conditions The original manufacturer's standard reaction system had a reaction volume of 100 µL, a reaction time of 10 min, and an addition of 10 µL of transposase, resulting in a final fragment length of 2-3 kJ, which did not meet the requirements of this invention. This test, through gradient testing of reaction system volume (100 µL / 50 µL), transposase interruption time (3 min / 5 min / 10 min), and transposase dosage (1 µL / 2 µL / 3 µL / 4 µL), screened for optimal conditions to achieve a fragment length of 6-7 kJ. Finally, a reaction system of 50 µL, an interruption time of 10 min, and a transposase dosage of 3 µL were determined to be the final fragmentation conditions. Specific results are as follows (Table 5): Table 5: Distribution of fragments after interruption under different conditions

[0049] Example 3: Conditions for the Number of Hot Cleaning Cycles We set up thermal cleaning cycles of 3 and 4 times to compare the capture efficiency of different cleaning cycles. Compared to 3 cleaning cycles, 4 cleaning cycles showed a slight improvement in capture efficiency.

[0050] Example 4: Optimized library preparation process: This invention is compatible with ONT (Oxford Nanopore Technologies, ONT) and PacBio (Pacific Biosciences). This process takes the PacBio sequencing platform as an example.

[0051] Part 1: Pre-capture Library Construction Tn5 transposase labeling (Reagent manufacturer: IGT® Long-Read Tagment DNA Library PrepKit# C10931) Prepare the following system on ice, gently tap or slowly blow with a pipette tip to mix, and then briefly centrifuge;

[0052] Place the PCR tube in the PCR instrument and perform the following reaction.

[0053] Transposase release After the reaction is complete, immediately add 10 μL of stop buffer to the reaction system, vortex to mix, and then centrifuge briefly.

[0054] Place the PCR tube in the PCR instrument and perform the following reaction:

[0055] purification Add 42 µL of 0.7X purified magnetic beads (AMPure XP) to the labeled product. Gently tap to mix, then react at room temperature for 10 minutes.

[0056] Insert the PCR tube into the well of the magnetic rack. After the magnetic beads are completely adsorbed and the solution becomes clear, remove the solution, being careful not to remove the magnetic beads (leaving about 5µL of solution). Leave the PCR tube on the magnetic rack.

[0057] Pipette 200 µL of 80% ethanol into a PCR tube and let it stand for 30 seconds. Aspirate the solution, being careful not to aspirate the magnetic beads. The 80% ethanol should be prepared on the same day.

[0058] Repeat the previous step.

[0059] Remove the PCR tube and centrifuge for 5 seconds. Insert the PCR tube back into the well of the magnetic holder. After the magnetic beads have completely adhered, use a pipette to remove any remaining solution, ensuring there is no residue on the tube wall. Open the PCR tube cap and allow it to air dry, taking care not to crack the magnetic beads.

[0060] Pipette 24 µL of nuclease-free water into a PCR tube, gently tap to mix, and react at room temperature for 5 minutes.

[0061] After a brief centrifugation, insert the PCR tube into the well of the magnetic rack. Once the magnetic beads have been completely adsorbed and the solution has become clear, use a pipette to transfer 22 µL into a new, correspondingly numbered PCR tube, being careful not to pick up the magnetic beads.

[0062] PCR amplification Prepare the following mixture on ice, gently tap or pipette tip to mix, and briefly centrifuge. (Reagent manufacturer: 2 × KeyPo SE Master Mix (Dye Plus #PK512-01 Novizan; IGT® UDI Primer 1-16 (20 μMeach, for Long-Read NXT Library, tube)# C11001 Agitac)

[0063] Place the PCR tubes in the PCR instrument and set it up as follows:

[0064] PCR product purification Add 40 µL of 0.8X purified magnetic beads (AMPure XP) to the PCR product. Gently tap to mix and incubate at room temperature for 10 minutes.

[0065] Insert the PCR tube into the well of the magnetic rack. After the magnetic beads are completely adsorbed and the solution becomes clear, remove the solution, being careful not to remove the magnetic beads (leaving about 5µL of solution). Leave the PCR tube on the magnetic rack.

[0066] Pipette 200 µL of 80% ethanol into a PCR tube and let it stand for 30 seconds. Aspirate the solution, being careful not to aspirate the magnetic beads. The 80% ethanol should be prepared on the same day.

[0067] Repeat the previous step.

[0068] Remove the PCR tube and centrifuge for 5 seconds. Insert the PCR tube back into the well of the magnetic holder. After the magnetic beads have completely adhered, use a pipette to remove any remaining solution, ensuring there is no residue on the tube walls. Open the PCR tube cap and allow it to air dry, being careful not to over-dry it.

[0069] Pipette 52µL of nuclease-free water into a PCR tube, gently tap to mix, and react at room temperature for 5 minutes.

[0070] After a brief centrifugation, insert the PCR tube into the well of the magnetic rack. Once the magnetic beads have completely adhered and the solution has become clear, use a pipette to transfer 50 µL into a new, correspondingly numbered PCR tube, being careful not to pick up the magnetic beads.

[0071] Take 1 µL and determine the concentration.

[0072] Product screening Dilute the AMPure PB magnetic beads to a 35% (v / v) screening solution using elution buffer (prepare the appropriate amount based on the actual number of samples). The diluted beads can be stored at 4°C for one month.

[0073] Take 2500 ng of the purified product from the previous step, add nuclease-free water to bring the volume to 50 µL, and add 155 µL of 35% magnetic screening beads.

[0074] Gently tap and invert to mix, centrifuge briefly at low speed for 1 second, and incubate at room temperature for 20 min.

[0075] Place the PCR tube on a magnetic rack and let it stand for 2 minutes until the liquid is clear. Carefully aspirate and discard the supernatant using a pipette.

[0076] Keep the PCR tube on the magnetic rack, add 200 µL of freshly prepared 80% ethanol to rinse the magnetic beads and tube walls, let stand for 30 seconds, then carefully aspirate and discard the supernatant.

[0077] Repeat the previous step.

[0078] Remove the PCR tube from the magnetic rack and place it on the centrifuge with the magnetic beads facing out. Centrifuge briefly at low speed for 1 second, then put it back on the magnetic rack. Let it stand for 5 seconds, then aspirate and discard the supernatant and let it air dry slightly.

[0079] Remove the PCR tube from the magnetic rack, add 32µL of nuclease-free water, and gently pipette 10 times or tap until completely mixed.

[0080] Incubate at room temperature for 5 minutes.

[0081] Briefly centrifuge the PCR tube and place it on a magnetic rack. Let it stand for 2 minutes until the liquid is clear. Slowly aspirate 30 µL of the supernatant and transfer it to a new PCR tube, ensuring that no magnetic beads remain in the supernatant. Aspirate 1 µL and determine the concentration.

[0082] Stop point: The completed library can be stored at -20℃ or used for overnight hybridization capture experiments.

[0083] Part Two: Hybrid Capture Magnetic bead concentration and probe hybridization For each embryo MDA sample, 1500 ng (single-mixed) of pre-capture library was added to a PCR tube, and nuclease-free water was added to bring the volume to 30 µL.

[0084] Add 30 µL of 1X purified magnetic beads (AMPure XP), gently mix, and incubate at room temperature for 10 min.

[0085] Place the PCR tube on a magnetic rack and let it stand for 2 minutes until the liquid is clear. Carefully aspirate and discard the supernatant using a pipette.

[0086] Keep the PCR tube on the magnetic rack, add 200µL of freshly prepared 80% ethanol to rinse the magnetic beads and tube walls, let stand for 30 seconds, then carefully aspirate and discard the supernatant.

[0087] Repeat the previous step.

[0088] Remove the PCR tube from the magnetic rack and place it on the centrifuge with the magnetic beads facing out. Centrifuge briefly at low speed for 1 second, then put it back on the magnetic rack. Let it stand for 5 seconds, then aspirate and discard the supernatant, and air dry to ensure no ethanol residue remains.

[0089] Remove the PCR tube from the magnetic rack, add 28 µL of hybridization reaction solution, and gently pipette 10 times or tap lightly until completely mixed. The hybridization reaction solution is prepared as follows: Prepare hybridization reaction solutions according to the table below (reagent catalog numbers and manufacturers: TargetSeq One® Hyb&Wash Kit v2.0 (for Illumina)# C10332; TargetSeq® Cap Beads&Nuclease-Free Water# C10422; TargetSeq® Target Probes (T917V3)# PT1012692; TargetSeq® Eco Universal Blocking Oligo (for Illumina Nextera)# C80512).

[0090] Add freshly prepared reaction solution to 1.6 to elute (make sure the ethanol has completely evaporated before adding), and incubate at room temperature for 5 min.

[0091] Transfer 28 µL of the eluted supernatant into a new PCR tube, and then add 2 µL of Target Probes.

[0092] Place the PCR tube in the PCR instrument and perform the following reaction.

[0093]

[0094] The recommended hybridization time is 12-18 hours. Before the end of the hybridization process, preparations should be made for the capture experiment.

[0095] Pre-capture experimental preparation Remove the Cap Beads from the 4°C refrigerator beforehand, mix them thoroughly, and allow them to equilibrate at room temperature for 30 minutes. Prepare 80% ethanol in advance with anhydrous ethanol and nuclease-free water, store it temporarily at room temperature, and use it in the purification step; Remove Wash Buffer 1 from Box 2 of TargetSeq One® Hybridization and Wash Kit v2.0. If any precipitate forms, heat Wash Buffer 1 in a 37°C water bath until the precipitate is completely dissolved before use. Remove the TargetSeqOne® Wash Buffer 2 v2 from TargetSeq One® Hybridization and Wash Kit v2.0 - Box 2 and preheat it in a 50 ℃ water bath; Add 50 μL of Cap Beads to a new PCR tube, place it on a magnetic rack for 1 min, and wait for the solution to become clear before discarding the supernatant. Remove the PCR tube from the magnetic rack, add 180 μL of Binding Buffer from TargetSeq One® Hybridization and WashKit v2.0 - Box2, and mix by pipetting or vortexing to resuspend the magnetic beads; After a brief centrifugation, place the PCR tube on a magnetic rack for 1 min until the solution becomes clear, then discard the supernatant. Repeat steps 2.6-2.7 twice, for a total of three cleanings of the magnetic beads using Binding Buffer; Remove the PCR tube from the magnetic rack, add 180 μL of Binding Buffer and mix by pipetting or vortexing, then proceed to the next step immediately.

[0096] Target region DNA capture Keep the hybridization product from STEP 1 on the PCR instrument, add 180 μL of Cap Beads prepared in STEP 2 to the hybridization product, and mix well by pipetting. After capping the tube, remove the PCR tube from the PCR instrument and place it on a vertical spin mixer at a speed not exceeding 10 rpm for 30 minutes at room temperature (if the laboratory does not have a vertical spin mixer, it can be incubated at room temperature for 30 minutes, inverting the tube several times every 5 minutes to mix). Remove the PCR tube, centrifuge briefly, place on a magnetic rack for 2 minutes, and discard the supernatant after the solution becomes clear. Remove the PCR tube from the magnetic rack, add 150 μL of Wash Buffer 1 to the PCR tube, gently aspirate and mix to resuspend the magnetic beads, replace the tube cap, and then place it on a vertical spin mixer to wash at room temperature for 15 min at a speed not exceeding 10 rpm. Remove the PCR tube, centrifuge briefly, place on a magnetic rack for 2 minutes, and discard the supernatant after the solution has clarified. Remove the PCR tube from the magnetic rack, add 150 μL of TargetSeq One® WashBuffer 2 v2 preheated to 50 ℃, gently pipette to mix, centrifuge briefly, and place on a constant temperature shaker or metal bath to incubate at 50 ℃ for 10 min. Remove the PCR tube, centrifuge briefly, place on a magnetic rack for 2 minutes, and discard the supernatant after the solution has clarified. Repeat steps 3.6-3.7 three times, for a total of four washes using TargetSeq One® Wash Buffer 2 v2; Keep the PCR tube on the magnetic rack, add 200 μL of 80% ethanol to the PCR tube, let it stand for 30 seconds, and then completely discard the ethanol solution (you can use a 10 μL pipette to discard the residual ethanol). Let the magnetic beads air dry at room temperature to allow the residual ethanol to evaporate completely. Add 23 μL of Nuclease Free Water to the PCR tube, remove the PCR tube from the magnetic rack, briefly vortex to resuspend and mix the magnetic beads, and proceed to the next step of the amplification reaction.

[0097] Post-capture PCR amplification Prepare the reaction system according to the table below.

[0098] PCR program

[0099] Purification after amplification Add 40 µL of 0.8X purification magnetic beads (AMPure XP) to the PCR product. Gently tap to mix and incubate at room temperature for 10 minutes.

[0100] Insert the PCR tube into the well of the magnetic rack. After the magnetic beads are completely adsorbed and the solution becomes clear, remove the solution, being careful not to remove the magnetic beads (leaving about 5µL of solution). Leave the PCR tube on the magnetic rack.

[0101] Pipette 200 µL of 80% ethanol into a PCR tube and let it stand for 30 seconds. Aspirate the solution, being careful not to aspirate the magnetic beads. The 80% ethanol should be prepared on the same day.

[0102] Repeat the previous step.

[0103] Remove the PCR tube and centrifuge for 5 seconds. Insert the PCR tube back into the well of the magnetic holder. After the magnetic beads have completely adhered, use a pipette to remove any remaining solution, ensuring there is no residue on the tube wall. Open the PCR tube cap and allow it to air dry.

[0104] Pipette 32µL of nuclease-free water into a PCR tube, gently tap to mix, and react at room temperature for 5 minutes.

[0105] Insert the PCR tube into the well of the magnetic holder. After the magnetic beads have been completely adsorbed and the solution has become clear, use a pipette to transfer 30µL into a new PCR tube with the corresponding number. Be careful not to pick up the magnetic beads.

[0106] Take 1 µL of the captured product and determine its concentration. A concentration of 10-20 ng / µL is acceptable.

[0107] Stop point: The captured library can be stored at -20℃ or used to continue experiments to build third-generation libraries.

[0108] Part 3: PacBio Amplicon Library Construction Mixing: Take each captured library and mix them at the same mass, with a total mass of 800-1000 ng.

[0109] If the mixed volume is greater than 46 µL, concentrate with 1X purification magnetic beads (XP) and elute with 46 µL of nuclease-free water; if the mixed volume is less than 46 µL, make up to 46 µL with nuclease-free water.

[0110] Damage repair & end repair & A addition The following system was prepared on ice.

[0111] Gently tap to mix, then briefly centrifuge.

[0112] Place the reaction tube in the PCR instrument and run the PCR instrument according to the following procedure (cover temperature 75℃, volume 60 µL):

[0113] Connector connection and purification Add 4 µL of SMRTbell adapter 3.0 with barcode to the product of the previous reaction step; Prepare the following components on ice.

[0114] Gently mix; Place the PCR tube on the PCR instrument and run the PCR instrument according to the following procedure (cover temperature OFF, volume 95 µL).

[0115] After the reaction was complete, 124 µL of SMRTbell cleanup beads were added to the ligation product; the mixture was gently shaken and inverted to mix, and then centrifuged briefly at low speed for 1 second.

[0116] Incubate at room temperature for 10 minutes.

[0117] Place the PCR tube on a magnetic rack and let it stand for 2 minutes until the liquid is clear. Carefully aspirate and discard the supernatant using a pipette.

[0118] Keep the PCR tube on the magnetic rack, add 200 µL of freshly prepared 80% ethanol to rinse the magnetic beads and tube walls, let stand for 30 seconds, then carefully aspirate and discard the supernatant.

[0119] Repeat the previous step.

[0120] Remove the PCR tube from the magnetic rack and place it on the centrifuge with the magnetic beads facing outwards. Centrifuge briefly at low speed for 1 second, then place it back on the magnetic rack. Let it stand for 5 seconds, then aspirate and discard the supernatant.

[0121] Remove the PCR tube from the magnetic rack and immediately add 40 µL of elution buffer. Use a wide-mouth pipette tip to slowly pipette 10 times until completely mixed.

[0122] Incubate at room temperature for 5 minutes.

[0123] After briefly centrifuging the PCR tube, place it on a magnetic rack and let it stand for 2 minutes until the liquid is clear. Slowly transfer 40 µL of supernatant to a new PCR tube, ensuring that there are no magnetic beads remaining in the supernatant.

[0124] Enzymatic digestion and product purification Prepare the following mixture on ice and gently stir to mix.

[0125]

[0126] Place the PCR tubes on the PCR instrument and run the PCR instrument according to the following procedure (cover temperature 75℃, volume 50 µL).

[0127] Add 65µL of SMRTbell cleanup beads to the digestion product. Gently tap and invert to mix, then centrifuge briefly at low speed for 1 second.

[0128] Incubate at room temperature for 10 minutes.

[0129] Place the PCR tube on a magnetic rack and let it stand for 2 minutes until the liquid is clear. Carefully aspirate and discard the supernatant using a pipette.

[0130] Keep the PCR tube on the magnetic rack, add 200µL of freshly prepared 80% ethanol to rinse the magnetic beads and tube walls, let stand for 30 seconds, then carefully aspirate and discard the supernatant.

[0131] Repeat the previous step.

[0132] Remove the PCR tube from the magnetic rack and place it on the centrifuge with the magnetic beads facing outwards. Centrifuge briefly at low speed for 1 second, then place it back on the magnetic rack. Let it stand for 5 seconds, then aspirate and discard the supernatant.

[0133] Remove the PCR tube from the magnetic rack, immediately add 15 µL of elution buffer, and gently tap to mix.

[0134] Incubate at room temperature for 5 minutes.

[0135] After briefly centrifuging the PCR tube, place it on a magnetic rack and let it stand for 2 minutes until the liquid is clear. Transfer 36 µL of supernatant to a new PCR tube, ensuring that there are no magnetic beads remaining in the supernatant.

[0136] Transfer the eluted library to a new 1.5 mL EP tube. Take 1 µL to determine the concentration. A total library volume of at least 300 ng is considered acceptable. Example 5:

[0137] Detection of positive samples: 32 positive embryo samples with known variant information were tested using the library construction kit of this invention. The overall performance met expectations, as shown in Table 6.

[0138] Table 6

[0139] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A primer kit for targeted amplification of WGA products from third-generation sequencing embryos, characterized in that, include: The targeted capture probe set includes specific probes targeting the full-length region, copy number variation region, and single nucleotide polymorphism sites within the 0-5Mb range upstream and downstream of the pathogenic gene of a single-gene genetic disease. The specific probe is 100nt in length, and the tiling spacing of the specific probe in the target area is one probe every 450bp. Library amplification primer set; Library construction reagents include fragmentation reagents containing Tn5 transposase, long-fragment PCR amplification enzymes, and fragment screening reagents for removing nucleic acid fragments shorter than 5kb.

2. The kit for targeted amplification primer set based on third-generation sequencing embryo WGA products according to claim 1, characterized in that: The targeted capture probe set has probe dose compensation in the high GC region of the target area, and the error tolerance of the sequence of the specific probe and the sequence of the target area is less than 5%.

3. The kit for targeted amplification primer set based on third-generation sequencing embryo WGA products according to claim 1, characterized in that: The pathogenic gene for the single-gene hereditary disease is selected from at least one of ASS1, ATP7B, CYP21A2, DMD, F8, F9, FMR1, FXN, GAA, GBA1, GJB2, GLA, HBA1 / HBA2, HBB, IDS, MMACHC, MMUT, PAH, SLC22A5, SLC25A13, SLC26A4, and STS.

4. The kit for targeted amplification primer set based on third-generation sequencing embryo WGA products according to claim 1, characterized in that: The fragment screening reagent is AMPure PB screening magnetic beads diluted to a volume concentration of 35%; the long fragment PCR amplification enzyme is PK512 DNA polymerase.

5. The kit for targeted amplification primer set based on third-generation sequencing embryo WGA products according to claim 1, characterized in that: The sequence of the specific probe is SEQ ID NO: 0001-1303.

6. A method for in vitro construction of a third-generation targeted sequencing library of embryonic WGA products, characterized in that, Using the targeted amplification primer kit based on third-generation sequencing embryo WGA products as described in any one of claims 1-5, the method comprises the following steps: Fragmentation and adapter introduction: The purified embryonic WGA product was fragmented and sequenced using the fragmentation reagent to obtain fragmented products; Pre-capture library amplification: The fragmented product was amplified by PCR using the library amplification primer set and the long fragment PCR amplification enzyme to obtain the original pre-capture library; Fragment screening: The original pre-capture library is screened using the fragment screening reagent to remove nucleic acid fragments with a length of less than 5kb, thereby obtaining the screened pre-capture library; Hybridization capture and elution: The screened pre-capture library and the target capture probe group were added to the hybridization reaction solution for liquid-phase hybridization incubation, streptavidin magnetic beads were added to bind the target fragment, and a washing solution was used for washing. Third-generation sequencing library construction: The target fragment after washing and elution was amplified using PCR primers to obtain a captured library, and the captured library was subjected to damage repair, end repair, A addition reaction and third-generation sequencing adapter ligation reaction in sequence.

7. The in vitro construction method according to claim 6, characterized in that, In the fragmentation and connector introduction steps: The total volume of the reaction system for a single interruption reaction is 50 μL, the amount of Tn5 transposase in the reaction system is 3 μL, and the reaction time of the interruption reaction is 10 minutes; the main peak length of the nucleic acid fragment of the obtained interruption product is distributed between 6kb and 7kb.

8. The in vitro construction method according to claim 6, characterized in that, In the hybridization capture and elution step: The cleaning operation is as follows: add a preheated hot cleaning buffer to the streptavidin magnetic beads, incubate and clean at a constant temperature of 50°C, and repeat the constant temperature incubation and cleaning operation a total of 4 times.

9. A data processing method, characterized in that, Performed by a computer system, including the following steps: Obtain circularly consistent sequence sequencing data generated from a third-generation sequencing library prepared by the in vitro construction method according to any one of claims 6-8; The circular consensus sequence sequencing data is aligned to the target region of the reference genome, and split reads are extracted; Assess read internal consistency and filter out chimeric molecular fragments and aberrant connections introduced by embryonic whole-genome amplification in the sequencing data; Based on the filtered sequencing data, local genome assembly and variant detection are performed, and local haplotypes are constructed based on the single nucleotide polymorphism sites to output genetic variation status data of the target embryo region.

10. The application of the kit for targeted amplification primer set based on third-generation sequencing embryo WGA product as described in any one of claims 1-5 in the preparation of in vitro detection products for preimplantation genetic diseases.

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