CN-122012701-A - Single-molecule sequencing detection kit and system for genetic integration before embryo implantation

Abstract

The invention belongs to the field of genetic detection, and particularly relates to a single-molecule sequencing detection kit and a single-molecule sequencing detection system for genetic integration before embryo implantation. Specifically, the kit and the system provided by the invention can realize synchronous detection of embryo aneuploidy, copy number variation, chromosome structure abnormality and monogenic diseases before human implantation. The invention carries out single-molecule long-reading long-sequencing on both the parent sample and the embryo sample, does not need a prover and family members except the parent, realizes direct detection on aneuploidy, copy number variation, chromosome structure abnormality and the like and indirect detection by monomer type inference, and has accurate, sensitive, visual and comprehensive detection results. According to the kit and the system, the PGT-A, PGT-M, PGT-SR and the monomer genetic condition detection can be completely supported only by the same experimental system and the same single-molecule long-fragment sequencing platform, the flow is simple and convenient, the application range is wide, and therefore, the kit and the system have good practical application value.

Inventors

• GAO YUAN
• GAO YANG
• WANG RONGYI
• GAO MING
• CHEN DI
• GAO XUAN
• MAO AIPING
• MA JINLONG
• LI SHUJUAN
• CHEN ZIJIANG

Assignees

• 山东大学
• 北京贝瑞和康生物技术有限公司

Dates

Publication Date

20260512

Application Date

20260415

Claims (20)

1. 1. A kit for integrated pre-implantation genetic testing of embryos based on long-read long single molecule sequencing, the kit comprising the following reagents: (1) Reagents for whole genome amplification of embryo samples; (2) Reagents for constructing a long fragment sequencing library of a parent sample genome; (3) Reagents for constructing a long fragment sequencing library of a genome of an embryo sample; Wherein the reagent for constructing the embryo sample genome long fragment sequencing library at least comprises an endonuclease, and the endonuclease is T7 endonuclease I and/or FEN1.
2. 2. The kit of claim 1, wherein the reagents for whole genome amplification of an embryo sample comprise a whole genome amplification buffer, random primers, and a whole genome amplification DNA polymerase.
3. 3. The kit of claim 1, wherein the reagents for constructing a long fragment sequencing library of a parent sample genome comprise a DNA disruption tube, a repair buffer, a DNA repair enzyme, a DNA end repair enzyme, a ligation buffer, a sequencing linker, a ligase, an exonuclease, and DNA purification magnetic beads.
4. 4. The kit of claim 1, wherein the reagents for constructing a long fragment sequencing library of a genome of an embryo sample comprise an endonuclease buffer, an amplification linker, an amplification primer, a PCR DNA polymerase, a repair buffer, a DNA repair enzyme, a DNA end repair enzyme, a ligation buffer, a sequencing linker, a ligase, an exonuclease, and a DNA purification bead.
5. 5. The kit of claim 1, wherein the parent sample comprises peripheral blood or tissue of parent origin.
6. 6. The kit of claim 1, wherein the embryo sample is a biopsy of an embryo at blastomere or blastocyst stage.
7. 7. The kit of claim 1, wherein the single molecule sequencing is a single molecule real-time sequencing or nanopore based technology platform.
8. 8. The kit of any one of claims 1-7, wherein the kit is used for pre-embryo implantation genetic integrated detection based on long-read long single molecule sequencing of a parental sample and an embryo sample.
9. 9. The kit of claim 8, wherein the pre-embryo implantation genetic integrated assay comprises a simultaneous assay of PGT-A, PGT-M, PGT-SR.
10. 10. The kit of claim 9, wherein the pre-embryo implantation genetic integrated detection comprises haplotype construction and linkage analysis and simultaneous detection of aneuploidy, copy number variation, single nucleotide variation, deletions, insertions, and chromosomal structural variations.
11. 11. A system for integrated pre-embryo implantation genetic testing based on long-read long single molecule sequencing, comprising the following modules: (1) A whole genome amplification module for obtaining a whole genome amplification product of the embryo sample; (2) A pretreatment module for converting the whole genome amplification product into a pretreatment product suitable for use in an amplification module; (3) An amplification module for amplifying and converting the pretreatment product into an amplification product suitable for constructing a long fragment single molecule sequencing library; (4) A fragmentation module for obtaining a genomic fragmentation product of a parent sample; (5) The library construction module is used for constructing a long fragment single molecule sequencing library; (6) The sequencing module is used for high-throughput long-reading long single-molecule sequencing; wherein the pretreatment module converts the whole genome amplification product into a pretreatment product suitable for the amplification module by adopting endonuclease, and the endonuclease is T7 endonuclease I and/or FEN1.
12. 12. The system of claim 11, wherein the whole genome amplification module obtains whole genome amplification products of an embryo sample using reagents comprising a whole genome amplification buffer, random primers, and a whole genome amplification DNA polymerase.
13. 13. The system of claim 11, wherein the amplification module uses reagents comprising amplification adaptors, amplification primers, and PCR DNA polymerase to amplify and convert the pretreatment products to amplification products suitable for use in constructing a long fragment single molecule sequencing library.
14. 14. The system of claim 11, wherein the fragmentation module obtains a genomic fragmentation product of the parent sample using a reagent comprising a DNA disruption tube.
15. 15. The system of claim 11, wherein the library construction module constructs a long fragment single molecule sequencing library using reagents comprising a repair buffer, a DNA repair enzyme, a DNA end repair enzyme, a ligation buffer, a sequencing linker, a ligase, an exonuclease, and DNA purification magnetic beads.
16. 16. The system of claim 11, wherein the sequencing module performs high throughput long read long single molecule sequencing using a single molecule real time sequencing or nanopore technology platform.
17. 17. The system of claim 11, wherein the parent sample comprises peripheral blood or tissue of parent origin.
18. 18. The system of claim 11, wherein the embryo sample is a biopsy of an embryo at blastomere or blastocyst stage.
19. 19. The system of any one of claims 11-18, wherein the system is used for pre-embryo implantation genetic integrated detection based on long-read long single molecule sequencing of a parent sample and an embryo sample.
20. 20. The system of claim 19, wherein the pre-embryo implantation genetic integrated test comprises a simultaneous test of PGT-A, PGT-M, PGT-SR.

Description

Single-molecule sequencing detection kit and system for genetic integration before embryo implantation Technical Field The invention belongs to the field of genetic detection, and particularly relates to a single-molecule sequencing detection kit and a single-molecule sequencing detection system for genetic integration before embryo implantation. Background The genetic test (preimplantation GENETIC TESTING, PGT) before embryo implantation is a kind of genetic test for in vitro fertilized embryo generated in auxiliary reproduction process, and aims to select embryo to be transplanted into mother uterus from genetic aspect, avoiding carrying part genetic variation and implantation failure, abortion or birth defect caused by the genetic variation. PGT is generally classified into one of three categories, chromosome aneuploidy detection before embryo implantation (PGT for aneuploidy, PGT-A), while embryo aneuploidy (aneuploidy) and copy number variation (copy number variant, CNV) are detected. Secondly, single-gene genetic disease detection (PGT for monogenic disease, PGT-M) before embryo implantation mainly aims at the situation that parents carry specific gene mutation. Third, detection of chromosomal structural abnormalities (PGT for structural rearrangement, PGT-SR) prior to embryo implantation, detection of chromosomal structural rearrangements, such as inversion, balanced translocation, rogowski translocation, insertion translocation, etc., for the parent. PGT techniques first obtain genetic material available for detection by biopsy, and embryo biopsies of blastocysts only obtain a few cells with limited genetic material, so whole genome amplification is typically performed prior to PGT (whole genome amplification, WGA). There are a number of WGA modes at present, of which MDA (multiple displacement amplification) is widely used due to its simplicity of operation, high yield, good coverage, high fidelity. The fragment of the WGA product of the MDA method is longer, and the fragment has the potential of adapting to a single-molecule long-reading long-sequencing platform, however, because the MDA product has a complex branched structure, when the single-molecule long-reading long library is constructed according to a general procedure, the sequencing yield is extremely low, and the application of the single-molecule long-reading long-sequencing technology on embryo biopsy samples is limited. In the case of PGT-M and PGT-SR, it is necessary to perform haplotype linkage analysis. The reason is that, firstly, there are unavoidable amplification bias, allele Drop (ADO) and possibility of introducing amplification errors in WGA, so that there is a risk of false detection and omission in the detection of embryo directly, and secondly, for some genetic variations, such as variations occurring in highly homologous regions or structural variations such as balanced translocation, it is more difficult for the prior art means to directly detect based on embryo WGA DNA, and indirect inference is necessary depending on linkage relationship. Currently, there are two types of linkage detection methods, short TANDEM REPEAT (STR) analysis, SNP (single-nucleotide polymorphism) analysis based on chip and second generation sequencing (next-generation sequencing, NGS) technology. The methods are limited to Indel detection, SNP detection and the like, do not directly provide haplotype information, rely on genetic lineage analysis when constructing linkage relationship, namely, detection of a ancestor or two-generation family member is required besides an embryo and a parent, and detection of new variation of the parent or homologous recombination near a target area of the embryo is difficult. In order to overcome the limitation, a series of PGT schemes based on single-molecule long-reading long-sequencing (also called three-generation sequencing) are developed in recent years, and the PGT schemes are mainly characterized in that high-throughput single-molecule long-reading long-sequencing is carried out on single-gene disease or structural variation carriers in parents, genotypes and haplotypes of target detection areas are obtained simultaneously, SNP information is provided for linkage analysis when PGT-A is realized by using NGS or chips for embryos, and genetic information of the embryos is obtained by linkage analysis. The method overcomes the dependence on the ancestor and the family members outside parents, has the capability of detecting new variation, and still stays on the NGS or chip level for embryo detection, namely the whole is still in a state inferred indirectly by the dependence linkage relationship, so that the direct detection of embryo genotypes and haplotypes cannot be comprehensively realized. In addition, the method needs means of integrating a plurality of technical platforms, such as 'third generation sequencing and second generation sequencing', or 'third generation sequencing and chip', and the like, r