Primer set, kit and application thereof for detecting multiple mutations of human embryonic α-thalassemia

Abstract

The present invention relates to a primer set, a kit and an application thereof for detecting multiple mutations of human embryonic α-thalassemia. The primer set includes a primer set for amplifying HBA 5 primers for multiple mutations in the gene region: HBA-F1, HBA-F2, HBA-F3, HBA-R1 and HBA-R2, whose sequences are shown in SEQ ID NO:1-5 respectively; and for amplifying HBA 8 primers for linked SNP sites within 1Mb upstream and downstream of the gene: L1-F, L1-R, L2-F, L2-R, L3-F, L3-R, L4-F and L4-R, whose sequences are SEQ ID NO: 6-13. The present invention based on a specific combination of long-fragment PCR amplification and long-fragment high-throughput sequencing can detect multiple mutations of α-thalassemia in multiple embryo samples simultaneously with high specificity, accuracy and rapidity.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and in particular to a primer set, a kit and applications thereof for detecting multiple mutations of human embryonic α-thalassemia using a long-fragment long-read sequencing platform. Background Art

[0002] Thalassemia is the most common single gene genetic disease in the world. It is an autosomal recessive disease. High-incidence areas include southern China, Southeast Asia, the Mediterranean region, India, the Middle East and Africa. Thalassemia is mainly divided into α-thalassemia and β-thalassemia, which are caused by the lack or deficiency of α-globin or β-globin synthesis. 1,2,3 .

[0003] The α-globin gene cluster is located on chromosome 16 and contains 7 gene loci: 5'- HBZ - HBZps - HBM - HBA1ps - HBA2 (α2)- HBA1 (α1)- HBQ -3'. Among them only HBA1 and HBA2 Both genes have the ability to encode globin in both embryos and adults. HBA1 and HBA2 Gene mutations include deletion mutations and non-deletion mutations. Based on the Ithanet, HbVar, LOVD and LOVD-China thalassemia databases, about 50 deletions in the α-globin gene cluster region and more than 900 non-deletion mutations in HBA1 and HBA2 genes have been found worldwide. In addition, there are some rare structural variations, such as ααα anti3.7 , ααα anti4.2 , HKαα and antiHKαα, etc. The β-globin gene cluster is located on chromosome 11 and contains 5 gene loci: 5'- HBE - HBG2 - HBG1 - HBD (δ)- HBB (β)-3'. HBB The gene encodes globin in adults, and the other four genes are expressed during embryonic development or are expressed at very low levels in adults. The vast majority of mutations that cause β-thalassemia are non-deletional, and a few are large deletions. 4,5 Based on the Ithanet, HbVar, LOVD and LOVD-China thalassemia databases, more than 1,000 non-deletion mutations of the HBB gene have been found worldwide. So far, 129 non-deletion mutations of the β-globin gene have been found in the Chinese population.

[0004] Thalassemia is congenital, lifelong and familial. There is no ideal treatment for it. Patients need lifelong treatment, which not only causes pain but also comes at a high cost. 3 . However, since genetic diseases usually occur through the reproductive behavior of gene carriers, assisted reproductive technology can be used to block their transmission to future generations and reduce the number of patients with genetic diseases. Preimplantation genetic testing (PGT) is a genetic test of embryos before they are implanted in the uterus, selecting normal or non-pathogenic embryos for transplantation.

[0005] PGT includes preimplantation chromosomal aneuploidy detection (PGT-A), preimplantation chromosomal structural rearrangements (PGT-SR) and preimplantation single-gene defects detection (PGT-M). PGT-A is for patients with advanced female age, recurrent miscarriage, repeated implantation failure, adverse pregnancy history and severe male teratozoospermia. PGT-SR is for patients with chromosomal structural abnormalities in both or one of the parents, such as inversion, balanced translocation, Robertsonian translocation, insertion translocation, etc. PGT-M is for parents who suffer from or carry known single-gene genetic diseases, such as thalassemia, and tests the embryos for single-gene genetic diseases before embryo implantation. 6,7,8 . PGT technology first needs to use biopsy technology to obtain embryonic genetic material that can be used for testing. Biopsy technology can be divided into polar body biopsy, blastomere biopsy and blastocyst trophoblast cell biopsy according to time: polar body biopsy is to obtain polar bodies on the day of egg retrieval (or the first day after fertilization) for genetic testing; blastomere biopsy is on the third day after fertilization (6-8 cell stage), to obtain 1-2 cells for genetic testing; blastocyst trophoblast cell biopsy is on the fifth or sixth day after fertilization, when the embryo develops to the blastocyst stage, to obtain 5-10 trophoblast cells for genetic testing 9 .

[0006] Since the number of cells obtained by PGT technology is very small and the available genetic material is limited, whole genome amplification (WGA) is generally required. Currently, whole genome amplification methods are mainly divided into three categories. One is based on polymerase chain reaction (PCR) technology, such as primer extension preamplification PCR (PEP-PCR) and degenerate oligomer primer PCR (DOP-PCR); the other is constant temperature whole genome amplification reaction, such as multiple displacement amplification (MDA); the third is multiple annealing and looping-based amplification cycles (MALBAC). Although there are many commercial kits now, and the key parameters are different, each method and each kit will have amplification preferences, allele dropout (ADO) and coefficient of variation, etc. 10 , resulting in false negatives or false positives, leading to diagnostic failure or even misdiagnosis.

[0007] In order to improve the accuracy of diagnosis, PGT-M needs to conduct haplotype linkage analysis at the same time. There are currently three mainstream linkage analysis methods: short tandem repeat (STR), SNP chips (single-nucleotide polymorphisms arrays) and next-generation sequencing (NGS) technology. 7. However, STR technology is relatively cumbersome and costly, and STR and SNP chips can only analyze known sites, which will miss some unknown potential SNP sites that can be used for haploid typing. NGS technology can only detect DNA fragments of about 300 bases, but the average interval of SNP sites in the human genome is about 1000 bases. Therefore, NGS can only obtain less than one SNP site from each DNA fragment on average, so it is difficult to find continuous SNPs, and only limited SNP sites can be selected to construct haplotypes, resulting in a waste of SNP sites. And the above three methods all require probands, but some families do not have probands or cannot obtain the genetic material of the probands. At present, there is also a third-generation whole genome sequencing method that does not require a proband, but this method is expensive and has poor market accessibility and promotion in clinical applications. For example, in the prior art, patent application CN108531582A discloses a primer combination and method for detecting human embryonic α-thalassemia gene mutations. Using a primer combination consisting of 1354 primers and based on NGS technology, the method realizes the detection of thalassemia-related gene mutations. In addition, this method uses the location and genotype of the polymorphic sites (SNPs) on the chromosomes that are closely linked to the α-thalassemia pathogenic gene HBA1 in the parents and the proband to construct a haplotype, and uses the constructed haplotype to determine whether the embryo is pathogenic.

[0008] Currently, methods based on STR, SNP chips, NGS and long-read whole genome sequencing have the following limitations:

[0009] 1. Dependence on the proband: However, some families do not have a proband or cannot obtain the proband's genetic material;

[0010] 2. Low efficiency of linkage analysis: only known loci can be used, or only limited loci can be selected to construct haplotypes, resulting in a waste of linkage loci and requiring many linkage fragments for analysis;

[0011] 3. High cost: Traditional methods are cumbersome, costly, and time-consuming, and third-generation whole genome sequencing is expensive. Summary of the invention

[0012] The purpose of the present invention is to solve the problems that the current detection of human embryonic α-thalassemia depends on the proband, the linkage analysis is inefficient, the detection and analysis are cumbersome, the cost is high, and the time is long. HBA Four linked fragments of linked SNP sites within 1Mb upstream and downstream of the gene, and preparation of long-fragment sequencing libraries, to achieve comprehensive, accurate, efficient and rapid detection of multiple samples HBAThe goal of detecting multiple gene mutations avoids the need for a proband while ensuring the accuracy and sensitivity of embryo testing, and reduces the cost pressure on patients.

[0013] In view of this, the present invention provides a method based on multiple long-fragment PCR amplification and long-fragment sequencing for detecting multiple mutations in human embryonic α-thalassemia without the need for a proband. The long-fragment PCR amplification is implemented in one reaction tube, and the amplification for detection is carried out separately. HBA1 / 2 Fragments for all point mutations within the primer range of the gene locus and gap fragments for detecting large deletions, and HBA Linked fragments of linked SNP sites within 1Mb upstream and downstream of the gene; combined with the read length measurement of the long-fragment sequencing platform, it can achieve accurate, rapid and high-throughput detection of gene mutations in human embryonic α-thalassemia. The method involved in the present invention is simple to operate, and the quality of long-fragment PCR and long-fragment library is reliable and reproducible, which is conducive to the application of long-fragment sequencing technology in clinical detection.

[0014] A first aspect of the present invention provides a primer set for simultaneously amplifying HBA Multiple mutations in the gene region and their linked single nucleotide polymorphism (SNP) sites within 1Mb upstream and downstream.

[0015] Used for amplification HBA The five primers for various mutations in the gene region are as follows: HBA-F1, HBA-F2, HBA-F3, HBA-R1, and HBA-R2, and their sequences are shown in SEQ ID NOs: 1-5, respectively;

[0016] Used for amplification HBA The eight primers for the linked SNP sites within 1 Mb upstream and downstream of the gene are as follows: L1-F, L1-R, L2-F, L2-R, L3-F, L3-R, L4-F and L4-R, and their sequences are shown in SEQ ID NOs: 6-13, respectively.

[0017] The positions of the 13 primers are as follows: Figure 1 The primers can simultaneously amplify HBA1 / 2 The complete sequence within the primer range of the gene locus, including any type of mutation sequence within the primer range, and the sequence of the linked fragment within 1Mb upstream and downstream.

[0018] In some embodiments, the primer set is used to simultaneously detect one or more of the following mutations: point mutations, structural variations, and SNP sites linked within the upstream and downstream 1Mb range within the primer range of the HBA1 / 2 gene locus; wherein the point mutations and the structural variations include those from the four thalassemia databases of Ithanet, HbVar, LOVD, and LOVD-China. HBA Gene mutation.

[0019] In some preferred embodiments, the structural variation comprises a large fragment deletion. For example, the large fragment deletion includes --SEA, -α3.7, -α4.2, --THAI, --FIL, -α-9.7, --11.1, 5.3kb_deletion, --BRIT, --CANT, --GEO, --JS, --MED-II, --NOR, Qinzhou type deletion, --SA, --SPAN, --THAI, -α0.8, -α1.2, -α2.4, -α2.7, -α2.8, -α20.5, -α3.5, -α3.7, -α5.2 and -α5.6.

[0020] In some embodiments, the size of the amplified fragment of the primer is 1k to 20kb, that is, the size of the amplified product is about 1k to 20kb.

[0021] In some preferred embodiments, if there is a SNP at the primer position, a degenerate base primer is used.

[0022] In a specific embodiment, the primer set is used to complete multiplex-long fragment PCR amplification in one reaction tube.

[0023] In a preferred experimental scheme, the primer set is used for multiplex-long fragment PCR amplification of a system HBA Gene fragments and concatenated fragments, totaling more than 5 target fragments.

[0024] In a preferred embodiment, the primer set amplifies more than 5 target fragments in one system, including HBA At least 1 fragment in the gene region and 4 fragments in the linkage region, so that at least: HBA1 / 2All point mutations within the primer range of the gene locus, 28 large deletions (including --SEA, -α3.7, -α4.2, --THAI, --FIL, -α-9.7, --11.1, 5.3kb deletion, --BRIT, --CANT, --GEO, --JS, --MED-II, --NOR, Qinzhou type deletion, --SA, --SPAN, --THAI, -α0.8, -α1.2, -α2.4, -α2.7, -α2.8, -α20.5, -α3.5, -α3.7, -α5.2 and -α5.6), and SNP sites linked within 1Mb upstream and downstream. Point mutations and structural variations include those from four thalassemia databases (Ithanet, HbVar, LOVD and LOVD-China) HBA Gene mutation.

[0025] In one embodiment, DNA of different sequences of 5-50 nt, i.e., DNA barcodes, can be added to the 5' end of the primers to distinguish different samples; preferably, the 5' end barcodes of the F and R primers can be the same or different, and those skilled in the art can choose as needed.

[0026] In one embodiment, the primer set is used to simultaneously directly detect haplotypes of the HBA gene region and long linkage fragments, the detection using blastocyst and parental samples to infer haplotype linkage and is a PGT-M test independent of the proband.

[0027] The primer set of the present invention can be used for multiple-long fragment PCR amplification of a system including all fragments within the primer range. Combined with the subsequent long fragment sequencing platform, it can directly detect HBA The haplotypes of gene fragments and long linkage fragments use blastocyst and parental samples to infer haplotype linkage, truly realizing PGT-M testing that is independent of the proband.

[0028] The second aspect of the present invention provides a kit for simultaneously detecting multiple mutations of human embryonic α-thalassemia, comprising the following reagents:

[0029] (1) Reagents for multiplex long-fragment PCR amplification;

[0030] (2) Reagents for constructing long-fragment sequencing libraries;

[0031] The reagents for multiple long-fragment PCR amplification include the primer set described in the first aspect of the present invention.

[0032] In a preferred embodiment, the primer set in the kit is selected from the following 13 (primer positions are as follows: Figure 1 Multiple primers in the primer set (shown):

[0033] For amplification HBA The five primers for various mutations in the gene region are as follows: HBA-F1, HBA-F2, HBA-F3, HBA-R1, and HBA-R2, and their sequences are shown in SEQ ID NOs: 1-5, respectively;

[0034] For amplification HBA The eight primers for the linked SNP sites within 1 Mb upstream and downstream of the gene are as follows: L1-F, L1-R, L2-F, L2-R, L3-F, L3-R, L4-F and L4-R, and their sequences are shown in SEQ ID NOs: 6-13, respectively.

[0035] The primers can simultaneously amplify HBA1 / 2 The complete sequence within the primer range of the gene locus, including any type of mutation sequence within the primer range, and the sequence of the linked fragment within 1Mb upstream and downstream.

[0036] In some embodiments, the kit is used to simultaneously detect one or more of the following mutations: point mutations within the primer range of the HBA1 / 2 gene locus, structural variations, and SNP sites linked within 1 Mb upstream and downstream; wherein the point mutations and the structural variations include mutations from four thalassemia databases, including Ithanet, HbVar, LOVD, and LOVD-China. HBA Gene mutation.

[0037] In some preferred embodiments, the structural variation comprises a large fragment deletion. For example, the large fragment deletion includes --SEA, -α3.7, -α4.2, --THAI, --FIL, -α-9.7, --11.1, 5.3kb_deletion, --BRIT, --CANT, --GEO, --JS, --MED-II, --NOR, Qinzhou type deletion, --SA, --SPAN, --THAI, -α0.8, -α1.2, -α2.4, -α2.7, -α2.8, -α20.5, -α3.5, -α3.7, -α5.2 and -α5.6.

[0038] In some embodiments, the size of the amplified fragment of the primer is 1k to 20kb, that is, the size of the amplified product is about 1k to 20kb.

[0039] In some preferred embodiments, if there is a SNP at the primer position, a degenerate base primer is used.

[0040] In one embodiment, DNA of different sequences of 5-50 nt, i.e., DNA barcodes, can be added to the 5' end of the primers to distinguish different samples; preferably, the 5' end barcodes of the F and R primers can be the same or different, and those skilled in the art can choose as needed.

[0041] In one embodiment, the reagents for multiple long-fragment PCR amplification further include DNA polymerase, reaction buffer and primers.

[0042] In one embodiment, the long-fragment PCR amplification product obtained using the kit may be purified or not before the next step of the reaction, and those skilled in the art may choose as needed.

[0043] In a specific embodiment, the multiplex-long fragment PCR amplification using the kit is completed in one reaction tube.

[0044] In a preferred experimental scheme, the kit is used for multiplex-long fragment PCR amplification of a system HBA Gene fragments and concatenated fragments, totaling more than 5 target gene fragments.

[0045] In a preferred embodiment, the kit amplifies more than 5 target fragments in one system, including HBA At least 1 fragment in the gene region and 4 fragments in the linkage region, so that at least: HBA1 / 2 All point mutations within the primer range of the gene locus, 28 large deletions (including --SEA, -α3.7, -α4.2, --THAI, --FIL, -α-9.7, --11.1, 5.3kb deletion, --BRIT, --CANT, --GEO, --JS, --MED-II, --NOR, Qinzhou type deletion, --SA, --SPAN, --THAI, -α0.8, -α1.2, -α2.4, -α2.7, -α2.8, -α20.5, -α3.5, -α3.7, -α5.2 and -α5.6), and SNP sites linked within 1Mb upstream and downstream. Point mutations and structural variations include those from four thalassemia databases (Ithanet, HbVar, LOVD and LOVD-China) HBA Gene mutation.

[0046] In one embodiment, the kit is used to directly detect the haplotype of the HBA gene region and the long linkage fragment simultaneously, the detection uses blastocyst and parental samples to infer the haplotype linkage and is a PGT-M test independent of the proband.

[0047] In a preferred embodiment, the long fragment sequencing is selected from the SMRT sequencing based on Pacific Biosciences (PacBio) or the Nanopore sequencing platform of ONT.

[0048] In a specific embodiment, the SMRT library adapter ligation can be performed using blunt-end ligation or TA ligation.

[0049] In a specific embodiment, the SMRT universal blunt-end adapter sequence is 5'-pATCTCTCTCTTTTCCTCCTCCTCCGTTGTTGTTGTTGAGAGAGAT-3' (SEQ ID NO: 14), and a blunt-end stem-loop structure adapter adapter is formed by annealing. DNA (Barcode) of different sequences of 5-50nt can be added to the stem to form adapter adapters with different Barcodes. SMRT libraries with different Barcodes can be mixed together for sequencing.

[0050] In a specific embodiment, the SMRT universal TA adapter sequence is 5'-pATCTCTCTCTTTTCCTCCTCCTCCGTTGTTGTTGTTGAGAGAGATT-3' (SEQ ID NO: 15), and a blunt-ended stem-loop structure adapter adapter is formed by annealing. DNA (Barcode) of different sequences of 5-50nt can be added to the stem to form adapter adapters with different Barcodes. SMRT libraries with different Barcodes can be mixed together for sequencing.

[0051] In one embodiment, the SMRT connector may or may not have a barcode. Preferably, the SMRT connector has a barcode designed by PacBio or a self-designed barcode, and those skilled in the art can select one according to their needs.

[0052] In a preferred embodiment, the SMRT library is compatible with the sequencing platform of PacBio.

[0053] In a preferred embodiment, the reagents used to construct the long-fragment Nanopore library include end-repair enzymes, linkers, ligases, DNA purification magnetic beads, 80% ethanol, and reaction buffer.

[0054] In one embodiment, Nanopore library adapter ligation can be performed using blunt-end ligation or TA ligation.

[0055] In one embodiment, the Nanopore connector may or may not have a barcode. Preferably, the Nanopore connector has a barcode designed by ONT or a self-designed barcode, and those skilled in the art can select one according to their needs.

[0056] In a preferred embodiment, the Nanopore library matches the sequencing platform of ONT.

[0057] The third aspect of the present invention provides a system for simultaneously detecting multiple mutations of human embryonic α-thalassemia, which comprises the following modules:

[0058] (1) Collection module: obtaining samples from subjects;

[0059] (2) Amplification module: The sample is subjected to multiple long-fragment PCR amplification, including HBA Related fragments of genes, such as detection HBA1 / 2 1 fragment for all point mutations within the primer range of the gene locus and a Gap fragment for detecting large deletions, and HBA Four linked fragments within 1 Mb upstream and downstream of the gene;

[0060] (3) Library construction module: construction of long-fragment sequencing library;

[0061] (4) Sequencing module: sequencing and analyzing gene mutation types and linkage relationships between different fragments;

[0062] The multiple long-fragment PCR amplification in the amplification module is performed using the primer set described in the first aspect of the present invention or the kit described in the second aspect of the present invention.

[0063] In one embodiment, a DNA of different sequences of 5-50 nt, ie, a DNA barcode, may be added to the 5' end of the primer to distinguish different samples.

[0064] In a preferred embodiment, the 5' end barcodes of the F and R primers may be the same or different, and those skilled in the art may select the barcodes as needed.

[0065] In a preferred embodiment, the system can be used to simultaneously detect at least one of the following mutations: point mutations within the primer range of the HBA1 / 2 gene locus, structural variations, and SNP sites linked within 1 Mb upstream and downstream; wherein the point mutations and structural variations include those from four thalassemia databases, namely, Ithanet, HbVar, LOVD, and LOVD-China. HBA Gene mutation.

[0066] In some preferred embodiments, the structural variation comprises a large fragment deletion. For example, the large fragment deletion includes --SEA, -α3.7, -α4.2, --THAI, --FIL, -α-9.7, --11.1, 5.3kb_deletion, --BRIT, --CANT, --GEO, --JS, --MED-II, --NOR, Qinzhou type deletion, --SA, --SPAN, --THAI, -α0.8, -α1.2, -α2.4, -α2.7, -α2.8, -α20.5, -α3.5, -α3.7, -α5.2 and -α5.6.

[0067] In a specific embodiment, the multiplex-long fragment PCR amplification in the system is completed in one reaction tube.

[0068] In a preferred experimental scheme, the system is used for multiplex-long fragment PCR amplification in one system HBA Gene fragments and concatenated fragments, totaling more than 5 target gene fragments.

[0069] In a preferred embodiment, the system amplifies more than 5 target fragments in one system, including HBA At least 1 fragment in the gene region and 4 fragments in the linkage region, so that at least: HBA1 / 2 All point mutations within the primer range of the gene locus, 28 large deletions (including --SEA, -α3.7, -α4.2, --THAI, --FIL, -α-9.7, --11.1, 5.3kb deletion, --BRIT, --CANT, --GEO, --JS, --MED-II, --NOR, Qinzhou type deletion, --SA, --SPAN, --THAI, -α0.8, -α1.2, -α2.4, -α2.7, -α2.8, -α20.5, -α3.5, -α3.7, -α5.2 and -α5.6), and SNP sites linked within 1Mb upstream and downstream. Point mutations and structural variations include those from four thalassemia databases (Ithanet, HbVar, LOVD and LOVD-China) HBA Gene mutation.

[0070] In one embodiment, the system is used to directly test the haplotype of the HBA gene region and long linkage segments simultaneously, using blastocyst and parental samples to infer haplotype linkage and is a proband-independent PGT-M test.

[0071] In a specific embodiment, the long fragment sequencing in the sequencing template of the system is selected from SMRT sequencing of PacBio or Nanopore sequencing of ONT.

[0072] In one embodiment, SMRT library adapter ligation can be performed using blunt-end ligation or TA ligation.

[0073] In one embodiment, the universal blunt-end adapter sequence of the SMRT library is 5'-pATCTCTCTCTTTTCCTCCTCCTCCGTTGTTGTTGTTGAGAGAGAT-3' (SEQ ID NO: 14), which is annealed to form a blunt-end stem-loop structure adapter adapter. DNA (barcode) with different sequences of 5-50nt can be added to the stem to form adapter adapters with different barcodes. PacBio libraries with different barcodes can be mixed together for sequencing.

[0074] In one embodiment, the universal TA adapter sequence of the SMRT library is 5'-pATCTCTCTCTTTTCCTCCTCCTCCGTTGTTGTTGTTGAGAGAGATT-3' (SEQ ID NO: 15), and a blunt-ended stem-loop structure adapter adapter is formed by annealing. 5-50nt of different sequence DNA (Barcode) can be added to the stem to form adapter adapters with different Barcodes, and PacBio libraries with different Barcodes can be mixed together for sequencing.

[0075] In one embodiment, the SMRT library adapter may or may not have a barcode. In a preferred embodiment, the SMRT library adapter has a barcode designed by PacBio or a self-designed barcode. Those skilled in the art can select as needed.

[0076] In a preferred embodiment, the SMRT library is compatible with the sequencing platform of PacBio.

[0077] In one embodiment, Nanopore library adapter ligation can be performed using blunt-end ligation or TA ligation.

[0078] In one embodiment, the Nanopore connector may be with or without a barcode, and those skilled in the art may select as needed. Preferably, the Nanopore connector has a barcode designed by ONT or a self-designed barcode, and those skilled in the art may select as needed.

[0079] In a preferred embodiment, the Nanopore library matches the sequencing platform of ONT.

[0080] The fourth aspect of the present invention provides use of the primer set described in the first aspect of the present invention in preparing a kit for detecting multiple mutations of human embryonic α-thalassemia.

[0081] A fifth aspect of the present invention provides a method for detecting multiple mutations of human embryonic α-thalassemia, comprising the following steps:

[0082] (i) Obtaining subject samples;

[0083] (ii) performing multiplex long-fragment PCR amplification on the sample;

[0084] (iii) constructing a long-fragment sequencing library;

[0085] (iv) sequencing and analyzing the types of gene mutations and the linkage relationships between different fragments;

[0086] The multiple long-fragment PCR amplification is performed using the primer set described in the first aspect of the present invention or the kit described in the second aspect of the present invention.

[0087] In one embodiment, a DNA of different sequences of 5-50 nt, ie, a DNA barcode, may be added to the 5' end of the primer to distinguish different samples.

[0088] In a preferred embodiment, the 5' end barcodes of the F and R primers may be the same or different, and those skilled in the art may select the barcodes as needed.

[0089] In a preferred embodiment, the method can be used to simultaneously detect at least one or more of the following mutations: point mutations within the primer range of the HBA1 / 2 gene locus, structural variations, and SNP sites linked within 1 Mb upstream and downstream; wherein the point mutations and the structural variations include those from four thalassemia databases, namely, Ithanet, HbVar, LOVD, and LOVD-China. HBA Gene mutation.

[0090] In some preferred embodiments, the structural variation comprises a large fragment deletion. For example, the large fragment deletion includes --SEA, -α3.7, -α4.2, --THAI, --FIL, -α-9.7, --11.1, 5.3kb_deletion, --BRIT, --CANT, --GEO, --JS, --MED-II, --NOR, Qinzhou type deletion, --SA, --SPAN, --THAI, -α0.8, -α1.2, -α2.4, -α2.7, -α2.8, -α20.5, -α3.5, -α3.7, -α5.2 and -α5.6.

[0091] In a specific embodiment, the multiplex-long fragment PCR amplification of the method is completed in one reaction tube.

[0092] In a preferred experimental scheme, the method is used for multiplex-long fragment PCR amplification of a system HBA Gene fragments and concatenated fragments, totaling more than 5 target gene fragments.

[0093] In a preferred embodiment, the method amplifies more than 5 target fragments in one system, including HBA At least 1 fragment in the gene region and 4 fragments in the linkage region, so that at least: HBA1 / 2 All point mutations within the primer range of the gene locus, 28 large deletions (including --SEA, -α3.7, -α4.2, --THAI, --FIL, -α-9.7, --11.1, 5.3kb deletion, --BRIT, --CANT, --GEO, --JS, --MED-II, --NOR, Qinzhou type deletion, --SA, --SPAN, --THAI, -α0.8, -α1.2, -α2.4, -α2.7, -α2.8, -α20.5, -α3.5, -α3.7, -α5.2 and -α5.6), and SNP sites linked within 1Mb upstream and downstream. Point mutations and structural variations include those from four thalassemia databases (Ithanet, HbVar, LOVD and LOVD-China) HBA Gene mutation.

[0094] In one embodiment, the method is used to simultaneously directly detect haplotypes of the HBA gene region and long linkage segments using blastocyst and parental samples to infer haplotype linkage and is a proband-independent PGT-M test.

[0095] In a specific embodiment, the long fragment sequencing of the method is selected from SMRT sequencing of PacBio or Nanopore sequencing of ONT.

[0096] The sixth aspect of the present invention also provides a method for constructing a haplotype linkage independent of a proband, comprising:

[0097] (1) Obtain parental samples and blastocyst samples;

[0098] (2) using the primer set described in any one of claims 1 to 6 or the kit described in claims 7 to 13 to perform multiple long-fragment PCR amplification on the parental sample and the blastocyst sample, respectively, wherein the amplified product comprises HBA At least 1 fragment of the gene region and HBA Four fragments of linkage regions within 1 Mb upstream and downstream of the gene region;

[0099] (3) Construction of long-fragment sequencing library and sequencing;

[0100] (4) According to the parental samples and blastocyst samples HBA Mutations in gene regions and HBA Haplotype linkage was constructed for SNP loci in the linkage region within 1Mb upstream and downstream of the gene region;

[0101] Among them, the method only performs multiple long-fragment PCR amplification on parental samples and blastocyst samples.

[0102] In some embodiments, the HBA At least 1 fragment of the gene region and HBA Four fragments of the linkage region within 1Mb upstream and downstream of the gene region contain one or more of the following mutations: HBA1 / 2 Point mutations, structural variations within the gene locus primer range, and SNP sites linked within 1Mb upstream and downstream; wherein the point mutations and structural variations include those from four thalassemia databases, Ithanet, HbVar, LOVD, and LOVD-China. HBA Gene mutation; preferably, the structural variation comprises a large deletion; more preferably, the large deletion includes --SEA, -α3.7, -α4.2, --THAI, --FIL, -α-9.7, --11.1, 5.3kb deletion, --BRIT, --CANT, --GEO, --JS, --MED-II, --NOR, Qinzhou type deletion, --SA, --SPAN, --THAI, -α0.8, -α1.2, -α2.4, -α2.7, -α2.8, -α20.5, -α3.5, -α3.7, -α5.2 and -α5.6.

[0103] The seventh aspect of the present invention provides a system for detecting human embryonic α-thalassemia by combining parental samples, which comprises the following modules:

[0104] (1) Collection module: used to obtain parental samples and blastocyst samples;

[0105] (2) Amplification module: used for multiple long-fragment PCR amplification, using the primer set described in any one of claims 1 to 6 or the kit described in claims 7 to 13 to perform multiple long-fragment PCR amplification on the parental sample and the blastocyst sample, respectively, wherein the amplified product comprises HBA At least 1 fragment of the gene region and HBA Four fragments of linkage regions within 1 Mb upstream and downstream of the gene region;

[0106] (3) Library construction module: used to construct long-fragment sequencing libraries;

[0107] (4) Sequencing module: used for sequencing;

[0108] (5) Analysis module: used for embryo pathogenicity analysis, based on the parental samples and blastocyst samples HBA Mutations in gene regions and HBA The SNP sites in the linkage region within 1Mb upstream and downstream of the gene region constructed haplotype linkage and analyzed whether the embryo was pathogenic;

[0109] Among them, the system only performs multiple long-fragment PCR amplification on parental samples and blastocyst samples.

[0110] In some embodiments, the HBA At least 1 fragment of the gene region and HBA Four fragments of the linkage region within 1Mb upstream and downstream of the gene region contain one or more of the following mutations: HBA1 / 2 Point mutations, structural variations within the gene locus primer range, and SNP sites linked within 1Mb upstream and downstream; wherein the point mutations and structural variations include those from four thalassemia databases, Ithanet, HbVar, LOVD, and LOVD-China. HBA Gene mutation; preferably, the structural variation comprises a large deletion; more preferably, the large deletion includes --SEA, -α3.7, -α4.2, --THAI, --FIL, -α-9.7, --11.1, 5.3kb deletion, --BRIT, --CANT, --GEO, --JS, --MED-II, --NOR, Qinzhou type deletion, --SA, --SPAN, --THAI, -α0.8, -α1.2, -α2.4, -α2.7, -α2.8, -α20.5, -α3.5, -α3.7, -α5.2 and -α5.6.

[0111] In some embodiments, the sequencing module utilizes a technology platform selected from single-molecule real-time sequencing (SMRT) and nanopore technology platforms.

[0112] In some embodiments, the amplification module is also used to perform whole genome amplification on the blastocyst sample.

[0113] The present invention based on a specific combination of long-fragment PCR amplification and long-fragment high-throughput sequencing can simultaneously detect multiple mutations of α-thalassemia in multiple embryo samples with high specificity, accuracy and speed.

[0114] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0115] Definition

[0116] In the present invention, the term " HBA "Gene" refers to a group of genes that encode α-globin in humans and other mammals. The HBA1 gene is located on chromosome 16 and encodes adult α-globin. The HBA2 gene is also located on chromosome 16 and is very close to HBA1. It encodes another α chain and is usually expressed more in the fetus. Mutations or abnormal expression of these genes can lead to a series of diseases, such as α-thalassemia, which is usually associated with genetic deletion or non-functionality of the α chain.

[0117] In the present invention, the term "linkage" refers to the phenomenon that two or more loci tend to be passed on to offspring together during inheritance. This phenomenon usually occurs between genes that are physically close on chromosomes.

[0118] In the present invention, the terms "SNP", "single nucleotide polymorphism" or "SNP site", "single nucleotide polymorphism site" are used interchangeably, which refers to the DNA sequence polymorphism caused by the variation of a single amino acid at the genomic level, including conversion, transversion, insertion and deletion of a single base.

[0119] In the present invention, the term "structural variation" refers to a structural variation in which at least two adjacent bases, preferably at least four bases, in a nucleic acid sequence are changed, and may refer to, for example, a deletion, insertion, substitution, duplication of a sequence, or translocation of a sequence of multiple nucleotides. According to certain embodiments, the structural variation affects a sequence length of at least about 50 bases, preferably at least about 100 bases, more preferably at least about 1 Kb (=1000 bases). According to certain embodiments, the structural variation affects a sequence length of at most 300 Mb (megabase=1000000 bases), for example at most 30 Mb, for example at most 3 Mb.

[0120] In the present invention, the "sample" can be a parental sample or a blastocyst sample. The parental sample can be derived from blood, oral swab, hair, urine, saliva, feces, joint fluid, cerebrospinal fluid, ascites, pleural effusion, bile or pancreatic fluid, etc. The blastocyst sample can be a blastocyst trophoblast cell. The PGT technology must first obtain the genetic material of the embryo, and the blastocyst trophoblast cells are commonly used. On the 5th to 6th day after fertilization, when the embryo develops to the blastocyst stage, there are about 100-150 cells, and 5 to 10 trophoblast cells are obtained for genetic testing.

[0121] In the present invention, the term "haplotype" refers to an individual's allele combination on a series of closely linked loci on a certain chromosome. In genetics, haplotype is usually used to describe a group of alleles on a specific locus that are inherited as a whole.

[0122] In the present invention, the term "proband" refers to a patient who is diagnosed as carrying a disease-causing gene and exhibiting symptoms of the disease, and is an organism that has a genetic relationship with the subject.

[0123] In the present invention, Ithanet, HbVar, LOVD and LOVD-China are four databases focusing on thalassemia, which provide genetic information and related data about the disease. Ithanet is an international database supported by the World Health Organization (WHO) to collect and share genetic variation information about thalassemia. It is a multi-ethnic and multi-national project that contains a wide range of genetic data, which helps researchers understand the genetic background of thalassemia in different populations. The HbVar database is a public and professional database focusing on hemoglobin variation, including thalassemia-related variation. It provides detailed variation information, including gene sequence changes, related phenotypes and literature citations, and is an important resource for studying hemoglobin diseases. LOVD is an open variation database developed by the Leiden University Medical Center in the Netherlands. It allows researchers to submit and share data on genetic variation. LOVD contains multiple sub-databases covering different genetic diseases, including thalassemia. LOVD-China is a branch of LOVD, focusing on genetic variation in the Chinese population. It provides variation information on thalassemia and other genetic diseases in the Chinese population, helping to understand the genetic characteristics and disease risks of the Chinese population.

[0124] The excellent technical effects of the present invention mainly lie in the following aspects:

[0125] (1) Not dependent on probands. The present invention can directly and simultaneously detect HBA The haplotypes of gene regions and long linkage fragment regions are combined with the genotypes of the parents for linkage analysis, avoiding the problem of having to have a proband.

[0126] (2) Detection of multiple mutation types with a single kit. The present invention can simultaneously detect multiple mutations, including SNVs, Indels, and structural variations, in one reaction primer system.

[0127] (3) High sensitivity and low cost. The present invention can efficiently utilize SNP sites for typing, greatly improving the simplicity of embryo genotype detection, greatly improving sensitivity and accuracy, preventing misdiagnosis caused by amplified allele dropout, and greatly reducing the time and labor costs of detection.

[0128] (4) High-throughput detection. Long-fragment sequencing can achieve 384 barcode adapters, and more barcode adapters can be designed according to needs. Alternatively, a dual barcode system with primer barcodes and adapter barcodes can be used to achieve more barcode combinations. The high-throughput characteristics of the long-fragment sequencing platform determine that high-throughput sample detection can be achieved.

[0129] (5) High accuracy. PacBio's SMRT dumbbell library can be read multiple times during sequencing, and the base accuracy of the sequencing results after correction is greater than 99%. In addition, SMRT sequencing errors are random, and the base accuracy is greater than 99.9% after sequencing depth correction. Therefore, gene mutations within the primer detection range can be accurately read.

[0130] (6) Flexible detection time. The Nanopore platform can generate data within minutes, and data analysis can be started within minutes or hours depending on the actual data volume required. When the detection timeliness requirements are relatively high, the Nanopore platform has a time advantage. BRIEF DESCRIPTION OF THE DRAWINGS

[0131] Figure 1 It is a schematic diagram of multiplex-long fragment PCR primer design.

[0132] Figure 2 It is a DNA gel electrophoresis diagram of a sample amplified by the multiplex-long fragment PCR method in Example 1.

[0133] Figure 3 This is a PacBio sequencing result diagram of a representative family testing result. DETAILED DESCRIPTION

[0134] Exemplary embodiments of the present invention will now be described in detail, which should not be considered as limiting the present invention but rather should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0135] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope. Although any methods and materials similar or equivalent to those described herein can be used in the practice or test of the present disclosure, what is described below is preferred materials and methods.

[0136] The sequences used in the present invention

[0137] Table 1 Primer sequences

[0138]

[0139] Example 1: Amplification using the multiplex-long fragment PCR method of the present invention HBA Gene fragments and linkage analysis fragments

[0140] Prepare the reaction system according to Table 2 below to amplify DNA samples of blastocyst samples (i.e., blastocyst trophoblast cells) and parental samples:

[0141] Table 2:

[0142]

[0143] On the PCR instrument, pre-amplification was performed according to the conditions shown in Table 3 below:

[0144] Table 3:

[0145]

[0146] After amplification, 5 ul of each sample was taken and tested on a 1% DNA gel. The results were as follows: Figure 2 As shown, different samples are used as templates. HBA Both genes and linkage fragments can be amplified efficiently.

[0147] Example 2: Construction of PacBio sequencing library using the long-fragment PCR method of the present invention

[0148] Step 1: Long-range PCR amplification

[0149] Prepare the reaction system according to Table 4 below to amplify the DNA samples of the blastocyst sample (i.e., blastocyst trophoblast cells) and the parental sample. HBA Gene fragments and linkage analysis fragments.

[0150] Table 4:

[0151]

[0152] On the PCR instrument, pre-amplification was performed according to the conditions shown in Table 5 below:

[0153] Table 5:

[0154]

[0155] After amplification, the amplified product was placed in a centrifuge at 10,000 rpm for 20 minutes. After centrifugation, the product was placed horizontally and 4 mL of supernatant was added to a new tube.

[0156] Step 2: Construction of PacBio sequencing library

[0157] Prepare the reaction system according to Table 6 below:

[0158] Table 6:

[0159]

[0160] In a PCR instrument, the reaction was carried out under the following conditions: 37ºC 20 min; 25ºC 15 min; 65ºC 10 min. After the reaction was completed, 0.5 mL Exonuclease III (NEB, Cat#M0206L) and 0.5 mL Exonuclease VII (NEB, Cat#M0379L) were added and the reaction was continued at 37ºC for 1 hour. The DNA was purified twice using 0.6x Ampure PB magnetic beads (PacBio, Cat#100-265-900) according to the manufacturer's instructions, and finally eluted with 10uL Elution Buffer. The obtained DNA eluate is the target DNA PacBio sequencing library. The DNA concentration was measured on the Qubit 3 Fluoromter (ThermoFisher, Cat#Q33216) using Qubit dsDNA HS reagent (ThermoFisher, Cat# Q32851). When there are multiple sample PacBio sequencing libraries, equal amounts of the libraries can be mixed together to prepare a mixed library.

[0161] Step 3: PacBio sequencing and analysis

[0162] According to the total concentration and molar concentration of the library, an appropriate volume of the library was reacted with the binding reagent (PacBio, Cat#101-820-200) and primer (PacBio, Cat#100-970-100) to prepare the final library that can be used in the machine. The sequencing results of the representative family are shown in Figure 3 As shown, A is the test results of the parents and some representative embryos of family 6 (mother--SEA heterozygous / father--SEA heterozygous) and the IGV schematic diagram of the linkage relationship, and B is the test results of the parents and some representative embryos of family 8 (mother--α3.7 heterozygous / father--SEA heterozygous) and the IGV schematic diagram of the linkage relationship.

[0163] Embodiment 3: HBA Detection and verification of gene mutations

[0164] 93 blastocyst samples (i.e., blastocyst trophoblast cells) and their parents' samples from 10 families were collected. Referring to Example 2, the method (and kit) of the present invention was used to simultaneously detect the genotypes of the parents and embryos. At the same time, the NGS+Gap+Sanger method was used as a control. The results obtained by the present invention were compared with the control results. The results are shown in Table 7, and the results of all samples are completely consistent.

[0165] Table 7

[0166]

[0167]

[0168]

[0169]

[0170]

[0171] Therefore, the results of the detection using the method of the present invention, after comparison with the NGS+Gap+Sanger method, both the specificity and sensitivity reached 100%.

[0172] It should be noted that although the above implementation cases have demonstrated a series of features of the present invention, researchers and technicians in this field can use the ideas of the present invention to adjust and change the reaction reagents, reaction conditions, etc. involved in the multiple + long fragment PCR reaction and long fragment sequencing library construction according to specific needs. Therefore, for those skilled in the art, several simple substitutions can be made without departing from the concept and principle of the present invention, which should be included in the protection scope of the present invention.

[0173] References

[0174] [1] Modell B, Darlison M. Bull World Health Organ. Globalepidemiology of haemoglobin disorders and derived service indicators. BullWorld Health Organ. 2008, 86(6):480-7. Doi:10.2471 / blt.06.036673.

[0175] [2] Chui DH. Alpha-thalassaemia and population health in SoutheastAsia. Ann Hum Biol. 2005, 32(2):123-30. Doi: 10.1080 / 03014460500075084.

[0176] [3] Xu Xiangmin. Guidelines for the prevention and control of thalassemia[M]. Beijing: People’s Military Medical Publishing House, 2011.

[0177] [4] Thein SL. Molecular basis of β thalassemia and potentialtherapeutic targets. Blood Cells Mol Dis. 2018, 70:54-65. Doi: 10.1016 / j.bcmd.2017.06.001.

[0178] [5] Shang X, Xu

[0179] [6] ESHRE PGT-M Working Group, Carvalho F, Moutou C, Dimitriadou E, Dreesen J, Giménez C, Goossens V, Kakourou G, Vermeulen N, Zuccarello D, DeRycke M. ESHRE PGT Consortium good practice recommendations for the detection of monogenic disorders. Human Reproduction Open. 2020, 1–18. Doi:10.1093 / hropen / hoaa018.

[0180] [7] De Rycke M, Berckmoes V. Preimplantation Genetic Testing for Monogenic Disorders. Genes. 2020, 11(8): 871. Doi:10.3390 / genes11080871.

[0181] [8] ESHRE PGT-SR / PGT-A Working Group, Coonen E, Rubio C, ChristopikouD, Dimitriadou E, Gontar J, Goossens V, Maurer M, Spinella F, Vermeulen N, DeRycke M. ESHRE PGT Consortium good practice recommendations for the detection of structural and numerical chromosomal aberrations. Human Reproduction Open.2020, 1-20. Doi: 10.1093 / hropen / hoaa017.

[0182] [9] Zhang Ningyuan, Huang Guoning, Fan Liqing, Feng Yun, Shen Huan, Liu Ping, Lu Wenhong, Zhang Yunshan, Wang Xiuxia, Zhang Songying, Huang Xuefeng, Wu Qiongfang, Quan Song, Zhou Canquan, Zhou Congrong, Shi Juanzi, Sun Yingpu, Sun Haixiang. Laboratory Technical Guidelines for Preimplantation Genetic Diagnosis and Screening. Journal of Reproductive Medicine, 2018, 29(9). doi:10.3969 / j.issn.1004-3845.2018.09.001.

[0183]

[10] Huang L, Ma F, Chapman A, Lu S, Xie XS. Sunneyal. Single-CellWhole-Genome Amplification and Sequencing: Methodology and Applications. AnnuRev Genomics Hum Genet. 2015, 16:79-102. Doi:10.1146 / annurev-genom-090413-025352.

[0184]

[11] Liang Q, Gu W, Chen P, Li Y, Liu Y, Tian M, Zhou Q, Qi H, Zhang Y, He J, Li Q, Tang L, Tang J, Teng Y, Zhou Y, Huang S, Lu Z, Xu M, Hou W,Huang T, Li Y, Li R, Hu L. Mo Z, Li Y S, Gu C Zo Q, MoreUniversal Approach to Comprehensive Analysis of Thalassemia Alleles (CATSA).J Mol Diagn. 2021 Sep;23(9):1195-1204. Doi: 10.1016 / j.jmoldx.2021.06.008.

Claims

1. A primer set for simultaneous amplification HBA Multiple mutations in gene regions and their linked single nucleotide polymorphism (SNP) sites within 1Mb upstream and downstream. Used for amplification HBA The five primers for multiple mutations in the gene region are as follows: HBA-F1, HBA-F2, HBA-F3, HBA-R1 and HBA-R2, whose sequences are shown in SEQ ID NOs: 1-5, respectively; Used for amplification HBA The 8 primers for the linked SNP sites within 1Mb upstream and downstream of the gene are as follows: L1-F, L1-R, L2-F, L2-R, L3-F, L3-R, L4-F and L4-R, whose sequences are shown in SEQ ID NOs: 6-13, respectively.

2. Use of the primer set according to claim 1 in preparing a kit for detecting multiple mutations of human embryonic α-thalassemia.

3. A kit for detecting multiple mutations of human embryonic α-thalassemia, comprising the following reagents: (1) Reagents for multiplex long-fragment PCR amplification; (2) Reagents for constructing long-fragment sequencing libraries; The reagents for multiple long-fragment PCR amplification comprise the primer set as claimed in claim 1.

4. The kit according to claim 3, wherein the reagents for multiple long-fragment PCR amplification further include DNA polymerase and reaction buffer.

5. The kit according to claim 3, wherein the reagents for constructing a long fragment sequencing library include adapters, ligases, DNA purification magnetic beads, reaction buffers and exonucleases.

6. The kit according to claim 3, wherein the long fragment sequencing is selected from the group consisting of single molecule real-time sequencing (SMRT) and nanopore technology platforms.

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