FSHD assisted reproduction system based on third-generation sequencing

Abstract

The invention provides an FSHD assisted reproduction system based on third-generation sequencing, and the system comprises a data comparison module which is used for comparing third-generation sequencing data of a family of an FSHD patient with a CHM13 reference genome, sorting the data and constructing an index; the sequence recognition module is used for recognizing D4Z4 region sequencing fragments from 4q35 on the chromosome Chr4 and 10q26 on the chromosome Chr10; the clustering and typing module is used for clustering and typing the sequencing fragments to obtain clustered and typed sequencing fragments; the variation detection module is used for comparing the sequencing fragment to the CHM13 reference genome in the index data and performing variation detection on upstream and downstream of a D4Z4 region to obtain a variation detection result; and the linkage variation information acquisition module is used for constructing an upstream and downstream linkage relationship of the D4Z4 region according to the variation detection result, and acquiring variation information linked with D4Z4, so that the variation information is used for downstream assisted reproduction.

Description

Technical Field

[0001] The present invention relates to the field of assisted reproductive technology, and in particular to a FSHD assisted reproductive system based on third-generation sequencing. Background Art

[0002] Facioscapulohumeral muscular dystrophy (FSHD) is a hereditary muscle disease. According to different pathogenic mechanisms, FSHD is divided into FSHD1 and FSHD2. Among them, FSHD1 accounts for 95% of patients and is autosomal dominant. FSHD1 patients are related to the deletion of 3.3kb D4Z4 repeat units in the 4q35 region. The abnormal shortening of D4Z4 in patients is 1-10, which is linked to the downstream 4qA to cause the disease. FSHD patients show muscle weakness and atrophy in the muscles of the face, shoulders and upper arms, which seriously affects the quality of life of patients and produces vertical transmission. If FSHD families choose to have normal children, birth defects will occur, which will bring serious psychological and economic burdens to the patient's family and society. Therefore, for FSHD patient families, preimplantation genetic testing (PGT) technology in assisted reproduction can be used to perform genetic analysis before the embryo is implanted in the uterus, and embryos that do not carry specific genetic disease genes can be selected for transplantation to achieve FSHD genetic blockade.

[0003] Genetic testing is the gold standard for the diagnosis of genetic diseases, but it is very difficult to perform molecular diagnosis on FSHD patients. This is mainly due to the presence of a repetitive sequence in the 10q26 subtelomeric region with a homology of up to 99% with 4q35 D4Z4, which interferes with the detection of the number of D4Z4 repeats and downstream haplotypes. Existing FSHD molecular diagnostic methods are based on enzyme-labeled Southern Blot and Bionano optical mapping technology, which require FSHD patients to provide a sufficient amount of high-quality DNA samples. This is very difficult for FSHD patient families with reproductive needs who want to directly perform D4Z4 repeat unit detection and downstream haplotype linkage analysis on in vitro cultured embryos through assisted reproductive technology.

[0004] At present, the third-generation long read sequencing (LRS) based on PacBio single-molecule real-time sequencing technology and Oxford Nanopore nanopore sequencing technology can have a sequencing read length of more than 15kb to 100kb, and the accuracy of sequencing single bases can reach 99%. The third-generation long read sequencing data can easily cross specific genomic regions, such as highly complex repetitive sequences, telomere regions, and high GC regions, and has significant advantages in the diagnosis of complex diseases caused by large fragment duplications and deletions on chromosomes. At the same time, long read sequencing technology can directly detect single nucleotide variants (SNV), small fragment insertions and deletions (INDEL), and structural variations (SV) and realize Phasing between variant information, clarify the linkage relationship between upstream and downstream variants of pathogenic haplotypes, making the application potential of LRS in the field of assisted reproduction more prominent.

[0005] Therefore, it is necessary to develop a system or product for assisted reproduction. Summary of the invention

[0006] The purpose of the present invention is to provide a system for FSHD assisted reproduction based on third-generation sequencing. The system performs haplotype analysis on the D4Z4 region of FSHD patients, detects upstream and downstream SNVs, INDELs, and SVs of the D4Z4 region, and determines the variation information linked to the pathogenic haplotype. This system can achieve haplotypes linked to the pathogenic chain without relying on proband and family information, provide accurate and reliable variation linkage information for downstream assisted reproductive embryo preimplantation screening, and provide markers for assisted reproductive screening of normal embryos.

[0007] To achieve the above object, the present invention adopts the following technical solution:

[0008] In a first aspect of the present invention, a system for FSHD assisted reproduction based on third-generation sequencing is provided, the system comprising:

[0009] The data comparison module is used to compare, sort and construct indexes for the third-generation sequencing data of FSHD patient families with the preset CHM13 reference genome to obtain index data;

[0010] A sequence recognition module, used to recognize and determine the sequencing fragments of the D4Z4 region from 4q35 on chromosome Chr4 and 10q26 on Chr10;

[0011] A cluster typing module is used to cluster and type each sequencing fragment from the D4Z4 region on Chr4 to obtain clustered and typed sequencing fragments;

[0012] A variation detection module is used to align the clustered and typed sequencing fragments to the CHM13 reference genome in the index data, and perform variation detection on the upstream and downstream of the D4Z4 region to obtain variation detection results;

[0013] The linkage variation information acquisition module is used to construct the upstream and downstream linkage relationship of the D4Z4 region according to the variation detection results, and obtain variation information linked to D4Z4 for use in downstream assisted reproduction.

[0014] Furthermore, the data comparison module includes:

[0015] The data initial screening unit is used to perform data quality control and filtering on the third-generation data of FSHD patient family samples to obtain third-generation data that pass quality control;

[0016] The index acquisition unit is used to align the third-generation data that has passed quality control to the reference genome, and locate, sort and construct indexes for the sequencing fragments to obtain index data.

[0017] Furthermore, the sequence recognition module includes:

[0018] A region extraction unit is used to extract the sequencing fragments aligned to the D4Z4 region on Chr4 (Chr4: 190065229-190093263) and the D4Z4 region on Chr10 (Chr10: 133664430-133761680);

[0019] The D4Z4 region sequencing fragment screening unit is used to screen out the sequencing fragments covering the 10-110bp position upstream of the D4Z4 repeat region on Chr4 (Chr4:190065119-190065219) and the sequencing fragments covering the 10-110bp position upstream of the D4Z4 repeat region on Chr10 (Chr10:133664320-133664420). These reads are the sequencing fragments covering the upstream of the D4Z4 repeat.

[0020] Furthermore, the cluster typing module includes:

[0021] A data calculation unit, used to identify and calculate the number of repeating units in the D4Z4 region on Chr4 and the downstream haplotype;

[0022] A sequencing fragment distinguishing unit, used to identify and distinguish the Chr4 sequencing fragments extending upstream and downstream of the D4Z4 region;

[0023] The clustering unit is used to cluster the fragments according to the number of D4Z4 repeating units, downstream haplotypes and sources of the sequencing fragments in each sequencing fragment list to obtain fragment clustering and typing results.

[0024] Further, the step of identifying and calculating the number of repeating units in the D4Z4 region on Chr4 and the downstream haplotype in the data calculation unit comprises:

[0025] The sequencing fragments covering the upstream of the D4Z4 region on Chr4 and Chr10 were aligned with a single D4Z4 repeat unit, and the number of D4Z4 repeat units in each sequencing fragment was determined and recorded;

[0026] The sequencing fragments with the determined number of D4Z4 repeat units are compared with the DUX4_marker, DUX4L_marker, markerB, and pLAM_marker sequences, and it is determined whether the sequencing fragments extend to the downstream haplotype A or B structural region according to the preset conditions;

[0027] The preset conditions include:

[0028] Judgment condition 1: If the alignment length of the D4Z4 downstream sequencing fragment representing the structure of haplotype A and the DUX4_marker or DUX4L_marker sequence exceeds 70% of the marker length, the sequencing fragment is marked as extending to the downstream haplotype region;

[0029] Judgment condition 2: If the length of the alignment between the downstream sequencing fragment of D4Z4 representing the structure of haplotype B and the markerB sequence exceeds 2000 bp, the marker sequencing fragment is extended to the downstream haplotype region;

[0030] Judgment condition three: If there is a non-D4Z4 heavy sequence of more than 2000bp downstream of the D4Z4 region in the sequencing fragment, then the sequencing fragment is marked as extending to the downstream haplotype region;

[0031] The sequencing fragments that meet any one of the conditions one, two, and three are compared with the pLAM_marker sequence. If the comparison length of the sequencing fragment exceeds 80% of the pLAM_marker length, the sequencing fragment is marked as the D4Z4 downstream haplotype A structure, and the others are marked as haplotype B structures. The comparison is cyclically performed in sequence, and the sequencing fragments that cannot be compared with the downstream maker are discarded. The sequencing fragments that can be compared to the haplotype extending downstream of the D4Z4 region are recorded, and the downstream haplotype structure is recorded.

[0032] Furthermore, the step of identifying and distinguishing the Chr4 sequencing fragments extending upstream and downstream of the D4Z4 region in the sequencing fragment distinguishing unit includes:

[0033] Select the sequence of the 4q35 region on chromosome Chr4 (Chr4: 190023373--190066146) and the sequence of the 10q26 region on chromosome Chr10 (Chr10: 133622203--133664430) in the reference genome version GRCh38 for comparison and search, and record the differential sites of Chr4 and Chr10 in the list;

[0034] Select the sequencing fragments that span the D4Z4 region and extend to the downstream haplotype region, and identify whether there is a Bln (CCTAGG) restriction site sequence in the 3216-3241bp interval in each D4Z4 repeat unit; at the same time, identify whether there is a XapI (AAATTC) restriction site sequence in the 1755-1780bp interval in each D4Z4 repeat unit; and record the identification results of each repeat unit of each sequencing fragment and the Bln / XapI site situation in the list;

[0035] Initialize the score of each sequencing fragment that crosses the D4Z4 region and extends to the downstream haplotype region to 0, judge each D4Z4 sequence on the sequencing fragment, if the specified region contains the Bln restriction site sequence, the score is -1, if the specified region contains the XapI restriction site sequence, the score is +1, and calculate the total score; if the total score>0, record the sequencing fragment score and source as Chr4 in the list; if the total score<0, record the sequencing fragment score and source as Chr10 in the list;

[0036] Initialize the score of each sequencing fragment that crosses the D4Z4 region and extends to the downstream haplotype region to 0, and judge according to the difference sites in Chr4 and Chr10 recorded in S3121. If the bases of the sequencing fragments aligned to these difference sites are consistent with the bases on Chr4, the score is +1, if they are consistent with the bases on Chr10, the score is -1, and no score is calculated for other cases. Calculate the total score. If the total score>0, the sequencing fragment score and source are recorded in the list as Chr4; if the total score<0, the sequencing fragment score and source are recorded in the list as Chr10;

[0037] According to the Score and source of each sequencing fragment in conditions S3123 and S3124, if both conditions determine that the source of the sequencing fragment is Chr4, then define the source of this sequencing fragment as Chr4 and record it in the list; if both conditions determine that the source of the sequencing fragment is Chr10, then define the source of this sequencing fragment as Chr10 and record it in the list;

[0038] Finally, the source of each sequencing fragment spanning D4Z4 was obtained, and the sequencing fragment information recorded as Chr4 was retained, and other inconsistencies were deleted.

[0039] Furthermore, the step of clustering in the clustering unit according to the number of D4Z4 repeating units, downstream haplotypes and sequencing fragment sources in each sequencing fragment list comprises:

[0040] Clustering was performed based on the number of D4Z4 repeat units, downstream haplotype structure, and sequencing fragment sources recorded in each sequencing fragment list;

[0041] Based on the clustered sequencing fragments in Chr4 and Chr10, each sequencing fragment was clustered into haplotypes, and each sequencing fragment in Chr4 was clustered into 4qA haplotype and 4qB haplotype.

[0042] The Chr4 4qA clustered haplotype sequencing fragments were clustered according to the number of D4Z4 repeat units, and the distribution of D4Z4 repeat numbers was counted and clustered;

[0043] If the sum of the number of sequenced fragments within the range of plus or minus 1 unit of the peak value in the D4Z4 repeat number distribution is greater than one-half of the total sequenced fragments, it is stored in the list as the number of Chr4 4qAD4Z4 repeat units;

[0044] The D4Z4 repeat count and storage were performed for the Chr4 4qB haplotype cluster sequencing fragments in the same manner to obtain the Chr4 cluster typing results based on the number of D4Z4 repeat units and downstream haplotypes.

[0045] Furthermore, the variation detection template includes:

[0046] The clustering and typing index acquisition unit is used to compare, sort and construct an index for the obtained clustering and typing sequencing fragments with the CHM13 T2T completed map reference genome;

[0047] A variation detection unit is used to perform variation detection on the upstream and downstream of the Chr4 D4Z4 region based on the obtained Chr4 haplotype typing and clustering results, and record the upstream and downstream SNV, INDEL, and SV information;

[0048] An information construction unit constructs a list based on the variant information sites, recording the number of sequencing fragments supporting each variant site, the total number of sequencing fragments, and homozygous and heterozygous information;

[0049] Marking unit, select variants with quality values ​​greater than 30 as High, select variants with quality values ​​between 15 and 30 as Medium, and variants with quality values ​​less than 15 as Low.

[0050] Furthermore, the linkage variation information acquisition module comprises

[0051] The variant information linkage relationship acquisition unit constructs the D4Z4 upstream and downstream variant information linkage relationship based on the variant detection information in S412 and the sequencing fragment haplotype clustering results in S31. The variant linked to the Chr4 4qA pathogenic haplotype is haplotype hap1, and the other haplotype is hap2.

[0052] The assisted reproductive PGT detection site acquisition unit selects the variant sites marked as High quality values ​​in hap1 and hap2 upstream and downstream of the Chr4 D4Z4 region as downstream assisted reproductive PGT detection sites.

[0053] In a second aspect of the present invention, a computer device is provided, comprising a memory, a processor and a computer program stored in the memory, wherein the processor executes the computer program to implement the steps in the system.

[0054] In a third aspect of the present invention, a computer storable medium is provided, on which a computer program / instruction is stored, which implements the steps of the system when executed by a processor.

[0055] In a fourth aspect of the present invention, there is provided a computer program product comprising a computer program / instructions which, when executed by a processor, implement the steps of the system.

[0056] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0057] The FSHD assisted reproduction system based on third-generation sequencing provided by the present invention performs third-generation sequencing on FSHD patient families, obtains sequencing fragments after D4Z4 region clustering and typing of FSHD patients, and performs variation detection based on the fragments combined with the CHM13 completion map, thereby realizing the detection of linkage information of upstream and downstream haplotypes of the D4Z4 pathogenic haplotype, providing reliable markers for downstream assisted reproduction PGT, and inferring whether the embryo contains the pathogenic haplotype by detecting the embryo marker, thereby realizing accurate genetic blocking of FSHD through pre-implantation screening, which has great clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0059] Figure 1 The present invention is a flow chart of a FSHD assisted reproductive method based on third-generation sequencing.

[0060] Figure 2 The present invention provides an FSHD assisted reproductive method based on third-generation sequencing and upstream variation information linked to the pathogenic chain D4Z4.

[0061] Figure 3 This is a structural diagram of the FSHD assisted reproduction system based on third-generation sequencing. DETAILED DESCRIPTION

[0062] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0063] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0064] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or obtained by existing methods.

[0065] The present application will be described in detail below with reference to embodiments and experimental data.

[0066] Example 1: FSHD assisted reproduction system based on third-generation sequencing

[0067] 1. FSHD Assisted Reproduction System Based on Third Generation Sequencing

[0068] (1) A data comparison module 100 is used to compare, sort and index the third-generation sequencing data of the FSHD patient family with the preset CHM13 reference genome to obtain index data; further, the data comparison module 100 includes:

[0069] The data initial screening unit is used to perform data quality control and filtering on the third-generation data of FSHD patient family samples to obtain third-generation data that pass quality control;

[0070] The index acquisition unit is used to align the third-generation data that has passed quality control to the reference genome, and locate, sort and construct indexes for the sequencing fragments to obtain index data.

[0071] (2) a sequence recognition module 200, for identifying and determining the sequenced fragments of the D4Z4 region from 4q35 on chromosome Chr4 and 10q26 on Chr10;

[0072] Furthermore, the sequence identification module 200 includes:

[0073] A region extraction unit is used to extract the sequencing fragments aligned to the D4Z4 region on Chr4 (Chr4: 190065229-190093263) and the D4Z4 region on Chr10 (Chr10: 133664430-133761680);

[0074] The D4Z4 region sequencing fragment screening unit is used to screen out the sequencing fragments covering the 10-110bp position upstream of the D4Z4 repeat region on Chr4 (Chr4:190065119-190065219) and the sequencing fragments covering the 10-110bp position upstream of the D4Z4 repeat region on Chr10 (Chr10:133664320-133664420). These reads are the sequencing fragments covering the upstream of the D4Z4 repeat.

[0075] (3) A cluster typing module 300, which is used to cluster and type each sequencing fragment from the D4Z4 region on Chr4 to obtain clustered and typed sequencing fragments; the cluster typing module 300 includes:

[0076] A data calculation unit, used to identify and calculate the number of repeating units in the D4Z4 region on Chr4 and the downstream haplotype;

[0077] A sequencing fragment distinguishing unit, used to identify and distinguish the Chr4 sequencing fragments extending upstream and downstream of the D4Z4 region;

[0078] The clustering unit is used to cluster the fragments according to the number of D4Z4 repeating units, downstream haplotypes and sources of the sequencing fragments in each sequencing fragment list to obtain fragment clustering and typing results.

[0079] (4) a variation detection module 400, which is used to align the clustered and typed sequencing fragments to the CHM13 reference genome in the index data, and perform variation detection on the upstream and downstream of the D4Z4 region to obtain a variation detection result; further, the variation detection module includes:

[0080] The clustering and typing index acquisition unit is used to compare, sort and construct an index for the obtained clustering and typing sequencing fragments with the CHM13 T2T completed map reference genome;

[0081] A variation detection unit is used to perform variation detection on the upstream and downstream of the Chr4 D4Z4 region based on the obtained Chr4 haplotype typing and clustering results, and record the upstream and downstream SNV, INDEL, and SV information;

[0082] An information construction unit constructs a list based on the variant information sites, recording the number of sequencing fragments supporting each variant site, the total number of sequencing fragments, and homozygous and heterozygous information;

[0083] For the labeling unit, select variants with a quality value greater than 30 as High, select variants with a quality value between 15 and 30 as Medium, and variants with a quality value less than 15 as Low.

[0084] (5) A linkage variation information acquisition module 500, which is used to construct the upstream and downstream linkage relationship of the D4Z4 region according to the variation detection results, and obtain variation information linked to D4Z4, so as to be used for downstream assisted reproduction. Further, the linkage variation information acquisition module includes

[0085] The variant information linkage relationship acquisition unit constructs the D4Z4 upstream and downstream variant information linkage relationship based on the variant detection information in S412 and the sequencing fragment haplotype clustering results in S31. The variant linked to the Chr4 4qA pathogenic haplotype is haplotype hap1, and the other haplotype is hap2.

[0086] The assisted reproductive PGT detection site acquisition unit selects the variant sites marked as High quality values ​​in hap1 and hap2 upstream and downstream of the Chr4 D4Z4 region as downstream assisted reproductive PGT detection sites.

[0087] Example 2: FSHD assisted reproductive method based on third-generation sequencing

[0088] S11, perform third-generation sequencing on the FSHD patient family, and align, sort, and index the sequencing data with the preset reference genome;

[0089] S21, identify and confirm the sequencing fragments from the D4Z4 region at 4q35 on chromosome Chr4 and 10q26 on Chr10;

[0090] S31, clustering and typing each sequencing fragment from the D4Z4 region on Chr4;

[0091] S41, based on the fragment clustering and typing results, the D4Z4 sequencing fragments were aligned to the CHM13 reference genome, and mutations were detected upstream and downstream of the D4Z4 region;

[0092] S51, construct the upstream and downstream linkage relationship of the D4Z4 region based on the variation detection results, and determine the variation information linked to D4Z4 for downstream assisted reproduction.

[0093] In Example 2 of the present invention, in a FSHD diagnosis method based on third-generation sequencing S11, a family of FSHD patients undergoes third-generation sequencing, and the sequencing data is compared, sorted, and indexed with a preset reference genome, including:

[0094] S111, extract long-fragment DNA from family samples of FSHD patients to be tested, construct high-quality third-generation sequencing libraries and sequence them on the sequencing platform;

[0095] S112, perform data quality control and filtering on the third-generation data of FSHD patient family samples off the machine, and use the third-generation data that pass the quality control for downstream data analysis;

[0096] S113, align the third-generation data that have passed quality control to the reference genome, and locate, sort and index the sequencing fragments.

[0097] In Example 2 of the present invention, in a method S21 for diagnosing FSHD based on third-generation sequencing, the sequenced fragments from the 4q35 region on chromosome Chr4 and the 10q26 D4Z4 region on Chr10 are identified and determined, comprising:

[0098] S211, intercepted the sequencing fragments aligned to the D4Z4 region on Chr4 (Chr4: 190065229-190093263) and the D4Z4 region on Chr10 (Chr10: 133664430-133761680);

[0099] S212, screen out the sequencing fragments covering the 10-110bp position upstream of the D4Z4 repeat region on Chr4 (Chr4:190065119-190065219), and the sequencing fragments covering the 10-110bp position upstream of the D4Z4 repeat region on Chr10 (Chr10:133664320-133664420). These reads are the sequencing fragments covering the upstream of D4Z4 repeat.

[0100] In Example 2 of the present invention, in a method S31 for diagnosing FSHD based on third-generation sequencing, each sequencing fragment from the D4Z4 region on Chr4 is clustered and typed, including:

[0101] S311, identify and calculate the number of repeat units in the D4Z4 region on Chr4 and the downstream haplotype;

[0102] S312, identification and differentiation of Chr4 sequencing fragments extending upstream and downstream of the D4Z4 region;

[0103] S313, clustering according to the number of D4Z4 repeating units, downstream haplotype structure and sequencing fragment sources recorded in each sequencing fragment list;

[0104] In Example 2 of the present invention, in a method for diagnosing FSHD based on third-generation sequencing S311, identifying and calculating the number of D4Z4 repeating units in the D4Z4 region on Chr4 includes:

[0105] S3111, aligning the sequencing fragments covering the upstream of the D4Z4 region on Chr4 and Chr10 with a single D4Z4 repeat unit, determining the number of D4Z4 repeat units in each sequencing fragment and recording it;

[0106] S3112, further aligning the sequencing fragments with the determined number of D4Z4 repeat units with the DUX4_marker, DUX4L_marker, markerB, and pLAM_marker sequences to determine whether the sequencing fragments extend to the downstream haplotype A or B structural region;

[0107] S3113, judgment condition 1: if the alignment length of the D4Z4 downstream sequencing fragment representing the haplotype A structure and the DUX4_marker or DUX4L_marker sequence exceeds 70% of the marker length, then mark this sequencing fragment as extending to the downstream haplotype region;

[0108] S3114, judgment condition 2: if the length of the alignment between the D4Z4 downstream sequencing fragment representing the structure of haplotype B and the markerB sequence exceeds 2000 bp, then mark this sequencing fragment as extending to the downstream haplotype region;

[0109] S3115, judgment condition three: if there is a non-D4Z4 heavy sequence of more than 2000 bp downstream of the D4Z4 region in the sequencing fragment, then mark this sequencing fragment as extending to the downstream haplotype region;

[0110] S3116, the sequencing fragments that meet any of the conditions 1, 2, and 3 are compared with the pLAM_marker sequence. If the comparison length of the sequencing fragment exceeds 80% of the length of pLAM_marker, the sequencing fragment is marked as the D4Z4 downstream haplotype A structure, and the others are marked as haplotype B structures.

[0111] S3117, cyclically align, discard the sequencing fragments that cannot be aligned to the downstream maker, record the sequencing fragments that can be aligned to the downstream haplotype extending to the D4Z4 region, and record the downstream haplotype structure.

[0112] In Example 1 of the present invention, in a method for diagnosing FSHD based on third-generation sequencing S312, identifying and distinguishing Chr4 sequencing fragments extending upstream and downstream of the D4Z4 region includes:

[0113] S3121, select the sequence of the 4q35 region on chromosome Chr4 (Chr4:190023373--190066146) and the sequence of the 10q26 region on chromosome Chr10 (Chr10:133622203--133664430) in the reference genome version GRCh38 for comparison and search, and record the differential sites between Chr4 and Chr10 in the list;

[0114] S3122, select the sequencing fragments that span the D4Z4 region and extend to the downstream haplotype region, identify whether there is a Bln (CCTAGG) restriction site sequence in the 3216-3241bp interval in each D4Z4 repeat unit; at the same time, identify whether there is a XapI (AAATTC) restriction site sequence in the 1755-1780bp interval in each D4Z4 repeat unit; and record the identification results of each repeat unit of each sequencing fragment and the Bln / XapI site situation in the list;

[0115] S3123, initialize the score of each sequencing fragment that crosses the D4Z4 region and extends to the downstream haplotype region to 0, judge each D4Z4 sequence on the sequencing fragment, if the specified region contains the Bln (CCTAGG) restriction site sequence, the score is -1, if the specified region contains the XapI (AAATTC) restriction site sequence, the score is +1, calculate the total score; if the total score>0, record the sequencing fragment score and source as Chr4 in the list; if the total score<0, record the sequencing fragment score and source as Chr10 in the list;

[0116] S3124, initialize the score of each sequencing fragment that crosses the D4Z4 region and extends to the downstream haplotype region to 0, and judge according to the difference sites in Chr4 and Chr10 recorded in S3121. If the bases of the sequencing fragments aligned to these difference sites are consistent with the bases on Chr4, the score is +1, if they are consistent with the bases on Chr10, the score is -1, and no score is calculated for other cases. Calculate the total score. If the total score>0, the sequencing fragment score and source are recorded in the list as Chr4; if the total score<0, the sequencing fragment score and source are recorded in the list as Chr10;

[0117] S3125, judging according to the score and source of each sequencing fragment in the conditions of S3123 and S3124, if the two conditions simultaneously judge that the source of the sequencing fragment is Chr4, then defining the source of this sequencing fragment as Chr4 and recording it in the list; if the two conditions simultaneously judge that the source of the sequencing fragment is Chr10, then defining the source of this sequencing fragment as Chr10 and recording it in the list;

[0118] S3126, finally obtain the source of each sequencing fragment spanning D4Z4, retain the sequencing fragment information recorded as Chr4, and delete other inconsistencies.

[0119] In Example 1 of the present invention, in a FSHD diagnostic method based on third-generation sequencing S313, clustering is performed according to the number of D4Z4 repeating units recorded in each sequencing fragment list, the downstream haplotype structure and the source of the sequencing fragments, including:

[0120] S3131, clustering according to the number of D4Z4 repeat units, downstream haplotype structure and sequencing fragment sources recorded in each sequencing fragment list;

[0121] S3132, based on the clustered sequencing fragments in Chr4 and Chr10, perform haplotype clustering on each sequencing fragment, and cluster each sequencing fragment in Chr4 into 4qA haplotype and 4qB haplotype,

[0122] S3133, clustering the Chr4 4qA clustered haplotype sequencing fragments according to the number of D4Z4 repeat units, and statistically analyzing the distribution of the number of D4Z4 repeats and clustering them;

[0123] S3134, if the sum of the number of sequenced fragments within the range of plus or minus 1 unit of the peak value in the D4Z4 repeat number distribution is greater than one-half of the total sequenced fragments, then store it in the list as the Chr4 4qAD4Z4 repeat unit number;

[0124] S3135, for the Chr4 4qB haplotype cluster sequencing fragments, the D4Z4 repeat counts and storage are performed according to the same method to obtain the Chr4 cluster typing results based on the number of D4Z4 repeat units and downstream haplotypes.

[0125] In Example 1 of the present invention, in a FSHD diagnostic method S41 based on third-generation sequencing, the D4Z4 sequencing fragment is aligned to the CHM13 reference genome according to the fragment clustering and typing results, and mutation detection is performed on the upstream and downstream of the D4Z4 region;

[0126] S411, the sequencing fragments clustered and typed in S31 were aligned, sorted and indexed with the CHM13 T2T completed map reference genome;

[0127] S412, based on the Chr4 haplotype typing and clustering results in S31, the upstream and downstream of the Chr4 D4Z4 region were subjected to mutation detection, and the upstream and downstream SNV, INDEL, and SV information were recorded;

[0128] S413, constructing a list according to the variant information sites, recording the number of sequencing fragments supporting each variant site, the total number of sequencing fragments, homozygous and heterozygous information;

[0129] S414, select the variants with a quality value greater than 30 and mark them as High, select the variants with a quality value between 15 and 30 and mark them as Medium; select the variants with a quality value less than 15 and mark them as Low;

[0130] In Example 1 of the present invention, in a FSHD diagnosis method based on third-generation sequencing S51, the upstream and downstream linkage relationship of the D4Z4 region is constructed according to the variation detection results, and the variation information linked to D4Z4 is determined for downstream assisted reproduction;

[0131] S511, based on the variant detection information in S412 and the haplotype clustering results of the sequencing fragments in S31, the linkage relationship of the upstream and downstream variant information of D4Z4 was constructed. The variant linked to the Chr4 4qA pathogenic haplotype was haplotype hap1, and the other haplotype was hap2;

[0132] S512, select the variant sites marked as High quality values ​​in hap1 and hap2 in the upstream and downstream of Chr4 D4Z4 region as downstream assisted reproductive PGT detection sites.

[0133] Example 3: FSHD assisted reproduction system based on third-generation sequencing

[0134] The embodiment of the present invention provides a system for FSHD assisted reproduction based on third-generation sequencing, the system comprising:

[0135] A processor and a memory, wherein the memory is coupled to the processor, and the memory stores instructions, and when the instructions are executed by the processor, the steps of the method described in Example 2 are performed.

[0136] Embodiment 4: Computer readable storage medium

[0137] An embodiment of the present invention provides a computer-readable storage medium on which a computer program is stored. When the computer program is executed by a processor, the steps of the method in the system of embodiment 1 and / or the method in embodiment 2 are implemented.

[0138] Of course, the computer executable instructions of a storage medium including computer executable instructions provided by an embodiment of the present invention are not limited to the operations of the method described above, but can also execute related operations in the method provided by any embodiment of the present invention.

[0139] Through the above description of the implementation methods, the technicians in the relevant field can clearly understand that the present invention can be implemented by means of software and necessary general hardware, and of course it can also be implemented by hardware, but in many cases the former is a better implementation method. Based on such an understanding, the technical solution of the present invention is essentially or the part that contributes to the prior art can be embodied in the form of a software product, and the computer software product can be stored in a computer-readable storage medium, such as a computer floppy disk, read-only memory (ROM), random access memory (RAM), flash memory (FLASH), hard disk or optical disk, etc., including a number of instructions for an electronic device (which can be a personal computer, a server, or a network device, etc.) to execute the methods described in each embodiment of the present invention.

[0140] It is worth noting that in the above embodiments, the various units and modules included are only divided according to functional logic, but are not limited to the above divisions, as long as the corresponding functions can be achieved; in addition, the specific names of the functional units are only for the convenience of distinguishing each other, and are not used to limit the scope of protection of the present invention.

[0141] Application Example 1: A method for assisted reproduction of FSHD based on third-generation sequencing

[0142] The FSHD patients in the FSHD patient family were subjected to third-generation sequencing and based on this method, the haplotype Hap1 linked to the pathogenic chain of FSHD patients was obtained. The upstream and downstream haplotype information of the pathogenic chain was obtained through linkage analysis, and the downstream assisted reproduction was used to screen embryos based on these marker information. The screening results are shown in the following table.

[0143] Table 1

[0144]

[0145]

[0146]

[0147]

[0148]

[0149] By selecting two highly feasible SNPs upstream of FSHDD4Z4 and two highly credible SNPs downstream for first-generation Sanger sequencing verification, the accuracy of SNPs detected after typing D4Z4 based on third-generation long-read sequencing was verified, and the accuracy of SNPs linked upstream and downstream of D4Z4 by third-generation sequencing was compared with the sequencing gold standard. The specific verification results are shown in Table 2. From Table 2, it can be seen that the consistency of SNP detection results based on the third-generation sequencing technology and the first-generation gold standard detection is 100%, which can provide a reliable SNP marker for subsequent embryo screening.

[0150] Table 2

[0151]

[0152] This embodiment provides a method based on the third-generation sequencing technology to achieve clustering and typing of D4Z4 haplotypes of FSHD patients, obtain marker information of the upstream and downstream of the D4Z4 region linked to the pathogenic haplotype, and provide a feasible and highly accurate assisted reproductive method for FSHD patient families, making it possible for FSHD patient families to conceive healthy babies through assisted reproductive PGT.

[0153] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0154] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0155] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A system for FSHD assisted reproduction based on third-generation sequencing, characterized in that: The system comprises: The data comparison module is used to compare, sort and construct indexes for the third-generation sequencing data of FSHD patient families with the preset CHM13 reference genome to obtain index data; A sequence recognition module, used to recognize and determine the sequencing fragments of the D4Z4 region from 4q35 on chromosome Chr4 and 10q26 on Chr10; A cluster typing module is used to cluster and type each sequencing fragment from the D4Z4 region on Chr4 to obtain clustered and typed sequencing fragments; A variation detection module is used to align the clustered and typed sequencing fragments to the CHM13 reference genome in the index data, and perform variation detection on the upstream and downstream of the D4Z4 region to obtain variation detection results; The linkage variation information acquisition module is used to construct the upstream and downstream linkage relationship of the D4Z4 region according to the variation detection results, and obtain variation information linked to D4Z4 for use in downstream assisted reproduction.

2. A system for FSHD assisted reproduction based on third-generation sequencing according to claim 1, characterized in that: The data comparison module comprises: The data initial screening unit is used to perform data quality control and filtering on the third-generation data of FSHD patient family samples to obtain third-generation data that pass quality control; The index acquisition unit is used to align the third-generation data that has passed quality control to the reference genome, and locate, sort and construct indexes for the sequencing fragments to obtain index data.

3. The FSHD assisted reproduction system based on third-generation sequencing according to claim 1, characterized in that: The sequence recognition module comprises: A region extraction unit is used to extract the sequencing fragments aligned to the D4Z4 region on Chr4 (Chr4: 190065229-190093263) and the D4Z4 region on Chr10 (Chr10: 133664430-133761680); The D4Z4 region sequencing fragment screening unit is used to screen out the sequencing fragments covering the 10-110bp position upstream of the D4Z4 repeat region on Chr4 (Chr4:190065119-190065219) and the sequencing fragments covering the 10-110bp position upstream of the D4Z4 repeat region on Chr10 (Chr10:133664320-133664420). These reads are the sequencing fragments covering the upstream of the D4Z4 repeat.

4. The FSHD assisted reproduction system based on third-generation sequencing according to claim 1, characterized in that: The cluster typing module includes: A data calculation unit, used to identify and calculate the number of repeating units in the D4Z4 region on Chr4 and the downstream haplotype; A sequencing fragment distinguishing unit, used to identify and distinguish the Chr4 sequencing fragments extending upstream and downstream of the D4Z4 region; The clustering unit is used to cluster the fragments according to the number of D4Z4 repeating units, downstream haplotypes and sources of the sequencing fragments in each sequencing fragment list to obtain fragment clustering and typing results.

5. The system for FSHD assisted reproduction based on third-generation sequencing according to claim 4, characterized in that: The step of identifying and calculating the number of repeating units in the D4Z4 region on Chr4 and the downstream haplotype in the data calculation unit comprises: The sequencing fragments covering the upstream of the D4Z4 region on Chr4 and Chr10 were aligned with a single D4Z4 repeat unit, and the number of D4Z4 repeat units in each sequencing fragment was determined and recorded; The sequencing fragments with the determined number of D4Z4 repeat units are compared with the DUX4_marker, DUX4L_marker, markerB, and pLAM_marker sequences, and it is determined whether the sequencing fragments extend to the downstream haplotype A or B structural region according to the preset conditions; The preset conditions include: Judgment condition 1: If the alignment length of the D4Z4 downstream sequencing fragment representing the structure of haplotype A and the DUX4_marker or DUX4L_marker sequence exceeds 70% of the marker length, the sequencing fragment is marked as extending to the downstream haplotype region; Judgment condition 2: If the length of the alignment between the downstream sequencing fragment of D4Z4 representing the structure of haplotype B and the markerB sequence exceeds 2000 bp, the marker sequencing fragment is extended to the downstream haplotype region; Judgment condition three: If there is a non-D4Z4 heavy sequence of more than 2000bp downstream of the D4Z4 region in the sequencing fragment, then the sequencing fragment is marked as extending to the downstream haplotype region; The sequencing fragments that meet any one of the conditions one, two, and three are compared with the pLAM_marker sequence. If the comparison length of the sequencing fragment exceeds 80% of the pLAM_marker length, the sequencing fragment is marked as the D4Z4 downstream haplotype A structure, and the others are marked as haplotype B structures. The comparison is cyclically performed in sequence, and the sequencing fragments that cannot be compared with the downstream maker are discarded. The sequencing fragments that can be compared to the haplotype extending downstream of the D4Z4 region are recorded, and the downstream haplotype structure is recorded.

6. The FSHD assisted reproduction system based on third-generation sequencing according to claim 4, characterized in that: The step of identifying and distinguishing the Chr4 sequencing fragments extending upstream and downstream of the D4Z4 region in the sequencing fragment distinguishing unit comprises: Select the sequence of the 4q35 region on chromosome Chr4 (Chr4: 190023373--190066146) and the sequence of the 10q26 region on chromosome Chr10 (Chr10: 133622203--133664430) in the reference genome version GRCh38 for comparison and search, and record the differential sites of Chr4 and Chr10 in the list; Select the sequencing fragments that span the D4Z4 region and extend to the downstream haplotype region, and identify whether there is a Bln (CCTAGG) restriction site sequence in the 3216-3241bp interval in each D4Z4 repeat unit; at the same time, identify whether there is a XapI (AAATTC) restriction site sequence in the 1755-1780bp interval in each D4Z4 repeat unit; and record the identification results of each repeat unit of each sequencing fragment and the Bln / XapI site situation in the list; Initialize the score of each sequencing fragment that crosses the D4Z4 region and extends to the downstream haplotype region to 0, judge each D4Z4 sequence on the sequencing fragment, if the specified region contains the Bln restriction site sequence, the score is -1, if the specified region contains the XapI restriction site sequence, the score is +1, and calculate the total score; if the total score>0, record the sequencing fragment score and source as Chr4 in the list; if the total score<0, record the sequencing fragment score and source as Chr10 in the list; Initialize the score of each sequencing fragment that crosses the D4Z4 region and extends to the downstream haplotype region to 0, and judge according to the difference sites in Chr4 and Chr10 recorded in S3121. If the bases of the sequencing fragments aligned to these difference sites are consistent with the bases on Chr4, the score is +1, if they are consistent with the bases on Chr10, the score is -1, and no score is calculated for other cases. Calculate the total score. If the total score>0, the sequencing fragment score and source are recorded in the list as Chr4; if the total score<0, the sequencing fragment score and source are recorded in the list as Chr10; According to the Score and source of each sequencing fragment in the conditions S3123 and S3124, if both conditions determine that the source of the sequencing fragment is Chr4, then define the source of this sequencing fragment as Chr4 and record it in the list; if both conditions determine that the source of the sequencing fragment is Chr10, then define the source of this sequencing fragment as Chr10 and record it in the list; Finally, the source of each sequencing fragment spanning D4Z4 was obtained, and the sequencing fragment information recorded as Chr4 was retained, and other inconsistencies were deleted.

7. The FSHD assisted reproduction system based on third-generation sequencing according to claim 4, characterized in that: The step of clustering in the clustering unit according to the number of D4Z4 repeating units, downstream haplotypes and sequencing fragment sources in each sequencing fragment list comprises: Clustering was performed based on the number of D4Z4 repeat units, downstream haplotype structure, and sequencing fragment sources recorded in each sequencing fragment list; Based on the clustered sequencing fragments in Chr4 and Chr10, each sequencing fragment was clustered into haplotypes, and each sequencing fragment in Chr4 was clustered into 4qA haplotype and 4qB haplotype. The Chr4 4qA clustered haplotype sequencing fragments were clustered according to the number of D4Z4 repeat units, and the distribution of D4Z4 repeat numbers was counted and clustered; If the sum of the number of sequenced fragments within the range of plus or minus 1 unit of the peak value in the D4Z4 repeat number distribution is greater than one-half of the total sequenced fragments, it is stored in the list as the number of Chr4 4qAD4Z4 repeat units; The D4Z4 repeat count and storage were performed for the Chr4 4qB haplotype cluster sequencing fragments in the same manner to obtain the Chr4 cluster typing results based on the number of D4Z4 repeat units and downstream haplotypes.

8. The FSHD assisted reproduction system based on third-generation sequencing according to claim 1, characterized in that: The variation detection module comprises: The clustering and typing index acquisition unit is used to compare, sort and construct an index for the obtained clustering and typing sequencing fragments with the CHM13 T2T completed map reference genome; A variation detection unit is used to perform variation detection on the upstream and downstream of the Chr4 D4Z4 region based on the obtained Chr4 haplotype typing and clustering results, and record the upstream and downstream SNV, INDEL, and SV information; An information construction unit constructs a list based on the variant information sites, recording the number of sequencing fragments supporting each variant site, the total number of sequencing fragments, and homozygous and heterozygous information; Marking unit, select variants with quality values ​​greater than 30 as High, select variants with quality values ​​between 15 and 30 as Medium, and variants with quality values ​​less than 15 as Low; The linkage variation information acquisition module comprises The variant information linkage relationship acquisition unit constructs the D4Z4 upstream and downstream variant information linkage relationship based on the variant detection information in S412 and the sequencing fragment haplotype clustering results in S31. The variant linked to the Chr4 4qA pathogenic haplotype is haplotype hap1, and the other haplotype is hap2. The assisted reproductive PGT detection site acquisition unit selects the variant sites marked as High quality values ​​in hap1 and hap2 upstream and downstream of the Chr4 D4Z4 region as downstream assisted reproductive PGT detection sites.

9. A system for FSHD assisted reproduction based on third-generation sequencing, comprising a memory, a processor, and a computer program stored in the memory, characterized in that: The processor executes the computer program to implement the steps in any one of claims 1-8.

10. A computer storable medium having a computer program / instruction stored thereon, characterized in that: When the computer program / instructions are executed by a processor, the steps of the system according to any one of claims 1 to 9 are implemented.

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