FSHD diagnosis system based on third-generation sequencing

Abstract

The invention provides an FSHD diagnosis system based on third-generation sequencing, and the system comprises an index construction module which is used for carrying out third-generation long-read-long sequencing on a to-be-detected sample, comparing sequencing data with a preset reference genome, carrying out sorting, and constructing an index to obtain index data; the sequence identification module is used for identifying and determining sequencing fragments from the 4q35 region on the chromosome Chr4 and the 10q26D4Z4 region on the chromosome Chr10; the sequence identification module is used for carrying out D4Z4 repetitive unit number and downstream haplotype identification on each sequencing fragment from D4Z4 regions on Chr4 and Chr10 to obtain an identification result; and the clustering and typing module is used for clustering and typing the sequencing fragments according to the identification result, and determining the number of D4Z4 on Chr4 and Chr10 and the downstream haplotype. The system can improve the FSHD molecular diagnosis accuracy, and a new clinical diagnosis product is provided for FSHD patients.

Description

Technical Field

[0001] The present invention relates to the technical field of medical genetic disease diagnosis, and in particular to a FSHD diagnosis system based on third-generation sequencing. Background Art

[0002] Facioscapulohumeral muscular dystrophy (FSHD) is a hereditary muscle disease with an incidence of about 1 in 20,000 people. Most patients will develop symptoms before the age of 20, affecting the muscles of the face, shoulders and upper arms, with muscle weakness and atrophy in the corresponding parts, which seriously affects the patient's quality of life and can be transmitted vertically, causing birth defects, and bringing serious psychological and economic burdens to the patient's family and society.

[0003] The pathogenesis of about 95% of FSHD patients is related to the deletion of 3.3kb D4Z4 repeat units in the 4q35 region. The patient's D4Z4 is abnormally shortened to 1-10, and is linked to the downstream 4qA to cause the disease. Genetic testing is the gold standard for the diagnosis of genetic diseases. Compared with other genetic diseases, molecular diagnosis of FSHD is very difficult, mainly because there are repetitive sequences with up to 99% homology to 4q35 D4Z4 in the 10q26 subtelomeric region of Chr10, which interfere with the detection of the number of D4Z4 repeats. The existing FSHD molecular diagnostic methods are based on Southern Blot and Bionano optical mapping technologies with enzyme cutting labels. The Southern Blot technology is relatively complex in experimental operation, has high requirements for personnel, is time-consuming and labor-intensive, and is difficult to be applied on a large scale in clinical practice; Bionano technology can accurately calculate the number of patients' D4Z4 repeat units and identify 4qA and 4qB monomer types based on the principle of enzyme cutting labels, but it has very high requirements for DNA quality and cannot obtain single base sequences.

[0004] The third-generation long read sequencing (LRS) technologies that are currently commercially successful include PacBio's single-molecule real-time sequencing technology and Oxford Nanopore's nanopore sequencing technology. The sequencing read length can exceed 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 segment duplications and deletions on chromosomes.

[0005] Therefore, it is necessary to develop a FSHD diagnostic system or product. Summary of the invention

[0006] The purpose of the present invention is to provide a FSHD diagnostic system based on third-generation sequencing. The system detects the number of D4Z4 repeat units and downstream linked haplotypes of FSHD patients through third-generation long-read sequencing technology to achieve FSHD diagnosis, improve the accuracy of FSHD molecular diagnosis, and provide a new clinical diagnostic method for FSHD patients.

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

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

[0009] The index building module is used to perform third-generation long-read sequencing on the sample to be tested, compare, sort and build an index with the preset reference genome to obtain index data;

[0010] A sequence recognition module is used to identify and determine the sequencing fragments from the 4q35 region on chromosome Chr4 and the 10q26D4Z4 region on Chr10;

[0011] A sequence identification module is used to identify the number of D4Z4 repeat units and downstream haplotypes of each sequenced fragment from the D4Z4 region on Chr4 and Chr10 to obtain an identification result;

[0012] The cluster typing module is used to cluster and type the sequencing fragments according to the identification results, and determine the number of D4Z4 on Chr4 and Chr10 and the downstream haplotype.

[0013] Furthermore, the index building module includes:

[0014] A sequencing data input unit is used to obtain data from a third-generation sequencing library of a long-fragment DNA extracted from a sample to be tested and sequenced on a third-generation long-read sequencing platform;

[0015] The data quality control unit is used to perform data quality control and filtering on the third-generation long-read sequencing fragments of the samples to be tested, and select the sequencing fragments that pass the quality control for downstream analysis;

[0016] The sequence alignment unit is used to align the third-generation long-read sequencing fragments that have passed quality control to the reference genome to locate and sort the sequencing fragments and build an index.

[0017] Furthermore, the sequence recognition module includes:

[0018] A sequence interception unit, used to intercept the sequencing fragments aligned to the D4Z4 region on Chr4 and the D4Z4 region on Chr10;

[0019] The sequence screening unit is used to screen out the sequencing fragments covering the 10-110 bp position upstream of the D4Z4 repeat region on Chr4 and the sequencing fragments covering the 10-110 bp position upstream of the D4Z4 repeat region on Chr10. These reads are the sequencing fragments covering the upstream of the D4Z4 repeat.

[0020] Furthermore, the sequence identification module comprises:

[0021] The region calculation unit is used to calculate the number of D4Z4 repeating units in the D4Z4 region on Chr4 and Chr10;

[0022] A downstream haplotype identification unit, used to identify and characterize the haplotypes downstream of D4Z4 on Chr4 and Chr10;

[0023] The sequencing fragment identification unit is used to identify and distinguish the Chr4 and Chr10 sequencing fragments extending upstream and downstream of the D4Z4 region.

[0024] Furthermore, the step of calculating the number of D4Z4 repeating units in the D4Z4 region on Chr4 and Chr10 in the region calculation unit specifically includes:

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

[0027] Further, the steps for identifying and identifying the downstream haplotype haplotypes of D4Z4 on Chr4 and Chr10 in the downstream haplotype identification unit specifically include:

[0028] 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;

[0029] The preset conditions include:

[0030] 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;

[0031] 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;

[0032] 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;

[0033] 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.

[0034] Furthermore, the steps for identifying and distinguishing the Chr4 and Chr10 sequencing fragments extending upstream and downstream of the D4Z4 region in the sequencing fragment identification unit specifically include:

[0035] Select the 4q35 region sequence on chromosome Chr4 and the 10q26 region sequence on chromosome Chr10 in the reference genome version GRCh38 for comparison and search, and record the differential sites between Chr4 and Chr10 in a list;

[0036] Select the sequencing fragments that span the D4Z4 region and extend to the downstream haplotype region, and identify whether there is a Bln restriction site sequence in the 3216-3241bp interval in each D4Z4 repeat unit; at the same time, identify whether there is a XapI restriction site sequence in the 1755-1780bp interval in each D4Z4 repeat unit; and record the identification results of each repeat unit and the Bln / XapI site situation of each sequencing fragment in a list;

[0037] 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;

[0038] 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 S3131. 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;

[0039] Judge according to the Score and source of each sequencing fragment in the conditions S3133 and S3134. If the two conditions simultaneously determine that the source of the sequencing fragment is Chr4, define the source of this sequencing fragment as Chr4 and record it in the list; if the two conditions simultaneously determine that the source of the sequencing fragment is Chr10, define the source of this sequencing fragment as Chr10 and record it in the list; delete the records in other inconsistent cases.

[0040] Furthermore, the cluster typing module clusters and types the sequencing fragments according to the identification results, and determines the number of D4Z4 on Chr4 and Chr10 and the downstream haplotype, including:

[0041] Clustering was performed based on the number of D4Z4 repeat units, downstream haplotype structure, and sequencing fragment sources recorded in each sequencing fragment list;

[0042] Cluster each sequencing fragment in the list into two categories according to Chr4 and Chr10;

[0043] 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, and each sequencing fragment in Chr10 was clustered into 4qA haplotype and 4qB haplotype;

[0044] Cluster the Chr4 4qA clustered haplotype sequencing fragments according to the D4Z4 repeat unit number, count the D4Z4 repeat number distribution and cluster them; if the sum of the number of sequencing fragments within the range of plus or minus 1 unit of the peak in the D4Z4 repeat number distribution is greater than half of the total sequencing fragments, then store them in the list as the Chr4 4qA D4Z4 repeat unit number;

[0045] The D4Z4 repeat count and storage were performed for the Chr4 4qB haplotype cluster sequencing fragment, the Chr10 4qA haplotype cluster sequencing fragment, and the Chr104qB haplotype cluster sequencing fragment in the same manner;

[0046] The results were judged based on the number of Chr4 D4Z4 repeat units and the downstream 4qA haplotype detection results.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0051] The FSHD diagnostic system based on third-generation sequencing provided by the present invention detects the number of D4Z4 repeat units and downstream linked haplotypes of FSHD patients through third-generation long-read sequencing technology to achieve FSHD diagnosis, improve the accuracy of FSHD molecular diagnosis, and provide a new clinical diagnostic product for FSHD patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] 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.

[0053] Figure 1 The present invention is a flow chart of a method for diagnosing FSHD based on third-generation sequencing.

[0054] Figure 2 This is a schematic diagram of D4Z4 repetition number and haplotype clustering in a FSHD diagnostic method based on third-generation sequencing of the present invention.

[0055] Figure 3 This is the result of the application example of the present invention.

[0056] Figure 4 This is a structural diagram of the FSHD diagnostic system based on third-generation sequencing. DETAILED DESCRIPTION

[0057] 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.

[0058] 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.

[0059] 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.

[0060] The present application will be described in detail below with reference to embodiments and experimental data.

[0061] Example 1: FSHD diagnostic system based on third-generation sequencing

[0062] 1. FSHD diagnostic system based on third-generation sequencing

[0063] (1) An index building module 100 is used to perform third-generation long-read sequencing on the sample to be tested, compare, sort and build an index for the sequencing data with a preset reference genome, and obtain index data; further, the index building module 100 includes:

[0064] A sequencing data input unit is used to obtain data from a third-generation sequencing library of a long-fragment DNA extracted from a sample to be tested and sequenced on a third-generation long-read sequencing platform;

[0065] The data quality control unit is used to perform data quality control and filtering on the third-generation long-read sequencing fragments of the samples to be tested, and select the sequencing fragments that pass the quality control for downstream analysis;

[0066] The sequence alignment unit is used to align the third-generation long-read sequencing fragments that have passed quality control to the reference genome to locate and sort the sequencing fragments and build an index.

[0067] (2) a sequence recognition module 200, for identifying and determining sequenced fragments from regions 4q35 on chromosome Chr4 and 10q26D4Z4 on Chr10; further, the sequence recognition module 200 comprises:

[0068] A sequence interception unit, used to intercept the sequencing fragments aligned to the D4Z4 region on Chr4 and the D4Z4 region on Chr10;

[0069] The sequence screening unit is used to screen out the sequencing fragments covering the 10-110 bp position upstream of the D4Z4 repeat region on Chr4 and the sequencing fragments covering the 10-110 bp position upstream of the D4Z4 repeat region on Chr10. These reads are the sequencing fragments covering the upstream of the D4Z4 repeat.

[0070] (3) a sequence identification module 300, for identifying the number of D4Z4 repeat units and downstream haplotypes of each sequenced fragment from the D4Z4 region on Chr4 and Chr10 to obtain an identification result; further, the sequence identification module includes:

[0071] The region calculation unit is used to calculate the number of D4Z4 repeating units in the D4Z4 region on Chr4 and Chr10;

[0072] A downstream haplotype identification unit, used to identify and characterize the haplotypes downstream of D4Z4 on Chr4 and Chr10;

[0073] The sequencing fragment identification unit is used to identify and distinguish the Chr4 and Chr10 sequencing fragments extending upstream and downstream of the D4Z4 region.

[0074] (4) A clustering and typing module, which is used to cluster and type the sequencing fragments according to the identification results, and determine the number of D4Z4 on Chr4 and Chr10 and the downstream haplotype.

[0075] Example 2: A method for FSHD diagnosis based on third-generation sequencing

[0076] The first embodiment of the present invention provides a FSHD diagnosis method based on third-generation sequencing, comprising:

[0077] S11, performing third-generation long-read sequencing on the sample to be tested, and aligning, sorting and indexing the sequencing data with the preset reference genome;

[0078] S21, identify and confirm the sequencing fragments from the D4Z4 region at 4q35 on chromosome Chr4 and 10q26 on Chr10;

[0079] S31, the number of D4Z4 repeat units and downstream haplotype identification were performed for each sequencing fragment from the D4Z4 region on Chr4 and Chr10;

[0080] S41, clustering and typing the sequenced fragments according to the identification results, and determining the number of D4Z4 on Chr4 and Chr10 and the downstream haplotype;

[0081] In Example 2 of the present invention, in a FSHD diagnosis method based on third-generation sequencing S11, the sample to be tested is subjected to third-generation long-read sequencing, and the sequencing data is compared, sorted, and indexed with a preset reference genome, including:

[0082] S111, extracting long DNA fragments from the sample to be tested, constructing a third-generation sequencing library, and sequencing it on a third-generation long-read sequencing platform;

[0083] S112, perform data quality control and filtering on the third-generation long-read sequencing fragments of the samples to be tested, and select the sequencing fragments that pass the quality control for downstream analysis;

[0084] S113, align the third-generation long-read sequencing fragments that have passed quality control to the reference genome to locate and sort the sequencing fragments and build an index.

[0085] 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:

[0086] S211, intercepted the sequencing fragments aligned to the D4Z4 region on Chr4 (Chr4: 190065229-190093263) and the D4Z4 region on Chr10 (Chr10: 133664430-133761680);

[0087] 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.

[0088] In Example 2 of the present invention, in a method S31 for diagnosing FSHD based on third-generation sequencing, the number of D4Z4 repeat units and downstream haplotype identification are performed on each sequencing fragment from the D4Z4 region on Chr4 and Chr10, including:

[0089] S311, calculate the number of D4Z4 repeating units in the D4Z4 region on Chr4 and Chr10;

[0090] S312, identify and characterize haplotypes downstream of D4Z4 on Chr4 and Chr10;

[0091] S313, identify and differentiate the Chr4 and Chr10 sequencing fragments extending upstream and downstream of the D4Z4 region.

[0092] In Example 2 of the present invention, in a method for diagnosing FSHD based on third-generation sequencing S311, the number of D4Z4 repeating units in the D4Z4 region on Chr4 and Chr10 is calculated, comprising:

[0093] S3111, aligning the sequencing fragments covering the upstream of the D4Z4 region on Chr4 with a single D4Z4 repeating unit, determining the number of D4Z4 repeating units in each sequencing fragment and recording it;

[0094] S3112, aligning the sequencing fragments covering the upstream of the D4Z4 region on Chr10 with a single D4Z4 repeat unit, determining the number of D4Z4 repeat units in each sequencing fragment and recording it in a list;

[0095] In Example 2 of the present invention, in a method for diagnosing FSHD based on third-generation sequencing S312, identifying and determining the haplotype downstream of D4Z4 on Chr4 and Chr10, comprises:

[0096] S3121, 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;

[0097] S3122, 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, then mark this sequencing fragment as extending to the downstream haplotype region;

[0098] S3123, 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, then mark this sequencing fragment as extending to the downstream haplotype region;

[0099] S3124, 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;

[0100] S3125, 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.

[0101] S3126, 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.

[0102] In Example 1 of the present invention, in a method for diagnosing FSHD based on third-generation sequencing S313, identifying and distinguishing Ch4 and Chr10 sequencing fragments extending upstream and downstream of the D4Z4 region comprises:

[0103] S3131, 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;

[0104] S3132, 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;

[0105] S3133, 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;

[0106] S3134, 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 S3131. 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;

[0107] S3135, judge according to the Score and source of each sequencing fragment in the conditions of S3133 and S3134. If the two conditions simultaneously determine that the source of the sequencing fragment is Chr4, define the source of this sequencing fragment as Chr4 and record it in the list; if the two conditions simultaneously determine that the source of the sequencing fragment is Chr10, define the source of this sequencing fragment as Chr10 and record it in the list; delete the record in other inconsistent cases.

[0108] In Example 2 of the present invention, in a method for diagnosing FSHD based on third-generation sequencing S41, the sequencing fragments are clustered and typed according to the identification results to determine the number of D4Z4 on Chr4 and Chr10 and the downstream haplotype, including:

[0109] S411, clustering according to the number of D4Z4 repeating units, downstream haplotype structure and sequencing fragment sources recorded in each sequencing fragment list;

[0110] S412, first, cluster each sequencing fragment in the list into two categories according to Chr4 and Chr10;

[0111] S413, based on the clustered sequencing fragments in Chr4 and Chr10, perform haplotype clustering on each sequencing fragment, clustering each sequencing fragment in Chr4 into 4qA haplotype and 4qB haplotype, and clustering each sequencing fragment in Chr10 into 4qA haplotype and 4qB haplotype;

[0112] S414, clustering the Chr4 4qA clustered haplotype sequencing fragments according to the number of D4Z4 repeating units, and statistically analyzing the distribution of the number of D4Z4 repeats and clustering them;

[0113] S415, 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 it is stored in the list as the Chr4 4qA D4Z4 repeat unit number;

[0114] S416, counting and storing the D4Z4 repeat times for the Chr4 4qB haplotype cluster sequencing fragments, the Chr10 4qA haplotype cluster sequencing fragments, and the Chr104qB haplotype cluster sequencing fragments according to the same method;

[0115] S417, judge the results according to the number of Chr4 D4Z4 repeat units and the downstream 4qA haplotype detection results.

[0116] Example 3: FSHD diagnostic system based on third-generation sequencing

[0117] An embodiment of the present invention provides a FSHD diagnosis system based on third-generation sequencing, the system comprising:

[0118] 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.

[0119] Embodiment 4: Computer readable storage medium

[0120] 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.

[0121] 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.

[0122] 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.

[0123] 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.

[0124] Application Example 1: A method for assisted reproduction of FSHD based on third-generation sequencing

[0125] A total of 4 clinically suspected FSHD samples and 5 normal samples were selected, for a total of 9 samples, and the number of D4Z4 repeat units and downstream haplotype detection were performed based on the third-generation sequencing technology according to the diagnostic process in Example 1. A total of 4 of the 9 samples were positive for FSHD, and the other 5 samples were negative for FSHD. The detailed test results are shown in the table below.

[0126] Table 1

[0127]

[0128]

[0129] As can be seen from the above table, the test results of the third-generation sequencing technology were further compared with the results of Bionano technology, the gold standard for FSHD clinical testing, and the consistency of the FSHD test results was 100%.

[0130] 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.

[0131] 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.

[0132] 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 FSHD diagnostic system based on third-generation sequencing, characterized in that: The system comprises: The index building module is used to perform third-generation long-read sequencing on the sample to be tested, compare, sort and build an index with the preset reference genome to obtain index data; A sequence recognition module is used to identify and determine the sequencing fragments from the 4q35 region on chromosome Chr4 and the 10q26D4Z4 region on Chr10; A sequence identification module is used to identify the number of D4Z4 repeat units and downstream haplotypes of each sequenced fragment from the D4Z4 region on Chr4 and Chr10 to obtain an identification result; The cluster typing module is used to cluster and type the sequencing fragments according to the identification results, and determine the number of D4Z4 on Chr4 and Chr10 and the downstream haplotype.

2. A FSHD diagnostic system based on third-generation sequencing according to claim 1, characterized in that: The index building module includes: A sequencing data input unit is used to obtain data from a third-generation sequencing library of a long-fragment DNA extracted from a sample to be tested and sequenced on a third-generation long-read sequencing platform; The data quality control unit is used to perform data quality control and filtering on the third-generation long-read sequencing fragments of the samples to be tested, and select the sequencing fragments that pass the quality control for downstream analysis; The sequence alignment unit is used to align the third-generation long-read sequencing fragments that have passed quality control to the reference genome to locate and sort the sequencing fragments and build an index.

3. The FSHD diagnostic system based on third-generation sequencing according to claim 1, characterized in that: The sequence recognition module comprises: A sequence interception unit, used to intercept the sequencing fragments aligned to the D4Z4 region on Chr4 and the D4Z4 region on Chr10; The sequence screening unit is used to screen out the sequencing fragments covering the 10-110 bp position upstream of the D4Z4 repeat region on Chr4 and the sequencing fragments covering the 10-110 bp position upstream of the D4Z4 repeat region on Chr10. These reads are the sequencing fragments covering the upstream of the D4Z4 repeat.

4. The FSHD diagnostic system based on third-generation sequencing according to claim 1, characterized in that: The sequence identification module comprises: The region calculation unit is used to calculate the number of D4Z4 repeating units in the D4Z4 region on Chr4 and Chr10; A downstream haplotype identification unit, used to identify and characterize the haplotypes downstream of D4Z4 on Chr4 and Chr10; The sequencing fragment identification unit is used to identify and distinguish the Chr4 and Chr10 sequencing fragments extending upstream and downstream of the D4Z4 region.

5. A FSHD diagnostic system based on third-generation sequencing according to claim 4, characterized in that: The step of calculating the number of D4Z4 repeating units in the D4Z4 region on Chr4 and Chr10 in the region calculation unit specifically includes: The sequencing fragments covering the upstream of the D4Z4 region on Chr4 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 covering the upstream of the D4Z4 region on Chr10 were aligned with a single D4Z4 repeat unit, and the number of D4Z4 repeat units in each sequencing fragment was determined and recorded in a list.

6. The FSHD diagnostic system based on third-generation sequencing according to claim 4, characterized in that: The steps for identifying and identifying the downstream haplotype haplotypes of D4Z4 on Chr4 and Chr10 in the downstream haplotype identification unit specifically include: 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.

7. The FSHD diagnostic system based on third-generation sequencing according to claim 4, characterized in that: The steps for identifying and distinguishing the Chr4 and Chr10 sequencing fragments extending upstream and downstream of the D4Z4 region in the sequencing fragment identification unit specifically include: Select the 4q35 region sequence on chromosome Chr4 and the 10q26 region sequence on chromosome Chr10 in the reference genome version GRCh38 for comparison and search, and record the differential sites between Chr4 and Chr10 in a list; Select the sequencing fragments that span the D4Z4 region and extend to the downstream haplotype region, and identify whether there is a Bln restriction site sequence in the 3216-3241bp interval in each D4Z4 repeat unit; at the same time, identify whether there is a XapI restriction site sequence in the 1755-1780bp interval in each D4Z4 repeat unit; and record the identification results of each repeat unit and the Bln / XapI site situation of each sequencing fragment in a 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 S3131. 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; Judge according to the Score and source of each sequencing fragment in the conditions S3133 and S3134. If the two conditions simultaneously determine that the source of the sequencing fragment is Chr4, define the source of this sequencing fragment as Chr4 and record it in the list; if the two conditions simultaneously determine that the source of the sequencing fragment is Chr10, define the source of this sequencing fragment as Chr10 and record it in the list; delete the records in other inconsistent cases.

8. The FSHD diagnostic system based on third-generation sequencing according to claim 1, characterized in that: The cluster typing module clusters and types the sequencing fragments according to the identification results, and determines the number of D4Z4 on Chr4 and Chr10 and the downstream haplotype in the following specific steps: Clustering was performed based on the number of D4Z4 repeat units, downstream haplotype structure, and sequencing fragment sources recorded in each sequencing fragment list; Cluster each sequencing fragment in the list into two categories according to Chr4 and Chr10; 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, and each sequencing fragment in Chr10 was clustered into 4qA haplotype and 4qB haplotype; Cluster the Chr4 4qA clustered haplotype sequencing fragments according to the D4Z4 repeat unit number, count the D4Z4 repeat number distribution and cluster them; if the sum of the number of sequencing fragments within the range of plus or minus 1 unit of the peak in the D4Z4 repeat number distribution is greater than half of the total sequencing fragments, then store them in the list as the Chr4 4qA D4Z4 repeat unit number; The D4Z4 repeat count and storage were performed for the Chr4 4qB haplotype cluster sequencing fragment, the Chr10 4qA haplotype cluster sequencing fragment, and the Chr104qB haplotype cluster sequencing fragment in the same manner; The results were judged based on the number of Chr4 D4Z4 repeat units and the downstream 4qA haplotype detection results.

9. A FSHD diagnostic system 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 of 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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