A method, system, and medium for double-end sequencing sample tag weight
By adding DNA fragment auxiliary field information during the alignment process and using sliding window technology to screen for repetitive sequences, the problems of decreased variation detection accuracy and high data throughput caused by repetitive nucleotide sequences in paired-end sequencing were solved, achieving an efficient sample reweighting process.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- GENETALKS BIO TECH (CHANGSHA) CO LTD
- Filing Date
- 2022-12-06
- Publication Date
- 2026-06-09
Smart Images
Figure CN115862741B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of bioinformatics, specifically to a method, system, and medium for weighing paired-end sequencing samples for gene sequencing. Background Technology
[0002] Next-generation sequencing (NGS) is a massively parallel gene sequencing technology capable of sequencing genes at extremely high throughput, scalability, and speed. This technology is used to determine the nucleotide sequence of a target region of the entire genome or DNA. Before determining the nucleotide sequence, a sequencing library needs to be constructed from the DNA sample. The original DNA sequence is broken down physically (by sonication) or chemically (by enzymes). Then, sequences of a specific length range are selected for polymerase chain reaction (PCR) amplification and sequencing. During PCR, the same DNA fragment is amplified several times or even tens of times to increase the density of these fragments in solution, making them easier to obtain during sampling. The sequencing instrument identifies the nucleotide sequences at both ends of the DNA fragment using optical imaging; this method is called paired-end sequencing. During optical imaging, due to the limitation of polymerase activity, paired-end sequencing cannot sequence the complete DNA fragment; the middle part of the DNA fragment is not sequenced, such as... Figure 1 As shown, DNA has a double helix structure with two main strands that are parallel to each other and run in opposite directions to form a double helix configuration. The main strand running from left to right is called the positive strand, and the main strand running from right to left is called the negative strand.
[0003] During the sequencing process, there are two pathways for introducing repetitive nucleotide sequences, known as PCR repeats and optical repeats. PCR repeats are introduced during the PCR process, where the same DNA fragment is amplified and then sequenced multiple times by the sequencing instrument, producing repetitive nucleotide sequences. Optical repeats are introduced during sequencing. To increase sequencing throughput, the density of the sequencing chip is increased. Due to light diffraction, the captured fluorescent bright spots cause ghosting, resulting in repetitive nucleotide sequences. In gene detection, a large number of repetitive nucleotide sequences can affect the test results, especially in variant detection. A large number of repetitive nucleotide sequences can introduce false positives and false negatives, significantly reducing the accuracy of variant detection. Therefore, it is necessary to identify repetitive sequences in the test sample, select one nucleotide sequence, and re-weight the repetitive data in other sequences, such as... Figure 2 As shown.
[0004] The main existing methods for weight calibration of paired-end sequencing samples are:
[0005] The reads are aligned to the whole-genome reference sequence, which is a positive strand sequence on the DNA double helix. During alignment, the negative strand is sequentially appended to the positive strand. The reads aligned to the negative strand are then converted to the positive strand, and only the starting position of the read in the reference sequence from left to right is recorded to simplify subsequent sorting, weighting, and other processing. Assuming the whole-genome reference sequence is L, after appending the negative strand to the reference sequence, positions 0 to L-1 are positive strand positions, and positions L to 2L-1 are negative strand positions. If the negative strand position is n, and L <= n < 2L, the method for converting the negative strand position n to the positive strand position p is: p = 2L - n - 1.
[0006] The main alignment information between the read length and the reference sequence includes: alignment position, read length direction (read length 1 on the positive chain in the figure is forward, and read length 2 is reverse) and matching result. The matching result records the relationship between the entire read length and the reference sequence, which mainly includes three types: one-to-one correspondence is called sequence matching; only the reference sequence exists, but the read length does not exist, which is called sequence missing; only the read length exists, but the reference sequence does not exist, which is called sequence insertion.
[0007] Next-generation sequencing produces nucleotide sequences stored in FASTQ format. Each consecutive nucleotide is considered a read, and the sequence includes the sequencing quality of each nucleotide and an identifier for the read. Reads with paired ends share the same identifier. An alignment tool is used to align the reads in the FASTQ file to their corresponding positions in the whole-genome reference sequence. The read length, nucleotide quality, alignment position, and other information are saved in SAM format. The SAM files are then sorted according to their alignment positions to generate a new SAM file sorted by the reference sequence sites. Possible duplicate reads are then identified from the ordered SAM files and marked. This process is repeated for each ordered SAM file, marking or deleting duplicate reads, and writing the results to a new SAM file.
[0008] In the fields of gene sequencing and bioinformatics analysis, the generated SAM file data is very large and cannot be buffered in computer memory at once. The performance of finding the other end of the read length is poor. Existing technologies require two readings of ordered SAM files, which places high demands on the throughput of stored read data. In sample population studies, distributed concurrent sequence weighting can easily create a performance bottleneck in shared storage read throughput. Summary of the Invention
[0009] The technical problem to be solved by this invention is to provide a method, system and medium for weight marking of paired-end sequencing samples, which reduces the throughput of data storage and retrieval during the weight marking process of paired-end sequencing samples, reduces running time and improves running efficiency.
[0010] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:
[0011] A method for weighting paired-end sequencing samples, comprising the following steps:
[0012] 1) Align the paired-end reads of each DNA fragment in the paired-end sequencing sample to the corresponding positions in the whole genome reference sequence. The paired-end reads of the fragments are in adjacent positions. Obtain the alignment information of the paired-end reads, including the alignment position, read direction and matching result. Add an auxiliary field to the alignment information of each read. The auxiliary field contains the starting position of the other end read corresponding to the read.
[0013] 2) Sort the aligned reads according to the reference sequence sites and save the ordered reads in the SAM file;
[0014] 3) Read the read lengths sequentially from the ordered SAM file, and take the read lengths at the same alignment position as a group of candidate read length repeat sequences. Then, filter the fragment repeat sequences from the candidate read length repeat sequences based on the auxiliary field.
[0015] 4) Delete duplicate sequences or mark them as duplicate sequences and write them to a new SAM file.
[0016] Optionally, the starting position in step 1) is: when the direction of the read length is positive, the starting position is its alignment position; when the direction of the read length is negative, the starting position is its alignment position plus the sum of the sequence matching length and the sequence missing length.
[0017] Optionally, in step 3), the sequential reading length from the ordered SAM file is performed using a sliding window technique.
[0018] Optionally, the specific steps for filtering fragment repeat sequences from candidate read-length repeat sequences based on auxiliary fields in step 3) are as follows:
[0019] 31) Calculate the position interval of the DNA fragment corresponding to the candidate read: Based on the auxiliary field information of the read, obtain the starting position of the other end of the DNA fragment. The starting position of the candidate read and the starting position of the other end of the DNA fragment are used as the position interval of the DNA fragment. The smaller value of the starting position is the starting position of the interval, and the larger value is the ending position of the interval.
[0020] 32) Finding repetitive sequences in fragments: Sort the DNA fragments by the starting position of the interval from smallest to largest. When the starting positions are the same, sort them by the ending position of the interval from smallest to largest. For multiple DNA fragments with the same interval, select one DNA fragment as the major fragment according to the preset strategy, and the read lengths corresponding to the other DNA fragments are the repetitive read lengths. Mark these repetitive read lengths.
[0021] Optionally, the preset strategy in step 32) is nucleotide quality value and maximum.
[0022] Optionally, in step 32), the marking method uses the Duplicate identifier in the SAM specification to mark repeated read lengths.
[0023] The present invention also provides a paired-end sequencing sample weighting system, including a computer device that is programmed or configured to perform the steps of the paired-end sequencing sample weighting method described above, or the computer device has a computer program programmed or configured to perform the paired-end sequencing sample weighting method stored in its memory.
[0024] The present invention also provides a computer-readable storage medium storing a computer program programmed or configured to perform the above-described paired-end sequencing sample weighting method.
[0025] Compared with the prior art, the present invention has the following advantages:
[0026] 1. This invention reduces runtime by adding auxiliary fields such as the location of the DNA fragment corresponding to the read length during the alignment process, eliminating the need to traverse and search for the DNA fragment corresponding to the paired-end read lengths during weighting;
[0027] 2. While traversing the read length alignment information in the SAM file, the present invention can calculate the DNA fragment repetitive sequence information and write it into a new SAM file. The weighting can be completed by traversing the SAM file once, which reduces the amount of data read by half compared with the existing methods and reduces the throughput of data storage and reading.
[0028] 3. This invention obtains candidate read length repeating sequences using sliding window technology, and performs concurrent search for repeating sequences based on candidate read length repeating sequences, thereby improving the utilization of computing resources and reducing running time. Attached Figure Description
[0029] Figure 1 This is a schematic diagram of DNA fragments in the paired-end sequencing sample described in this invention;
[0030] Figure 2 This is a schematic diagram of the weighting of paired-end sequencing samples described in this invention. Detailed Implementation
[0031] The implementation steps of the paired-end sequencing sample weighting method in this embodiment include:
[0032] 1) Align the paired-end reads of each DNA fragment in the paired-end sequencing sample to the corresponding positions in the whole genome reference sequence. The paired-end reads of the fragments are in adjacent positions. Obtain the alignment information of the paired-end reads, including the alignment position, read direction and matching result. Add an auxiliary field to the alignment information of each read. The auxiliary field contains the starting position of the other end read corresponding to the read.
[0033] 2) Sort the aligned reads according to the reference sequence sites and save the ordered reads in the SAM file;
[0034] 3) Read the read lengths sequentially from the ordered SAM file, and take the read lengths at the same alignment position as a group of candidate read length repeat sequences. Then, filter the fragment repeat sequences from the candidate read length repeat sequences based on the auxiliary field.
[0035] 4) Delete duplicate sequences or mark them as duplicate sequences and write them to a new SAM file.
[0036] This embodiment reduces running time by adding auxiliary fields such as the location of the DNA fragment corresponding to the read length during the alignment process, thus eliminating the process of traversing and searching for the DNA fragment corresponding to the paired-end read lengths during weighting.
[0037] Furthermore, the starting position in step 1) is: when the direction of the read length is positive, the starting position is its alignment position; when the direction of the read length is negative, the starting position is its alignment position plus the sum of the sequence matching length and the sequence missing length.
[0038] like Figure 1 The diagram shows a DNA fragment in a paired-end sequencing sample. The alignment information of read length 1 is supplemented with the start position of read length 2, and the alignment information of read length 2 is supplemented with the start position of read length 1. Read length 1 is forward and read length 2 is reverse. The start position of read length 1 is its alignment position, and the start position of read length 2 is its alignment position plus the sum of the sequence matching length and the sequence deletion length.
[0039] Furthermore, in step 3), the sequential reading length from the ordered SAM file is performed using a sliding window technique.
[0040] This embodiment uses sliding window technology to obtain candidate read length repeating sequences, and performs concurrent search for repeating sequences according to the candidate read length repeating sequences, which improves the utilization of computing resources and reduces running time.
[0041] Furthermore, the specific steps for filtering fragment repeat sequences from candidate read-length repeat sequences based on auxiliary fields in step 3) are as follows:
[0042] 31) Calculate the position interval of the DNA fragment corresponding to the candidate read: Based on the auxiliary field information of the read, obtain the starting position of the other end of the DNA fragment. The starting position of the candidate read and the starting position of the other end of the DNA fragment are used as the position interval of the DNA fragment. The smaller value of the starting position is the starting position of the interval, and the larger value is the ending position of the interval.
[0043] 32) Finding repetitive sequences in fragments: Sort the DNA fragments by the starting position of the interval from smallest to largest. When the starting positions are the same, sort them by the ending position of the interval from smallest to largest. For multiple DNA fragments with the same interval, select one DNA fragment as the major fragment according to the preset strategy, and the read lengths corresponding to the other DNA fragments are the repetitive read lengths. Mark these repetitive read lengths.
[0044] This embodiment calculates DNA fragment repetitive sequence information and writes it into a new SAM file while simultaneously traversing the read length alignment information in the SAM file. Weighting can be completed with only one traversal of the SAM file, reducing the amount of data read by half compared to existing methods and lowering the throughput of data storage and reading.
[0045] Furthermore, the preset strategy in step 32) is nucleotide quality value and maximum.
[0046] Furthermore, in step 32), the marking method uses the Duplicate identifier in the SAM specification to mark the repeated read length.
[0047] In the SAM specification, "duplicate" refers to PCR or optical replication. The operation in the SAM specification is to set the flag to 0x400, which means that the read can be discarded or not used, and is an invalid read. Alternatively, the read can be deleted. You can choose to mark or delete it.
[0048] This embodiment also provides a paired-end sequencing sample weighting system, including a computer device that is programmed or configured to perform the steps of the paired-end sequencing sample weighting method described above, or the computer device has a computer program programmed or configured to perform the paired-end sequencing sample weighting method stored in its memory.
[0049] This embodiment also provides a computer-readable storage medium storing a computer program programmed or configured to perform the above-described paired-end sequencing sample weighting method.
[0050] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.
Claims
1. A method for weighting paired-end sequencing samples, characterized in that, The implementation steps include: 1) Align the paired-end reads of each DNA fragment in the paired-end sequencing sample to their corresponding positions in the whole-genome reference sequence. The paired-end reads of the fragments are in adjacent positions. Obtain the alignment information of the paired-end reads, including the alignment position, read direction, and matching result. Add an auxiliary field to the alignment information of each read. The auxiliary field contains the starting position of the other end read corresponding to that read. The starting position is: when the read direction is positive, the starting position is its alignment position; when the read direction is negative, the starting position is its alignment position plus the sum of the sequence matching length and the sequence deletion length. 2) Sort the aligned reads according to the reference sequence sites and save the ordered reads in the SAM file; 3) Read the read lengths sequentially from the ordered SAM file, and treat read lengths at the same alignment position as a group of candidate read length repeat sequences. Then, filter the fragment repeat sequences from the candidate read length repeat sequences based on auxiliary fields. The specific steps are as follows: 31) Calculate the position interval of the DNA fragment corresponding to the candidate read: Based on the auxiliary field information of the read, obtain the starting position of the other end of the DNA fragment. The starting position of the candidate read and the starting position of the other end of the DNA fragment are used as the position interval of the DNA fragment. The smaller value of the starting position is the starting position of the interval, and the larger value is the ending position of the interval. 32) Finding repetitive sequences in fragments: Sort DNA fragments by the starting position of the interval from smallest to largest. When the starting positions are the same, sort them by the ending position of the interval from smallest to largest. For multiple DNA fragments with the same interval, select one DNA fragment as the major fragment according to the preset strategy, and the read lengths corresponding to the other DNA fragments are the repetitive read lengths. Mark these repetitive read lengths. 4) Delete duplicate sequences or mark them as duplicate sequences and write them to a new SAM file.
2. The method for weight labeling paired-end sequencing samples according to claim 1, characterized in that, In step 3), the reading length is sequentially read from the ordered SAM file using a sliding window technique.
3. The method for weighting paired-end sequencing samples according to claim 1, characterized in that, The preset strategy in step 32) is nucleotide quality value and maximum.
4. The method for weighting paired-end sequencing samples according to claim 1, characterized in that, In step 32), the marking method uses the Duplicate identifier in the SAM specification to mark the repeated read length.
5. A paired-end sequencing sample weighting system, comprising a computer device, characterized in that, The computer device is programmed or configured to perform the steps of the paired-end sequencing sample weighting method according to any one of claims 1 to 4, or the computer device has a computer program stored in its memory that is programmed or configured to perform the paired-end sequencing sample weighting method according to any one of claims 1 to 4.
6. A computer-readable storage medium, characterized in that, The computer-readable storage medium stores a computer program that is programmed or configured to perform the paired-end sequencing sample weighting method according to any one of claims 1 to 4.