Method for identifying and correcting half-double peak conditions of ab1 file based on Sanger sequencing

Abstract

The invention provides an ab1 file half-bimodal condition identification and correction method based on Sanger sequencing, and the method comprises the steps: carrying out the reading of the sequences and related peak height information of a first peak and a second peak of an ab1 file, and comparing the first peak with the second peak to screen out a file which may have a half-bimodal condition; and then correcting corresponding basic groups and peak heights to finally obtain corrected scf files and fasta files for later sequence analysis. The invention provides a method for effectively solving the half-bimodal phenomenon of an ab1 file in Sanger sequencing, so that the accuracy and reliability of sequencing data are improved, and the sequencing cost, the analysis cost and the time cost caused by repeated retesting are avoided.

Description

Technical Field

[0001] The present invention relates to the field of bioinformatics, and in particular to a method for identifying and correcting semi-double peaks of ab1 files based on Sanger sequencing. Background Art

[0002] With the emergence of the next-generation sequencing technologies 454 (Roche), Solexa (Illumina) and SOLiD (ABI), sequencing throughput has been rapidly increased, while sequencing costs have dropped dramatically. This breakthrough has greatly promoted the development of genomic science. Bacterial species identification through first-generation sequencing technology is a faster and more accurate identification method than traditional biochemical identification.

[0003] In Sanger sequencing, the ab1 file contains the raw electrophoretic data of the sequencing reaction. In some cases, due to the heterozygosity of the template DNA or other factors, two similar peaks may appear in the peak graph, i.e., the semi-doublet phenomenon, which may lead to incorrect base recognition and bring difficulties to subsequent sequence analysis. Traditional sequencing analysis software often cannot accurately identify and correct these semi-doublet situations, thus affecting the accuracy of sequencing results. Summary of the invention

[0004] In order to overcome the shortcomings of the prior art, the purpose of the present invention is to provide a method for identifying and correcting the semi-double peak situation of the ab1 file based on Sanger sequencing, by reading the sequence and related peak height information of the first peak and the second peak of the ab1 file, and screening out files that may have semi-double peak situations by comparing the first peak and the second peak, and then correcting the corresponding bases and peak heights, and finally obtaining the corrected scf file and fasta file for subsequent sequence analysis.

[0005] The technical solution for achieving the technical purpose of the present invention is as follows: A method for identifying and correcting semi-double peaks based on Sanger sequencing ab1 files, comprising: 1) Read the ab1 file to obtain the base and corresponding peak height information, and then obtain the top and bottom positions of each peak, thereby obtaining the sequence files of the first peak and the second peak; 2) Compare and analyze the sequences of the first peak and the second peak, output the comparison result file, and determine whether the file contains a semi-double peak; 3) For sequences with semi-double peaks, base correction and peak height correction are performed on the original sequence from the starting position to the ending position of the semi-double peak; 4) Write all relevant information after correction into new scf files and fasta files.

[0006] In step 1), at the peak top position obtained, if the height of the second peak is not enough to be recognized as a clear peak, that is, the height of the second peak is less than 10% of the height of the first peak, the first peak base sequence is used to replace the corresponding position of the second peak.

[0007] In step 2), the first peak and the second peak are aligned based on the Needleman-Wunsch (NW) algorithm, and the penalty rule is: Match = 1 Mismatch = -1 Gap = -10.

[0008] The alignment result file is output in a six-line structure, the first line is the ab1 file name, the second line is the sequence alignment identify, the third line is the gap number, the fourth line is the first peak sequence, including the introduced GAP; the fifth line is the comparison result, where "|" indicates a base match, "." indicates a base mismatch, and " " indicates the introduction of a GAP; the sixth line is the second peak sequence, including the introduced GAP.

[0009] In step 2), the following conditions must be met to determine if there is a semi-bimodal situation: 2.1) There are gaps in the alignment; 2.2) The sequence identity is identify> 90% or the sequence identity in the gap interval is identify> 93%.

[0010] In step 3), the base correction step comprises: 3.1) Correct the annexed bases in the original sequence within the start and end positions of the semi-doublet; 3.2) Correct base recognition errors in the original sequence caused by the second peak being higher than the first peak within the start and end positions of the semi-doublet.

[0011] Beneficial effects of the present invention: The first peak and the second peak sequence and related peak height information are read from the ab1 file, and the first peak and the second peak sequence are compared and analyzed to determine the semi-double peak situation. If it is a semi-double peak, the peak height of the corresponding base is corrected, and finally the corrected scf file and fasta file are obtained for later sequence analysis. This method can improve the accuracy and reliability of sequencing data by correcting the semi-double peak phenomenon of the ab1 file in Sanger sequencing, and avoid the sequencing cost, analysis cost and time cost caused by repeated re-testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 4 is a flowchart of semi-bimodal correction of ab1 file in an embodiment of the present invention.

[0013] Figure 2 Schematic diagram of the scoring matrix in the embodiment of the present invention.

[0014] Figure 3 It is the output result of the bimodal sequence alignment in the embodiment of the present invention.

[0015] Figure 4 It is a schematic diagram of comparing the correction result with the original file for the semi-bimodal situation in an embodiment of the present invention. DETAILED DESCRIPTION

[0016] In order to better illustrate the present invention, the following is further described in conjunction with embodiments, examples of which are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to be used to explain the present invention, but should not be understood as limiting the present invention.

[0017] A method for identifying and correcting the semi-double peak situation based on Sanger sequencing ab1 file, comprising the following steps: Step 1: Read the ab1 file, parse the bases and corresponding peak height information contained in the file and the original sequence file, calculate the top and bottom positions of each peak based on the base peak height information, and thus obtain the sequence files of the first peak and the second peak. At the calculated peak top position, if the height of the second peak is not enough to be recognized as a clear peak (the height of the second peak is less than 10% of the height of the first peak), use the first peak base sequence to replace the corresponding position of the second peak; Step 2: Based on the Needleman-Wunsch (NW) algorithm (such as Figure 2 As shown in FIG. 1 ), the present invention sets the penalty rule as 1 point for Match, -1 point for Mismatch, and -10 points for Gap to complete the sequence alignment of the first peak and the second peak, and outputs the alignment result, as shown in FIG. Figure 3 As shown, the alignment result is output in a six-line structure. The first line is the ab1 file name, the second line is the sequence alignment identify, the third line is the number of gaps, the fourth line is the first peak sequence, including the introduced GAP; the fifth line is the comparison result, where "|" indicates base match, "." indicates base mismatch, and a space indicates the introduction of a GAP; the sixth line is the second peak sequence, including the introduced GAP. Whether there is a gap in the alignment result and whether the sequence identity (identify) is greater than 90% or whether the sequence identity (identify) in the interval where the gap occurs is greater than 93%, etc., is used to determine whether the file has a semi-double peak. Step 3: If Figure 4 The upper part of the graph shows the ab1 peak diagram with a semi-double peak. Figure 4The lower part of the figure shows the corrected ab1 peak. For sequences with semi-double peaks, base correction is performed on the original sequence from the start position to the end position of the semi-double peak, including correction of the merged bases in the original sequence (such as Figure 4 yellow box), and to correct base errors in the original sequence caused by the second peak being higher than the first peak (as shown in Figure 4 The peak height is corrected according to the offset peak height, and the peak height caused by the offset is deleted; Step 4: Write all relevant information after correction into a new scf file, and write the corrected sequence into a fasta file. The scf file can be opened using software such as Chromas or Bioedit to view the new peak graph, or it can be aligned with the corresponding file for phred sequence.

[0018] Through the above steps, the present invention compares the first peak and the second peak obtained by reading and analyzing the ab1 file and calculating it, screens out files that may have semi-double peaks, and corrects the corresponding bases and peak heights, and finally obtains the corrected scf file, which is convenient for subsequent analysis, improves the accuracy and reliability of sequencing data, and avoids the sequencing cost, analysis cost and time cost caused by repeated retesting.

[0019] The embodiments described above can be further combined or replaced, and the embodiments are only descriptions of preferred embodiments of the present invention, and do not limit the concept and scope of the present invention. Without departing from the design concept of the present invention, various changes and improvements made by ordinary technicians in this field to the technical solution of the present invention belong to the protection scope of the present invention. The protection scope of the present invention is given by the attached claims and any equivalents thereof.

Claims

1. A method for identifying and correcting semi-double peaks based on Sanger sequencing ab1 files, characterized in that: include: 1) Read the ab1 file to obtain the base and corresponding peak height information, and then obtain the top and bottom positions of each peak, thereby obtaining the sequence files of the first peak and the second peak; 2) Compare and analyze the sequences of the first peak and the second peak, output the comparison result file, and determine whether the file contains a semi-double peak; 3) For sequences with semi-double peaks, base correction and peak height correction are performed on the original sequence from the starting position to the ending position of the semi-double peak; 4) Write all relevant information after correction into new scf files and fasta files.

2. The method according to claim 1, characterized in that In step 1), at the peak top position obtained, if the height of the second peak is not enough to be recognized as a clear peak, that is, the height of the second peak is less than 10% of the height of the first peak, the first peak base sequence is used to replace the corresponding position of the second peak.

3. The method according to claim 1, characterized in that In step 2), the first peak and the second peak are aligned based on the Needleman-Wunsch (NW) algorithm, and the penalty rule is: Match = 1 Mismatch = -1 Gap = -10.

4. The method according to claim 1 or 3, characterized in that: The alignment result file is output in a six-line structure, the first line is the ab1 file name, the second line is the sequence alignment identify, the third line is the gap number, the fourth line is the first peak sequence, including the introduced GAP; the fifth line is the comparison result, where "|" indicates a base match, ". " indicates a base mismatch, and " " indicates an introduced GAP; the sixth line is the second peak sequence, including the introduced GAP.

5. The method according to claim 1, characterized in that In step 2), the following conditions must be met to determine if there is a semi-bimodal situation: 2.1) There are gaps in the alignment; 2.2) The sequence identity is identify> 90% or the sequence identity in the gap interval is identify> 93%.

6. The method according to claim 1, characterized in that In step 3), the base correction step comprises: 3.1) Correct the annexed bases in the original sequence within the start and end positions of the semi-doublet; 3.2) Correct base recognition errors in the original sequence caused by the second peak being higher than the first peak within the start and end positions of the semi-doublet.

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