Method for recognizing and correcting half-peak condition based on sanger sequencing ab1 file

Abstract

The application provides a recognition correction method based on Sanger sequencing ab1 file half-peak condition, the application can read sequence and related peak height information of a first peak and a second peak of the ab1 file, screen out files possibly having the half-peak condition through comparison of the first peak and the second peak, then correct corresponding bases and peak heights, and finally obtain corrected scf files and fasta files for later sequence analysis. The application provides an effective method for solving the ab1 file half-peak phenomenon in Sanger sequencing, improves the accuracy and reliability of sequencing data, and avoids sequencing cost, analysis cost and time cost caused by repeated resequencing.

Description

Technical Field

[0001] This invention relates to the field of bioinformatics, and more specifically, to a method for identifying and correcting the half-double peak pattern based on Sanger sequencing ab1 files. Background Technology

[0002] With the advent of next-generation sequencing technologies such as 454 (Roche), Solexa (Illumina), and SOLiD (ABI), sequencing throughput has increased rapidly while sequencing costs have decreased dramatically. This breakthrough has greatly promoted the development of genomics. First-generation sequencing technology is a faster and more accurate method for identifying bacterial species than traditional biochemical identification.

[0003] In Sanger sequencing, the ab1 file contains the raw electrophoresis data of the sequencing reaction. In some cases, due to the heterozygosity of the template DNA or other factors, two closely spaced peaks (a semi-doublet phenomenon) may appear in the sequencing graph. This can lead to incorrect base identification, making subsequent sequence analysis difficult. Traditional sequencing analysis software often cannot accurately identify and correct these semi-doublet conditions, thus affecting the accuracy of the sequencing results. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies, the purpose of this invention is to provide a method for identifying and correcting the semi-double peak situation in Sanger sequencing ab1 files. This method involves reading the sequences and related peak height information of the first and second peaks in the ab1 file, comparing the first and second peaks to screen out files that may have a semi-double peak situation, and then correcting the corresponding bases and peak heights to finally obtain the corrected scf and fasta files for subsequent sequence analysis.

[0005] The technical solution for achieving the technical objective of this invention is as follows:

[0006] A method for identifying and correcting the half-double peak pattern based on Sanger sequencing ab1 files includes:

[0007] 1) Read the ab1 file to obtain the base and corresponding peak height information, then obtain the position of each peak top and bottom, thus obtaining the sequence files of the first and second peaks;

[0008] 2) Perform comparative analysis on the sequences of the first and second peaks, output the comparison result file, and determine whether the file contains a semi-double peak.

[0009] 3) For sequences with a half-doublet, perform base correction and peak height correction on the original sequence from the start position to the end position of the half-doublet;

[0010] 4) Write all relevant information after correction to new scf and fasta files.

[0011] In step 1), if the height of the second peak is insufficient to be considered a distinct peak at the obtained peak position, i.e., the peak height of the second peak is less than 10% of the peak height of the first peak, then the base sequence of the first peak is used to replace the corresponding position of the second peak.

[0012] In step 2), the first and second peaks are sequence aligned based on the Needleman-Wunsch (NW) algorithm, with the penalty rule as follows:

[0013] Match = 1

[0014] Mismatch = -1

[0015] Gap = -10.

[0016] The alignment result file is output in a six-line structure. The first line is the file name of ab1, 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 alignment result, where "|" indicates a base match, "." indicates a base mismatch, and "" indicates the introduced GAP; the sixth line is the second peak sequence, including the introduced GAP.

[0017] In step 2), the following conditions must be met to determine if a semi-bimodal case exists:

[0018] 2.1) A gap exists in the comparison;

[0019] 2.2) Sequence consistency (identify) > 90% or sequence consistency (identify) > 93% in intervals with gaps.

[0020] In step 3), the base correction step includes:

[0021] 3.1) Correct degenerate bases appearing in the original sequence within the range of the start and end positions of the half-doublet;

[0022] 3.2) Correct base recognition errors in the original sequence caused by the shifted second peak being higher than the first peak within the range of the start and end positions of the half-double peak.

[0023] The beneficial effects of this invention are:

[0024] By reading the sequences and peak height information of the first and second peaks in the ab1 file, and comparing and analyzing the sequences of the first and second peaks to determine the presence of a semi-double peak, the peak height of the corresponding bases is corrected if a semi-double peak is found. The resulting corrected SCF and FASTA files are then used for subsequent sequence analysis. This method of correcting the semi-double peak phenomenon in the ab1 file during Sanger sequencing can improve the accuracy and reliability of sequencing data and avoid the sequencing, analysis, and time costs associated with repeated retesting. Attached Figure Description

[0025] Figure 1 This is a flowchart of the semi-bimodal correction of the ab1 file in an embodiment of the present invention.

[0026] Figure 2 This is a schematic diagram of the scoring matrix in an embodiment of the present invention.

[0027] Figure 3 This is the output result of bimodal sequence alignment in the embodiments of the present invention.

[0028] Figure 4 This is a schematic diagram comparing the correction result for the semi-bimodal case with the original file in an embodiment of the present invention. Detailed Implementation

[0029] To better illustrate the present invention, further description is provided below with reference to 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 explain the present invention, and should not be construed as limiting the present invention.

[0030] A method for identifying and correcting the half-double peak pattern based on Sanger sequencing ab1 files includes the following steps:

[0031] Step 1: Read the ab1 file, parse it to obtain the bases and corresponding peak height information and the original sequence file contained in the file. Based on the base peak height information, calculate the position of each peak top and bottom, thereby obtaining 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 insufficient to be identified as a clear peak (the peak height of the second peak is less than 10% of the peak height of the first peak), then use the base sequence of the first peak to replace the corresponding position of the second peak.

[0032] Step 2: Based on the Needleman-Wunsch (NW) algorithm (e.g.) Figure 2 As shown), this invention sets the penalty scoring rules as follows: Match = 1 point, Mismatch = -1 point, and gap = -10 points, in order to complete the sequence alignment of the first peak and the second peak, and output the alignment results, as shown. Figure 3As shown, the alignment results are output in a six-line structure. The first line is the file name of ab1, 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 alignment result, where "|" indicates a base match, "." indicates a base mismatch, and a space indicates the introduction of a GAP; the sixth line is the second peak sequence, including the introduced GAP. The presence of a semi-double peak in the file is determined based on the presence of gaps in the alignment results and whether the sequence identity (identify) is greater than 90% or whether the sequence identity (identify) in the gap interval is greater than 93%.

[0033] Step 3: As Figure 4 The upper part shows the ab1 peak diagram with a semi-double peak configuration, as shown below. Figure 4 The lower part shows the corrected ab1 peak diagram. For sequences with a semi-doublet configuration, base correction was performed on the original sequence from the start to the end of the semi-doublet, including correcting degenerate bases in the original sequence (such as...). Figure 4 (as shown in the yellow box), and correcting base errors in the original sequence caused by the shifted second peak being higher than the first peak (such as...). Figure 4 (As shown in the red box), at the same time, the peak height is corrected according to the offset peak height, and the peak height caused by the offset is deleted;

[0034] Step 4: Write all relevant information after correction into a new SCF file, and write the corrected sequences into a FASTA file. The SCF file can be opened and viewed using software such as Chromas or Bioedit to view the new peak diagram, or it can be used for phred sequence alignment with the corresponding file.

[0035] Through the above steps, this invention compares the first and second peaks obtained by reading and parsing the ab1 file, filters out files that may have a semi-double peak situation, corrects the corresponding bases and peak heights, and finally obtains the corrected scf file, which facilitates subsequent analysis, improves the accuracy and reliability of sequencing data, and avoids the sequencing cost, analysis cost and time cost caused by repeated retesting.

[0036] The embodiments described above can be further combined or replaced, and these embodiments are merely descriptions of preferred embodiments of the present invention, not limitations on the concept and scope of the present invention. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the inventive concept are all within the protection scope of the present invention. The protection scope of the present invention is given by the appended claims and any equivalents.

Claims

1. A method for identifying and correcting the semi-double peak situation based on Sanger sequencing ab1 files, characterized in that, include: 1) Read the ab1 file to obtain the base and corresponding peak height information, then obtain the position of each peak top and bottom, thus obtaining the sequence files of the first and second peaks; 2) Perform comparative analysis on the sequences of the first and second peaks, output the comparison result file, and determine whether the file contains a semi-double peak. 3) For sequences with a half-doublet, perform base correction and peak height correction on the original sequence from the start position to the end position of the half-doublet; 4) Write all relevant information after correction to new scf and fasta files; In step 1), if the height of the second peak is insufficient to be considered a distinct peak at the obtained peak position, that is, the peak height of the second peak is less than 10% of the peak height of the first peak, then the base sequence of the first peak is used to replace the corresponding position of the second peak. In step 2), the first and second peaks are sequence aligned based on the Needleman-Wunsch (NW) algorithm, with the penalty rule as follows: Match = 1 Mismatch = -1 Gap = -10 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 alignment 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. In step 2), the following conditions must be met to determine if a semi-bimodal case exists: 2.1) A gap exists in the comparison; 2.2) Sequence consistency (identify) > 90% or sequence consistency (identify) > 93% in intervals with gaps.

2. The method according to claim 1, characterized in that, In step 3), the base correction step includes: 3.1) Correct degenerate bases appearing in the original sequence within the range of the start and end positions of the half-doublet; 3.2) Correct base recognition errors in the original sequence caused by the shifted second peak being higher than the first peak within the range of the start and end positions of the half-double peak.

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