Analysis method of insertion / deletion mutations in Sanger sequencing results
By identifying the DNA sequence to be analyzed from the Sanger sequencing results and performing comparison and disassembly operations, the problem of difficult to accurately determine the specific regions, sequence changes and site coordinates of the insertion/deletion mutations in the prior art is solved, and efficient and accurate mutation analysis is achieved, reducing interpretation errors and missed detection.
Patent Information
- Application Number
- CN202411935065.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-26
- Publication Date
- 2025-04-08
AI Technical Summary
The prior art is difficult to accurately determine the specific region, sequence changes and site coordinates of the insertion/deletion mutations from the results of the Sanger sequencing. Especially when the heterozygous mutations exist, it leads to difficult interpretation, low efficiency and prone to errors.
The DNA sequence to be analyzed is identified from the base signal peak map data obtained by sequencing by Sanger method, and is aligned with the reference DNA sequence. The wild strand and variant strand are disassembled using base and operation and base minus operation, and then the DNA sequence matching supports degenerate matching is compared to accurately analyze the specific regions, sequence changes and site coordinates of the insertion/deletion mutations.
It greatly reduces the difficulty and time of interpretation of insertion/deletion mutations, improves the accuracy and efficiency of interpretation, especially when low-proportion heterozygous mutations, improves the sensitivity of detection and reduces missed detection.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of biotechnology, and in particular to determining specific regions, sequence changes and site coordinates where insertion / deletion mutations occur from Sanger sequencing results. Background Art
[0002] Among many biotechnologies, gene mutation detection is an important need in the current prevention and treatment of human diseases.
[0003] In actual clinical applications, first-generation sequencing based on the Sanger method is currently an important technology for detecting gene mutations.
[0004] Due to the widespread existence of heterozygous mutations, Sanger sequencing often detects a mixture of multiple heterologous amplicons.
[0005] Taking the detection of gene mutations on the autosomes of the human nuclear genome as an example, when a heterozygous mutation occurs in the gene to be tested, the amplicon of the wild chain (the DNA chain that has not mutated) and the amplicon of the mutant chain (the DNA chain that has mutated) exist simultaneously in the amplicons detected by Sanger sequencing.
[0006] Taking human somatic gene mutation detection as an example, due to the variable heterozygous ratio, multiple amplicons with different sequences exist simultaneously in the amplicons detected by Sanger sequencing in an unpredictable ratio.
[0007] Taking human mitochondrial gene mutation detection as an example, due to the existence of heterogeneous variation, multiple amplicons with different sequences exist simultaneously in the amplicons detected by Sanger sequencing in an unpredictable proportion.
[0008] Taking the detection of chimera gene mutation as an example, due to the existence of chimera, multiple amplicons with different sequences exist simultaneously in the amplicons detected by Sanger sequencing in an unpredictable ratio.
[0009] The presence of heterozygous mutations leads to the mixing of heterologous amplicons, which in turn causes the appearance of heterozygous peaks in the base signal peak graph obtained by Sanger sequencing.
[0010] If the type of heterozygous mutation is a variation that does not change the number of bases (including inversion and substitution), the heterozygous peak and base changes can be seen in the specific region where the variation occurs.
[0011] If the type of heterozygous mutation is a variation that changes the number of bases (i.e., the insertion / deletion mutation, including insertion, deletion, indel, duplication, and inversion duplication), constrained by the Sanger sequencing principle, heterozygous peaks and base changes will appear not only in the specific region where the mutation occurs, but also in most regions after this region.
[0012] For heterozygous peaks caused by mutations that do not change the number of bases, they can be easily interpreted manually or using existing software to obtain the specific region where the mutation occurred, the sequence changes, and the site coordinates.
[0013] For heterozygous peaks caused by mutations that change the number of bases, due to the large number of heterozygous peaks, humans can only identify the insertion / deletion mutation to confirm that it has occurred, but cannot simply determine the specific region where the mutation occurred, the sequence changes, and the site coordinates.
[0014] Moreover, the current universal sequence alignment tools are designed to align homozygous sequences, not heterozygous sequences. After aligning the heterozygous sequence with the reference DNA sequence, satisfactory alignment results cannot be obtained or even no alignment results are obtained. It is also impossible to give the specific region, sequence changes and site coordinates where the insertion / deletion mutation occurs.
[0015] In actual clinical work, insertion / deletion mutations usually rely on manual experience and a lot of time to visually disassemble a small part of the heterozygous peaks near the region where the hypothetical mutation occurs to infer the specific region where the mutation occurs. This manual visual disassembly process is not only prone to errors and inefficient, but also sometimes has subtle errors. For example, there may be other mutations downstream of the insertion / deletion mutation. Such mutations are hidden in a bunch of heterozygous peaks caused by the insertion / deletion mutation, and are often difficult to be detected manually, resulting in missed detection.
[0016] Therefore, a method is needed to determine the specific region, sequence changes and site coordinates of the insertion / deletion mutation from the Sanger sequencing results. Summary of the invention
[0017] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention provides a method for effectively analyzing the specific region, sequence changes and site coordinates of insertion / deletion mutations in Sanger sequencing results, which greatly reduces the difficulty and time of interpreting insertion / deletion mutations and improves the accuracy and efficiency of interpretation.
[0018] In one embodiment of the present invention, a method for analyzing insertion / deletion mutations in Sanger sequencing results is provided. The method comprises the following steps: Step 1: Identify the DNA sequence to be analyzed from the base signal peak data obtained by Sanger sequencing; Step 2: Compare the DNA sequence to be analyzed with the reference DNA sequence; Step 3: Use base AND operations on the DNA sequence to be analyzed and the reference DNA sequence in the comparison to obtain the DNA sequence called the wild chain; Step 4: Perform base subtraction operation on the DNA sequence to be analyzed and the wild chain to obtain the DNA sequence called variant chain; Step 5: Align the wild chain and variant chain with the reference DNA sequence respectively to obtain the sequence changes and site coordinates of the mutation regions on the wild chain and variant chain.
[0019] In one embodiment of the present invention, the insertion / deletion mutation refers to a variation that changes the number of bases, which includes insertion, deletion, indel, duplication, inversion duplication, and other mutation forms that appear in any number, combination, order, and position.
[0020] In one embodiment of the present invention, in step 1, hybrid base recognition with an adjustable threshold is used to identify the base corresponding to each base position; when the base is a hybrid base, it is represented by one of 11 single letters, such as the 11 IUPAC standard hybrid base symbols (R, Y, M, K, S, W, H, B, V, D, N).
[0021] In one embodiment of the present invention, when a base signal peak graph cannot be provided, a ready-made DNA sequence is also applicable to steps 2 to 5 of the method.
[0022] In one embodiment of the present invention, a DNA sequence alignment supporting degenerate matching is performed in step 2 and step 5; the missing bases appearing in the alignment result are represented by a single letter, such as -; the rule of degenerate matching is: when at least one of the bases contained in base A (referring to one or more of A, C, G, T) and the bases contained in base B (referring to one or more of A, C, G, T) are the same, base A and base B are considered to match, otherwise they are considered to be mismatched.
[0023] In one embodiment of the present invention, the specific rule of the base AND operation in step 3 is: when base A and base B are ANDed, the base obtained is the base contained in common by base A and base B (referring to one or more of A, C, G, T); but when base B is a missing base, base A is obtained.
[0024] In one embodiment of the present invention, the specific rule of the base subtraction operation in step 4 is: subtracting base A from base B, the base obtained is the base contained in base A but not contained in base B (referring to one or more of A, C, G, T); but when base A and base B are the same, base A is obtained.
[0025] In one embodiment of the present invention, the mutations analyzed in step 5 include both mutations that change the number of bases and mutations that do not change the number of bases, including insertions, deletions, indels, duplications, inversion duplications, substitutions, and inversions in any number, combination, order, and position.
[0026] The analysis method provided by the present invention has at least the following beneficial effects: In one embodiment of the present invention, bases are identified according to an adjustable threshold, which not only reduces the errors in manual interpretation of heterozygous peaks, but also avoids the defects of general software using a pre-set fixed threshold; especially in the case of low-proportion heterozygous variants, such as some somatic variants, heteroplasmic variants, and chimeras, this adjustable strategy can improve the sensitivity of detection and reduce missed detections; In one embodiment of the present invention, a wild chain and a variant chain are disassembled from the DNA sequence to be analyzed by using a DNA sequence alignment supporting degenerate matching, a base addition operation and a base subtraction operation, and a DNA sequence alignment supporting degenerate matching is used again to obtain the variation of each of the wild chain and the variant chain relative to the reference DNA sequence; This strategy of disassembly and then analysis can not only accurately, quickly and effectively analyze the specific regions, sequence changes and site coordinates where insertion / deletion variations occur, but also reveal various variations that exist simultaneously in other regions to avoid missed detections. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] The present invention can be more easily understood with reference to the following drawings.
[0028] The following drawings are exemplary and are only used to illustrate one embodiment of the present invention, and are not to be construed as limiting the present invention.
[0029] Figure 1 It is a schematic diagram of the results of step 1 of an implementation method, from which it can be seen that the DNA sequence read out by the random software of the Sanger sequencer, a large number of heterozygous peaks and the DNA sequence to be analyzed obtained by the heterozygous base recognition with an adjustable threshold; it can be seen from the figure that the DNA sequence to be analyzed obtained by the heterozygous base recognition with an adjustable threshold is better than the DNA sequence read out by the random software of the Sanger sequencer.
[0030] Figure 2 This is a schematic diagram of the result of step 2 of an implementation method, from which the reference genomic DNA sequence on which the DNA sequence to be analyzed is aligned can be seen.
[0031] Figure 3 This is a schematic diagram of the result of step 3 of an implementation method. As can be seen from the figure, the DNA sequence to be analyzed and the reference genome DNA sequence on the comparison are operated to obtain the wild chain.
[0032] Figure 4 This is a schematic diagram of the result of step 4 of an implementation method. As can be seen from the figure, the DNA sequence to be analyzed and the wild chain are subjected to a subtraction operation to obtain the mutant chain.
[0033] Figure 5This is a schematic diagram of the results of step 5 of an implementation method. It can be seen from the figure that no mutation occurs in the wild chain in the region in the figure; the genomic coordinates and intragenic coordinates of the wild chain are also shown in the figure.
[0034] Figure 6 This is a schematic diagram of the results of step 5 of an implementation method. It can be seen from the figure that an insertion mutation is detected in the region of the variant chain in the figure, the inserted DNA sequence is TA, and the genomic coordinates of the insertion site are g.233760248_233760249; the genomic coordinates and intragenic coordinates of the variant chain are also shown in the figure. DETAILED DESCRIPTION
[0035] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is further described below in conjunction with the accompanying drawings and an implementation method.
[0036] An embodiment described below is exemplary and is only used to explain the present invention, and should not be construed as limiting the present invention.
[0037] One embodiment of the present invention provides a method for analyzing insertion / deletion mutations in Sanger sequencing results.
[0038] Implementation Step 1: Prepare the Sanger sequencing base signal peak file, which is the .ab1 file downloaded from the Sanger sequencer; Use BioPython to read the .ab1 file, set the peak feature used for the heterozygous base recognition threshold as the peak height ratio, and the threshold is generally 0.2. Manually visually observe the peak features, set a specific threshold, and parse the DNA sequence to be analyzed; Each base identified is represented using the 15 IUPAC standard base symbols, which include A, C, G, T, R, Y, M, K, S, W, H, B, V, D, N; The results are as follows Figure 1 As shown; From the figure, we can see the DNA sequence read out by the random software of the Sanger sequencer, a large number of heterozygous peaks, and the DNA sequence to be analyzed obtained by heterozygous base recognition with an adjustable threshold; As can be seen from the figure, the DNA sequence to be analyzed obtained by heterozygous base recognition with adjustable threshold is better than the DNA sequence read out by the random software of the Sanger sequencer.
[0039] Implementation Step 2: The reference sequence was derived from NCBI, and the sequence segment was NC_000002.12:g.233759270_233774300; The coordinate annotations of the custom sequence are: gene: UGT1A1, transcript: NM_000463.3, assembly: GRCh38.p13, chromosome: chr2, feature: upstream (NC_000002.12:g.233759270_233760269); Use the edlib dynamic programming alignment algorithm library, set degenerate matching, and align the DNA sequence to be analyzed with the reference DNA sequence; The rule of degenerate matching is: if at least one of the bases contained in base A (referring to one or more of A, C, G, T) and the base contained in base B (referring to one or more of A, C, G, T) is the same, base A and base B are considered to match, otherwise they are considered to be mismatched; Missing bases in the alignment results are indicated by -; The results are as follows Figure 2 As shown; From the figure, it can be seen that the DNA sequence to be analyzed is aligned with the reference genomic DNA sequence.
[0040] Implementation Step 3: The DNA sequence to be analyzed is subjected to base AND operation with the reference DNA sequence in the comparison to obtain a DNA sequence called wild strand; The specific rules of the base AND operation are: when base A and base B are ANDed, the bases obtained are the bases that base A and base B contain together (referring to one or more of A, C, G, T); but when base B is a missing base, base A is obtained; The results are as follows Figure 3 As shown; As can be seen from the figure, the DNA sequence to be analyzed and the reference genomic DNA sequence on the comparison are operated to obtain the wild chain.
[0041] Implementation Step 4: The DNA sequence to be analyzed and the wild chain are subjected to base subtraction operation to obtain a DNA sequence called a variant chain; The specific rules of the base subtraction operation are: when base A and base B are subtracted, the base obtained is the base contained in base A but not contained in base B (referring to one or more of A, C, G, T); but when base A and base B are the same, base A is obtained; The results are as follows Figure 4 As shown; As can be seen from the figure, the DNA sequence to be analyzed and the wild chain are subtracted to obtain the mutant chain.
[0042] Implementation Step 5: Using a DNA sequence alignment that supports degenerate matching, the wild-type chain and the variant chain are aligned with the reference DNA sequence respectively; Use edlib dynamic programming comparison algorithm library and set degenerate matching; The rule of degenerate matching is: if at least one of the bases contained in base A (referring to one or more of A, C, G, T) and the base contained in base B (referring to one or more of A, C, G, T) is the same, base A and base B are considered to match, otherwise they are considered to be mismatched; Missing bases in the alignment results are indicated by -; The results are as follows Figure 5 , Figure 6 As shown; from Figure 5 As can be seen, there is no mutation in the wild chain in the region of the figure, and the genomic coordinates and intragenic coordinates of the wild chain are also shown in Figure 5 middle; from Figure 6 As can be seen, an insertion mutation was detected in the mutant chain in the region of the figure. The inserted DNA sequence is TA, and the genomic coordinates of the insertion site are g.233760248_233760249. The genomic coordinates and intragenic coordinates of the mutant chain are also shown in Figure 6 middle.
Claims
1. A method for analyzing insertion / deletion mutations in Sanger sequencing results, characterized in that: The method comprises the following steps: Step 1: Identify the DNA sequence to be analyzed from the base signal peak data obtained by Sanger sequencing; Step 2: Compare the DNA sequence to be analyzed with the reference DNA sequence; Step 3: Use base AND operations on the DNA sequence to be analyzed and the reference DNA sequence in the comparison to obtain the DNA sequence called the wild chain; Step 4: Perform base subtraction operation on the DNA sequence to be analyzed and the wild chain to obtain the DNA sequence called variant chain; Step 5: Align the wild chain and variant chain with the reference DNA sequence respectively to obtain the sequence changes and site coordinates of the mutation regions on the wild chain and variant chain.
2. The method according to claim 1, characterized in that: The insertion / deletion mutation refers to a variation that changes the number of bases, which includes insertion, deletion, indel, duplication, inversion duplication, and these mutation forms appearing in any number, any combination, any order, and any position.
3. The method according to claim 1, characterized in that: In step 1, a heterozygous base recognition with an adjustable threshold is used to identify the base corresponding to each base position; when the base is a heterozygous base, it is represented by one of 11 single letters, such as the 11 IUPAC standard heterozygous base symbols (R, Y, M, K, S, W, H, B, V, D, N).
4. The method according to claim 1, characterized in that: When base signal peak graphs are unavailable, ready-made DNA sequences are also suitable for steps 2 to 5 of the method.
5. The method according to claim 1, characterized in that In step 2 and step 5, a DNA sequence alignment supporting degenerate matching is performed; missing bases appearing in the alignment result are represented by single letters, such as -; the rule for degenerate matching is: when at least one of the bases contained in base A (referring to one or more of A, C, G, T) and the bases contained in base B (referring to one or more of A, C, G, T) are the same, base A and base B are considered to match, otherwise they are considered to be mismatched.
6. The method according to claim 1, characterized in that The specific rule of the base AND operation in step 3 is: when base A and base B are ANDed, the base obtained is the base contained in both base A and base B (referring to one or more of A, C, G, T); but when base B is a missing base, the base obtained is base A.
7. The method according to claim 1, characterized in that The specific rules of the base subtraction operation in step 4 are: when base A and base B are subtracted, the base obtained is the base contained in base A but not contained in base B (referring to one or more of A, C, G, and T); but when base A and base B are the same, base A is obtained.
8. The method according to claim 1, characterized in that The mutations analyzed in step 5 include both mutations that change the number of bases and mutations that do not change the number of bases, including insertions, deletions, indels, duplications, inversion duplications, substitutions, and inversions in any number, combination, order, and position.