A method for analyzing multiplex amplified STR data

Abstract

The present invention discloses an analysis method for composite amplified STR data, comprising the following steps: 1. Site reference information: input data; 2. Quality control (preprocessing): filter data; 3. STR typing method: a) find the best match; b) refer to internal reference data. Our method can not only ensure high typing accuracy and timeliness, but also realize automatic conversion of the CODIS system of STR sites. In terms of accuracy, our consistency with capillary electrophoresis data is as high as 100%, and in terms of timeliness, our analysis time is significantly less than the mainstream analysis software on the market. Therefore, as an important supplementary method for whole genome sequence analysis, this process has great practical significance for forensic genetics.

Description

Technical Field

[0001] The present invention relates to the technical field of STR data analysis, and in particular to an analysis method for composite amplification STR data. Background Art

[0002] In recent years, deoxyribonucleic acid (DNA) sequencing technology has developed by leaps and bounds, and the continuous development of the second generation sequencing technology (Next generation sequencing) has made whole genome sequencing possible. However, whole genome sequencing is not suitable for analyzing complex repetitive DNA regions, including what we call short tandem repeat sequences (STR), due to its limitations such as high cost and large data consumption. Therefore, taking multiplex PCR and probe capture as examples, many methods for obtaining target sequences that match the second generation sequencing are also constantly maturing and developing. Compared with probe capture, multiplex polymerase chain reaction (PCR) has better acquisition efficiency for STR loci. Therefore, the multiplex PCR technology based on the NGS technology platform improves the detection efficiency of STR locus polymorphism, making it show better forensic application value.

[0003] Since 1990, with the launch of various genome sequencing projects, breakthroughs in molecular structure determination technology and the popularization of the Internet, hundreds of biological analysis methods have emerged. Most of the methods and software are aimed at whole genome sequencing data, and analysis software specific to STR loci is not common.

[0004] Take the STR analysis software lobSTR as an example. lobSTR is a tool for analyzing short tandem repeat sequences (STR) from high-throughput sequencing data. It has a built-in CODIS system and 88 STR internal reference data. In theory, it can be applied to a variety of second-generation sequencing data. Short tandem repeat sequences (STR) have a wide range of applications, including medical genetics, forensics, and genetic genealogy. In the application of second-generation sequencing, hundreds of thousands of STR loci may be sequenced at the same time. This will result in a lot of time spent in the analysis of offline data and biased sampling of STR alleles. lobSTR uses signal processing and statistics to a certain extent to solve the problem of sequencing noise generated during STR sequencing. The speed and reliability of its data analysis exceed the performance of the mainstream algorithms previously used for STR analysis. Its STR detection rate and genome matching rate are lower than the mainstream whole genome analysis method. The disadvantages are:

[0005] 1. Poor adaptability: Existing sequence analysis technologies are only used for whole genome data and lack specific processing of multiplex PCR sequencing data.

[0006] 2. Accuracy: The STR database is based on the typing standards of the CODIS system, and the existing analysis data cannot match the capillary electrophoresis data. For example, the mainstream technical software lobSTR has the problem of low genome matching rate and low accuracy.

[0007] 3. Coverage: There are few analytical software for STR, and the update time is long, which cannot solve forensic practice problems in a timely manner. For example, the mainstream technical software lobSTR has a built-in CODIS system and 88 STR internal reference data.

[0008] Therefore, it is urgent to develop a software that is suitable for the multiplex PCR offline data of the second-generation sequencing platform and can analyze STR sequences. Summary of the invention

[0009] To achieve the above object, the present invention provides the following technical solutions:

[0010] A method for analyzing composite amplified STR data, comprising the following steps:

[0011] 1. Site reference information:

[0012] Input data: In addition to the CODIS locus information that comes with the software, users can customize STR loci for typing detection by entering custom site information.

[0013] 2. Quality control-pretreatment:

[0014] Filtering data: To ensure the accuracy of typing results, the original sequencing data needs to be filtered for adapter sequences and low-quality bases. The adapter filtering software AdapterRemoval is used to achieve quality control of the second-generation sequencing data. Sequencing fragments with a sequencing depth lower than 5X are removed from subsequent analysis.

[0015] 3. STR typing method:

[0016] a) Finding the best match: The Hamming distance between the sequencing fragment and the STR locus flanking reference sequence is used to measure the similarity between sequences, thereby locating the flanking sequence position of each STR locus. If it completely matches the target sequence, the length is calculated and then the depth is counted. If it does not completely match the target sequence, the Hamming distance is calculated and the best match is found, and then the length is calculated and then the depth is counted.

[0017] b) Calculate STR typing with reference to internal reference data: After locating the flanking sequence, extract the predicted repeat unit region and compare it with the internal parameters of the corresponding STR locus. Calculate all possible typing results and their depth for the locus based on the difference between the repeat unit region and the reference coverage length based on the hg38 reference genome. If the CODIS locus is not included, the genotype structure is directly output. If the CODIS locus is included, the genotype structure is output after automatic conversion by the CODIS system.

[0018] As a further solution of the present invention, the customized locus information needs to include the following: 1) non-homologous regions of at least 30 bp flanking the target STR locus in a unified direction; 2) the repeat unit sequence of the corresponding STR locus; 3) each target STR locus needs to provide the number of repeats of the repeat unit based on the hg38 reference genome and the target region coverage length as an internal reference.

[0019] As a further solution of the present invention, the maximum value of the Hamming distance should not exceed 1 / 15 of the length of the flanking sequence.

[0020] As a further solution of the present invention, the specific parameter settings of the adapter filtering software AdapterRemoval include:

[0021] 1) Trim the 5' or 3' end of the sequencing fragments with a quality score less than 2 or unclear bases;

[0022] 2) Filter sequencing fragments with read length shorter than 50 bp;

[0023] 3) Set the remaining parameters to their default values.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] (1) Adaptability: It can supplement the whole genome sequencing analysis method. The existing STR locus analysis method is mainly aimed at whole genome sequencing. When processing data with a particularly high repetitive ratio such as composite amplification, it will filter out most of the repetitive sequences, which makes the sequencing accuracy and timeliness have certain limitations.

[0026] (2) Accuracy: The analysis results of the data generated by the composite amplification provided by the software of the present invention are completely consistent with the results of the first-generation capillary electrophoresis sequencing, so it has high accuracy and provides an accurate STR locus typing method.

[0027] (3) Timeliness: The software provided by the present invention takes a short time and requires a small amount of memory in the process of analyzing the data generated by composite amplification, which makes it have the advantages of high efficiency and strong flexibility in typing composite amplification data.

[0028] We have developed a unique method for STR multiplex PCR off-machine data. Our method can not only ensure high typing accuracy and timeliness, but also realize the automatic conversion of STR loci in the CODIS system. In terms of accuracy, our consistency with capillary electrophoresis data is as high as 100%, and in terms of timeliness, our analysis time is significantly less than the mainstream analysis software on the market. Therefore, this process, as an important supplementary method for whole genome sequence analysis, has great practical significance for forensic genetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 A technical roadmap for the analysis of multiplex amplified STR data.

[0030] Figure 2 A schematic diagram of data input for an analysis method for multiplex amplified STR data.

[0031] Figure 3 Schematic diagram of Hamming distance in an analysis method for composite amplified STR data.

[0032] Figure 4 Schematic diagram of the experimental process of an analysis method for composite amplified STR data.

[0033] Figure 5 Table 1a shows a comparison of the results of sequencing 9947a using the method of the present invention and capillary electrophoresis.

[0034] Figure 6 Table 1b shows a comparison of the results of sequencing 9948 using the method of the present invention and capillary electrophoresis.

[0035] Figure 7 Table 2 shows the time consumption and memory usage of the method of the present invention. DETAILED DESCRIPTION

[0036] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] STR loci are important genetic markers in human genetics, with an average of one every 6,000-10,000 bases in the human genome. They are widely used in individual identification and kinship identification because of their rich polymorphism, large number, easy amplification and convenient detection. It is reported that more than 8,000 STR loci have been found in the human genome, and all of them have genetic polymorphism. Therefore, the test of STR sequence polymorphism and length polymorphism is becoming the most important technical means for forensic evidence identification. Relying on the second-generation sequencing platform, not only can the same length information as capillary electrophoresis be obtained, but also the sequence information of STR can be obtained, which makes it possible to determine difficult relationships, detect genetic mutations and build STR databases.

[0038] Multiplex PCR, also known as multiple primer PCR or composite PCR, refers to the introduction of two or more pairs of primers in a certain primer ratio in the same PCR reaction system to achieve a fast and efficient amplification reaction. The technical elements of multiplex PCR mainly include target fragment selection, primer design, renaturation temperature and time, extension temperature and time, and the amount of each reaction component. The goal of multiplex PCR is to amplify multiple target fragments, so the selection of target fragments is the core. The fragments must be highly specific to ensure the accuracy of gene detection, avoid competitive amplification between target fragments, and achieve efficient and sensitive amplification. As early as 1988, Chamberlian et al. began to apply multiplex PCR in the field of STR amplification, laying the theoretical foundation for STR fluorescent PCR. The new generation of sequencing methods has expanded the application of multiplex PCR as an efficient library preparation method in forensic genetics.

[0039] There are significant differences in the composition of sequencing data between multiplex PCR, probe capture, and whole genome sequencing. In conventional sequencing data, repeated sequencing fragments are generally used as a source of PCR bias or contamination. However, for the offline data of multiplex PCR, the main component is the repeated fragments. Obviously, multiplex PCR data requires a more professional analysis process, which illustrates the necessity of the present invention as a supplement to the analysis of whole genome sequencing data.

[0040] See also Figures 1 to 7 In an embodiment of the present invention, a method for analyzing composite amplified STR data includes the following steps:

[0041] 1. Site reference information:

[0042] Input data: In addition to the CODIS locus information provided by the software, the present invention also supports users to customize the STR locus typing test of their interest by inputting custom locus information. The custom locus information must include the following: 1) At least 30 bp of non-homologous regions flanking the target STR locus and in the same direction (see Figure 2 ) 2) The repeat unit sequence of the corresponding STR locus; 3) Each target STR locus needs to provide the number of repeat units based on the hg38 reference genome and the target region coverage length as an internal reference.

[0043] 2. Quality control-pretreatment:

[0044] Filter data: To ensure the accuracy of the typing results, the original sequencing data needs to filter the adapter sequences and low-quality bases. The adapter filtering software AdapterRemoval included in the process can simultaneously achieve quality control of the second-generation sequencing data: 1) trim the 5' or 3' end of the sequencing fragment with a quality score less than 2 or unclear bases; 2) filter the sequencing fragments with a read length shorter than 50bp; 3) set the remaining parameters to the default values. In addition, sequencing fragments with a sequencing depth lower than 5X are also removed from subsequent analysis;

[0045] 3. STR typing method:

[0046] a) Finding the best match: Performing STR analysis according to the technical route of the present invention ( Figure 1 ). The Hamming distance between the sequencing fragment and the STR locus flanking reference sequence is used to measure the similarity between sequences, so as to locate the flanking sequence position of each STR locus. If it completely matches the target sequence, the length is calculated and then the depth is counted. If it does not completely match the target sequence, the Hamming distance is calculated and the best match is found, and then the length is calculated and then the depth is counted. Figure 3 As shown in Figure 3, taking the amplicon of STR locus CSF1P0 as an example, a window with a width equal to the upstream flanking reference sequence of the STR region is defined. Starting from the first position of the amplicon, the Hamming distance between the partial amplicon sequence and the reference sequence in each window is measured. The Hamming distance is equal to the number of bases that differ between the two sequences. The partial amplicon sequence corresponding to the window with the shortest Hamming distance is the actual upstream flanking sequence of the STR region on the amplicon. Figure 3 The different bases are marked inside the box, and the matching bases are marked outside the box.

[0047] In addition, the latest genetic data show that there are about 3 billion bases in the human genome, including 84.7 million SNPs, and the SNP frequency of STR flanking is relatively higher. The maximum value of the Hamming distance suggested by the present invention should not exceed 1 / 15 of the length of the flanking sequence.

[0048] b) Calculate STR typing with reference to internal reference data: After locating the flanking sequence, extract the predicted repeat unit region, compare it with the internal parameters of the corresponding STR locus, calculate all possible typing results and their depths of the locus by the difference between the repeat unit region and the reference coverage length based on the hg38 reference genome, and directly output the genotype structure if the CODIS locus is not included; if the CODIS locus is included, the genotype structure is output after the CODIS system automatically converts it. The present invention provides accurate typing results based on each typing and depth of the STR locus and filtering based on calculated indicators (such as allele coverage rate ACR).

[0049] Experimental process of the present invention:

[0050] (1) Amplification of specific target: In this part, the DNA sample and the specific primer pair mixture are mixed so that the universal sequences of the forward and reverse primers are connected to the two sides of the specific amplicon determined by the specific primers. This part of the reaction is also called the first step of PCR. In this part, magnetic beads are used for one or two purifications to eliminate primer dimers and excess primers.

[0051] (2) Target enrichment: In the target enrichment part, which is the second PCR part, we connected the adapter after the universal sequence connected in the first step. The connected adapters were amplified and purified by the second PCR, mixed into a single test tube and purified. The adapters added in this part enable the data of different samples to be distinguished during the sequencing process.

[0052] (3) Mixed library: This part uses the fluorescence quantitative method to quantify the enriched products and record the sample information and concentration information. The quantified samples are mixed in equal proportions.

[0053] (4) Library dilution: Use fluorescence quantitative methods to quantify the mixed library product and dilute it to 10 ng / μL, and prepare for sequencing according to the on-line process of different sequencing platforms.

[0054] (5) The NGS library was prepared using the BGISEQ-500 kit and then sequenced using the BGISEQ sequencing platform. Considering that STR loci may have relatively long repeat units, we chose the SE200 sequencing type.

[0055] 2. Bioinformatics Process Construction

[0056] (1) Quality control: The raw sequencing data needs to be filtered for adapter sequences and low-quality bases. The process includes adapter filtering and removal of sequencing fragments with a sequencing depth less than 5X.

[0057] (2) Anchoring the target sequence: Match the target sequence with the sequencing data after quality control. If the match is unsuccessful, the length of the target sequence is used as the window length by default, and the Hamming distance is calculated according to the dynamic programming algorithm to find the optimal match with a distance less than 1 / 15 of the target sequence length. After the match is completed, the length is calculated and the depth is counted.

[0058] (3) STR typing results: Based on the existing internal reference data and the offline data, the predicted repeat unit region is extracted and aligned to the internal parameters of the corresponding STR locus. The difference between the repeat unit region and the reference coverage length based on the hg38 reference genome is used to calculate all possible typing results and their depths for the locus. The STR typing result file contains the following contents: locus name, typing 1, typing 2 (optional), total typing ratio, total depth of the locus, depth of each typing, and specific sequence of the STR locus; for STRs judged to be homozygous loci, the typing 2 content can be omitted; the specific typing results of samples 9947a and 9948 are summarized in Table 1, which compares the results of the method of the present invention with those of capillary electrophoresis sequencing. Table 1 shows the typing results of STR loci by the method of the present invention and the first-generation capillary electrophoresis method. Tables 1a and 1b are based on 9947a and 9948 as input samples, respectively. The columns from left to right in this table are STR locus name, STR typing result of the first generation capillary electrophoresis method, STR typing result of the method of the present invention, and depth corresponding to the STR typing result of the method of the present invention.

[0059] 3. Accuracy Verification

[0060] In terms of the accuracy of STR typing, the method of the present invention has excellent accuracy and detection rate. In this section, we selected standard DNA: 9947a and 9948 for multiplex PCR and BGISEQ-500 sequencing, and then used the data generated by the method of the present invention to type the STR loci. The capillary electrophoresis typing results are from the STR database. In this section, we use sites with a depth greater than 100X by default for accuracy verification. From Tables 1a and b, whether 9947a or 9948 is used as the input sample, the consistency of the STR typing results using the method of the present invention and the capillary electrophoresis sequencing results is 100%.

[0061] 4. Timeliness Verification

[0062] In this section, it can be seen from Table 2 that the method of the present invention also performs well in terms of timeliness and maximum memory usage. Table 2 shows the time consumption and memory usage of the method of the present invention, showing the time consumed by each step of the method of the present invention and the maximum memory usage. In this section, when the input multiplex PCR data is 3773.26Mbp, excluding the data pre-processing time, it only takes about 19 minutes to complete STR typing by the method of the present invention, which is significantly better than similar products on the market. At the same time, our maximum memory usage is only 533.496M / 5 threads.

[0063] The method can accurately perform genotyping of STR using multiplex PCR data and realize automatic conversion of the CODIS system. The most important thing is that the method of the present invention can make up for the shortcomings of the targeted sequencing data analysis method in terms of accuracy and timeliness, broaden the scope of application of targeted sequencing, and provide new possibilities for the combined use of NGS and composite amplification technology.

[0064] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for analyzing composite amplified STR data, characterized in that: The steps include:

1. Site reference information: Input data: In addition to the CODIS locus information that comes with the software, users can customize STR loci for typing detection by inputting custom site information; 2. Quality control-pretreatment: Filtering data: To ensure the accuracy of typing results, the original sequencing data needs to be filtered for adapter sequences and low-quality bases. The adapter filtering software AdapterRemoval is used to achieve quality control of the second-generation sequencing data. Sequencing fragments with a sequencing depth lower than 5X are removed from subsequent analysis.

3. STR typing method: a) Find the best match: The Hamming distance between the sequenced fragment and the STR locus flanking reference sequence is used to measure the similarity between sequences, thereby locating the flanking sequence position of each STR locus. If it completely matches the target sequence, the length is calculated and then the depth is counted. If it does not completely match the target sequence, the Hamming distance is calculated and the best match is found, and then the length is calculated and then the depth is counted. b) Calculate STR typing with reference to internal reference data: After locating the flanking sequence, extract the predicted repeat unit region and align it with the internal parameters of the corresponding STR locus. Calculate all possible typing results and their depth for the locus based on the difference between the repeat unit region and the reference coverage length based on the hg38 reference genome. If the CODIS locus is not included, the genotype structure is directly output. If the CODIS locus is included, the genotype structure is output after automatic conversion by the CODIS system.

2. The method for analyzing composite amplified STR data according to claim 1, characterized in that: The customized site information includes the following contents: The target STR locus is flanked by at least 30 bp of non-homologous regions in the same direction; 2) The repeating unit sequence of the corresponding STR locus; 3) Each target STR locus needs to provide the number of repeat units based on the hg38 reference genome and the target region coverage length as an internal reference.

3. The method for analyzing composite amplified STR data according to claim 1, characterized in that: The maximum value of the Hamming distance should not exceed 1 / 15 of the length of the flanking sequence.

4. The method for analyzing composite amplified STR data according to claim 1, characterized in that: The specific parameter settings of the adapter filtering software AdapterRemoval include: Trim the bases with a quality score less than 2 at the 5' or 3' end of the sequencing fragment; Filter sequencing fragments with read length shorter than 50 bp; 3) Set the remaining parameters to their default values.

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