Analysis method, device and equipment for insertion information of transposition element and computer readable storage medium

Abstract

The invention discloses an analysis method, device and equipment for insertion information of a transposition element and a computer readable storage medium. The analysis method comprises the steps of screening candidate read pairs, selecting target read pairs, predicting the insertion interval of the transposition element, determining the insertion interval of the transposition element and determining the insertion position of the transposition element. According to the present application, a more practical and effective method or product for analyzing the swivel element can be provided.

Description

Technical Field

[0001] This application relates to the field of sequencing data analysis technology, and in particular to a method, apparatus, device, and computer-readable storage medium for analyzing insertion information of transposable elements. Background Technology

[0002] Transposable elements (TEs), as an important component of the genome, are related to genome evolution and have a significant impact on biological phenotypes. In the course of biological evolution, the complex enzyme systems and regulatory elements encoded by transposable elements, such as the RAG protein in the immunoglobulin V(D)J recombinant system, derive their functions from transposases. Furthermore, the polymorphism of transposable elements is a rich source of genetic variation. For example, variations in grape color and fruit cluster shape in plants, and alterations in gene expression patterns caused by L1 retrotransposons during human neuronal development, are closely related to the insertion of transposable elements. Abnormal activity of transposable elements is also associated with various diseases, including schizophrenia, hepatocellular carcinoma, and other cancers. Therefore, the analysis of transposable elements is of great significance for genome function research, evolutionary analysis, and the analysis of disease genetic mechanisms.

[0003] Currently, methods for detecting transposon insertion sites have many limitations. Traditional molecular biology techniques (such as semi-specific PCR) are only applicable to specific individuals or species, making large-scale application difficult. Assembly-based methods are limited by the quantity and quality of genome assemblies, facing difficulties when processing large samples or highly repetitive genomes. Chromosome array-based methods are ineffective at detecting repetitive regions of the genome, and neither of these methods can effectively identify heterozygosity. Next-Generation Sequencing (NGS) data analysis of transposons is an important research direction, but existing software tools for this purpose have limitations, focusing only on specific types of human transposons and failing to analyze all transposons.

[0004] Therefore, it is necessary to develop a more practical and effective method or product for analyzing transposers. Summary of the Invention

[0005] In view of the above problems, this application provides a method, apparatus, device and computer-readable storage medium for analyzing the insertion information of a transpose element.

[0006] The following technical solution is adopted in this application:

[0007] The first aspect of this application discloses a method for analyzing the insertion information of transposable elements, comprising: screening candidate read pairs: obtaining read pairs from paired-end sequencing data, each read pair including a forward read and a reverse read; removing read pairs that cannot be used to analyze the insertion information of the transposable elements from all read pairs to obtain the candidate read pairs; selecting target read pairs: selecting target read pairs from the candidate read pairs, wherein the target read pair refers to a read pair in which one read uniquely aligns to a non-transposable element position and the other read aligns to a transposable element position, wherein the read uniquely aligned to a non-transposable element position is denoted as an anchored read; predicting the insertion interval of the transposable element: if the anchored read is a forward read, the predicted insertion interval of the transposable element starts at position A and ends at position A + (L + n × S); if the If the anchored read is a reverse read, the predicted start position of the transposon element insertion interval is: B - (L + n × S), and the end position is B; where A refers to the starting coordinate of the anchored read, B refers to the ending coordinate of the anchored read, L refers to the length of the inserted fragment between the read pairs corresponding to the anchored read, S refers to the standard deviation of the length of the inserted fragment of the read pairs correctly aligned to the genome in the paired-end sequencing data, and n is a natural number greater than 1; Determine the insertion interval of the transposon element: Based on the overlap between the insertion intervals of the transposon element predicted by the forward read and the insertion intervals of the transposon element predicted by the reverse read, the determined insertion interval of the transposon element is obtained; Determine the insertion position of the transposon element: The position where multiple reads undergo soft shearing at the same location within the determined insertion interval of the transposon element is the insertion position of the transposon element.

[0008] In one implementation of this application, the step of screening candidate read pairs includes: removing read pairs from the paired-end sequencing data that meet at least one of the following conditions: both the forward and reverse reads of the read pair are aligned to the genome; both the forward and reverse reads of the read pair are aligned to transposable elements; and the length of the insert fragment corresponding to the read pair is less than the average length of the insert fragment of the read pair that is correctly aligned to the genome.

[0009] In one implementation of this application, the step of determining the insertion interval of the transpose element includes: clustering the anchoring read segments according to the predicted overlap of the insertion intervals of the transpose element; pairing the forward and reverse read segments that overlap the predicted insertion intervals of the transpose element in the anchoring read segments; the starting position of the determined insertion interval of the transpose element is: the ending coordinate of the anchoring read segment with the largest coordinate in the paired forward read segment cluster; the ending position of the determined insertion interval of the transpose element is: the ending coordinate of the anchoring read segment with the smallest coordinate in the paired reverse read segment cluster.

[0010] In one implementation of this application, the step of determining the insertion interval of the transpose element includes: searching for soft-shear segments within the determined insertion interval of the transpose element; if three or more segments within the determined insertion interval of the transpose element undergo soft shearing at the same position, then that position is the insertion position of the transpose element.

[0011] In one implementation of this application, the method further includes determining the mate type of the transposable element insertion: based on the determined insertion position of the transposable element, the number of read segments crossing the position is counted and denoted as M, and the number of soft shear read segments crossing the position is counted and denoted as N. Then the insertion frequency VAF of the transposable element is N / M, and the mate type of the transposable element insertion is determined based on the VAF value.

[0012] In one implementation of this application, if the VAF value is greater than or equal to 0.99, it is homozygous; if the VAF value is less than 0.99, it is heterozygous.

[0013] The second aspect of this application discloses an apparatus for analyzing the insertion information of transposable elements, comprising: a candidate read pair screening module for acquiring read pairs in paired-end sequencing data, each read pair including a forward read and a reverse read, removing read pairs that cannot be used to analyze the insertion information of the transposable elements from all read pairs to obtain the candidate read pairs; a target read pair selection module for selecting target read pairs from the candidate read pairs, wherein the target read pair refers to a read pair in which one read uniquely aligns to a non-transposable element position and the other read aligns to a transposable element position, wherein the read uniquely aligned to a non-transposable element position is denoted as an anchor read; and a transposable element insertion interval prediction module for predicting the insertion interval of the transposable element, wherein if the anchor read is a forward read, the starting position of the insertion interval of the transposable element is A, and the ending position is A + (L + n × S). If the anchored read is a reversed read, the starting position of the insertion interval of the transposon element is B - (L + n × S), and the ending position is B; where A refers to the starting coordinate of the anchored read, B refers to the ending coordinate of the anchored read, L refers to the length of the inserted fragment between the read pairs corresponding to the anchored read, S refers to the standard deviation of the length of the inserted fragment of the read pairs correctly aligned to the genome in the paired-end sequencing data, and n is a natural number greater than 1; the transposon element insertion interval determination module is used to determine the insertion interval of the transposon element based on the overlap between the insertion intervals of the transposon element predicted by the forward read and the insertion intervals of the transposon element predicted by the reverse read; the transposon element insertion position determination module is used to determine the insertion position of the transposon element. The position where multiple reads undergo soft shearing at the same location within the determined insertion interval of the transposon element is the insertion position of the transposon element.

[0014] In one implementation of this application, a transposable element insertion mate type determination module is further included: a module for determining the mate type of the transposable element insertion, which, based on the determined insertion position of the transposable element, counts the number of read segments crossing the position, denoted as M, and counts the number of soft shear read segments crossing the position, denoted as N. The insertion frequency VAF of the transposable element is then N / M, and the mate type of the transposable element insertion is determined based on the VAF value.

[0015] A third aspect of this application discloses an apparatus for analyzing insertion information of a transpose element, comprising a memory and a processor, the memory for storing a program, and the processor executing the program stored in the memory to implement the analysis method as described in the first aspect of this application.

[0016] The fourth aspect of this application discloses a computer-readable storage medium storing a program that can be executed by a processor to implement the analysis method as described in the first aspect of this application.

[0017] The beneficial effects of this application are as follows:

[0018] This application utilizes the different comparison characteristics of the forward and reverse read segments in paired-end reads to analyze the insertion position of the transpose element, enabling more practical and effective analysis of the transpose element. Attached Figure Description

[0019] Figure 1 This is a flowchart illustrating the method for analyzing the insertion information of the transpose element involved in this application.

[0020] Figure 2 This is a schematic diagram illustrating the principle of the method for analyzing the insertion information of the transducer element involved in this application.

[0021] Figure 3 This is a schematic diagram of the apparatus for analyzing insertion information of the rotary element involved in this application.

[0022] Figure 4 This is a schematic diagram of the device for analyzing the insertion information of the rotary element involved in this application. Detailed Implementation

[0023] The present invention will now be described in further detail with reference to specific embodiments and accompanying drawings. In the following embodiments, many details are described to facilitate a better understanding of the present application. However, those skilled in the art will readily recognize that some features may be omitted in different situations, or may be replaced by other materials or methods. In some cases, certain operations related to the present application are not shown or described in the specification. This is to avoid obscuring the core parts of the present application with excessive description. For those skilled in the art, detailed description of these related operations is not necessary; the relevant operations can be fully understood based on the description in the specification and general technical knowledge in the art.

[0024] Furthermore, the features, operations, or characteristics described in the specification can be combined in any suitable manner to form various embodiments. At the same time, the steps or actions in the method description can be rearranged or adjusted in a manner obvious to those skilled in the art. Therefore, the various orders in the specification and drawings are only for the clear description of a particular embodiment and do not imply a necessary order, unless otherwise stated that a particular order must be followed.

[0025] Currently, methods for detecting transposon insertion sites have many shortcomings. Traditional molecular biology techniques (such as semi-specific PCR) are only applicable to specific individuals or breeds, making large-scale application difficult. Assembly-based methods are limited by the quantity and quality of genome assemblies, facing difficulties when processing large numbers of samples or highly repetitive genomes. Chromosome array-based methods are ineffective at detecting repetitive regions of the genome, and neither of these methods can effectively identify heterozygosity. Existing software tools for analyzing transposon elements based on next-generation sequencing data also have limitations, focusing only on human-specific transposon types and failing to predict the zygotic type of the insertion.

[0026] Given the importance of transposon element research and the limitations of existing detection methods, the development of a more practical and effective detection technology is urgently needed. This application features broad applicability, ease of operation, and powerful functionality. It can be applied to various species and can identify different types of transposon elements. It directly uses standard BAM files to accurately identify insertion sites and predict allele frequencies, bringing new breakthroughs to genomics research and disease diagnosis and treatment. Specifically, this application has the following advantages: 1. Wide applicability: Traditional molecular biology techniques (such as semi-specific PCR) are only applicable to specific species, limiting their application scenarios. Assembly and array-based methods face difficulties when processing large numbers of samples or highly repetitive genomes and cannot effectively identify heterozygosity. 2. Convenient to use, directly using standard data formats: Most existing software tools cannot directly use standard BAM files generated by commonly used alignment tools, requiring additional alignment of reads, which is complex. This invention can directly use BAM files generated by any alignment software. 3. Ability to calculate mutation ratios: Existing tools have limitations; most cannot predict the zygotic type of the insertion. This invention calculates the mutation frequency through soft-splitting reads, thereby determining the zygotic type of the mutation.

[0027] This application provides a method, apparatus, device, and computer-readable storage medium for analyzing insertion information of a transpose element.

[0028] As described above, this application relates to a method for analyzing insertion information of a transducer element (which may be simply referred to as the "analysis method").

[0029] In some examples, the method for analyzing the insertion information of transposable elements in this application is based on the analysis of sequencing data from paired-end sequencing.

[0030] The core principle of the method for analyzing transposon insertion information in this application is as follows: when a transposon is inserted into the genome, in a pair of reads from paired-end sequencing, one read will align to a genomic location near the insertion site, which is usually unique on the genome; while the other read will align to another location on the same chromosome or another chromosome, where the sequence is usually annotated as a repetitive sequence (transposon) on the genome. Multiple such pairs of anomalous reads can be used to predict potential transposon insertion sites. The alignment software performs soft splicing on reads that cross the insertion boundary (the start or end point of the insertion). (Soft splicing refers to the process where, during sequence alignment, some reads cannot precisely match the reference genome; these reads are marked as unused but retained in the SAM / BAM file. Unlike hard splicing, soft splicing does not physically delete these bases from the reads; it simply marks them as not participating in the alignment.) These soft-spliced ​​reads can be used to determine the insertion site. Reads that are fully aligned and cross the predicted insertion breakpoint indicate the presence of wild-type alleles at that site. If both soft splicing and full alignment are present, it indicates the presence of heterozygous mutations. By calculating the ratio of soft-spliced ​​reads to fully aligned reads at the insertion site, the frequency of the inserted alleles can be determined.

[0031] Figure 1 This is a flowchart illustrating the method for analyzing the insertion information of the transpose element involved in this application. Figure 2 This is a schematic diagram illustrating the principle of the method for analyzing the insertion information of the transducer element involved in this application.

[0032] like Figure 1 As shown, in some examples, the method for analyzing the insertion information of the transpose element may include: filtering candidate read segment pairs (step S100), selecting target read segment pairs (step S200), predicting the insertion interval of the transpose element (step S300), determining the insertion interval of the transpose element (step S400), and determining the insertion position of the transpose element (step S500). Further, the method for analyzing the insertion information of the transpose element may also include: determining the type of transpose element insertion (step S600).

[0033] In some examples, screening candidate read pairs includes: obtaining read pairs from paired-end sequencing data, each read pair consisting of a forward read and a reverse read; and removing read pairs from all read pairs that cannot be used to analyze transposon insertion information to obtain candidate read pairs. Specifically, read pairs that meet any of the following conditions can be removed from all read pairs to obtain candidate read pairs: both the forward and reverse reads of the read pair are aligned to the genome; both the forward and reverse reads of the read pair are aligned to transposon elements; and the length of the corresponding insert fragment is less than the Mean_S value. The Mean_S value is the average length of the insert fragment of read pairs that are correctly aligned to the genome. Specifically, the obtained paired-end sequencing data can be in the form of a BAM file, and the Mean_S value is the average size of the insert fragment in correctly paired (FLAG value 0x2) reads in the BAM file. Accordingly, the standard deviation of the insert size in the correctly paired (FLAG value 0x2) reads in the BAM file can be calculated, and this standard deviation can be denoted as S (which will be used later). It should be noted that the insert refers to the sequence between a pair of reads during the sequencing process.

[0034] In some examples, selecting target read pairs includes selecting target read pairs from candidate read pairs. A target read pair is a read pair in which one read is uniquely aligned to a non-transposon element position (i.e., aligned to the genome) and the other read is aligned to a transposon element position. The read that is uniquely aligned to a non-transposon element position is denoted as an anchor read.

[0035] In some examples, the predicted insertion interval for transposable elements includes: if the anchored read is a forward read, the predicted insertion interval starts at position A and ends at position A + (L + n × S); if the anchored read is a reverse read, the predicted insertion interval starts at position B - (L + n × S) and ends at position B; where A is the starting coordinate of the anchored read, B is the ending coordinate of the anchored read, L is the length of the inserted fragment between the read pairs corresponding to the anchored read, S is the standard deviation of the length of the inserted fragment of the read pairs correctly aligned to the genome in the paired-end sequencing data, and n is a natural number greater than 1. It should be noted that the insertion interval of the transpose element is predicted by normal distribution. In the formula, when the number of standard deviations n is 1, it corresponds to a probability of 68.27%; when n is 2, it corresponds to a probability of 95.45%; when n is 3, it corresponds to a probability of 99.73%. In this application, the value of n is preferably a natural number greater than 1, such as 2, 3, or 4.

[0036] In some examples, determining the insertion interval of the transposable element includes: obtaining the determined insertion interval of the transposable element based on the overlap between the insertion intervals predicted by the forward read segments and the insertion intervals predicted by the reverse read segments. Specifically, based on the overlap of the predicted insertion intervals of the transposable element, the anchor read segments are clustered, and the forward and reverse read segments with overlapping predicted insertion intervals of the transposable element in the anchor read segments are paired. The starting position of the determined insertion interval of the transposable element is: the ending coordinate of the anchor read segment with the largest coordinate in the paired forward read segment cluster; the ending position of the determined insertion interval of the transposable element is: the ending coordinate of the anchor read segment with the smallest coordinate in the paired reverse read segment cluster.

[0037] In some examples, determining the insertion position of a transpose element may include identifying a location within the defined insertion range where multiple read segments undergo soft shearing at the same position. Specifically, a soft shearing read segment can be searched within the defined insertion range; if three or more read segments undergo soft shearing at the same position within the defined insertion range, that position is considered the insertion position of the transpose element.

[0038] In some examples, determining the zygotic type of the transposable element insertion may include: based on the determined insertion position of the transposable element, counting the number of reads crossing that position, denoted as M, and counting the number of soft-shear reads crossing that position, denoted as N. The insertion frequency VAF of the transposable element is then N / M. The zygotic type of the transposable element insertion is determined based on the VAF value. If the VAF value is greater than or equal to 0.99, it is homozygous; if the VAF value is less than 0.99, it is heterozygous.

[0039] Can be combined Figure 2 To understand, such as Figure 2 As shown, Figure 2 In the diagram, black reads represent unique reads aligned to the insertion site, while red reads represent reads of the aligned transposable element. Reads on the same horizontal line are paired, including a forward read (to the right) and a reverse read (to the left). Reads within boxes are anchor reads obtained from clustering, including both forward and reverse reads. The insertion interval of the transposable element can be determined based on the clustered reads. The starting position of the insertion interval is the anchor read with the largest coordinate in the paired forward read cluster (i.e., the...). Figure 2 The termination coordinates of the topmost forward read segment within the box, with the endpoint position being: the anchor read segment with the smallest coordinates in the paired reverse read segment cluster (i.e., Figure 2 The termination coordinates of the bottommost reverse read segment within the box. Subsequently, the soft-shear read segment (i.e., Figure 2 The two reading segments marked with a cross determine the insertion position of the rotary element.

[0040] This application also relates to an apparatus 800 for analyzing insertion information of a transpose element.

[0041] Figure 3 This is a schematic diagram of the apparatus 800 for analyzing the insertion information of the transducer element involved in this application. Figure 3 As shown, the apparatus 800 for analyzing the insertion information of transposable elements may include a candidate read segment pair filtering module 801, a target read segment pair selection module 802, a transposable element insertion interval prediction module 803, a transposable element insertion interval determination module 804, and a transposable element insertion position determination module 805. The apparatus 800 may also include a transposable element insertion mate type judgment module 806. The functions of each module can be referred to the corresponding content in the aforementioned method for analyzing the insertion information of transposable elements, and will not be repeated here.

[0042] This application also relates to a device 100 for analyzing insertion information of a transpose element.

[0043] Figure 4 This is a schematic diagram of the device 100 for analyzing the insertion information of the transducer element involved in this application. Figure 4 As shown, the device 100 for analyzing the insertion information of the transpose element may include a processor 10, a memory 20, and a computer program 21 (also referred to as a computer-readable storage medium 21) stored in the memory 20. The computer program 21 can run on the processor 10, and when the processor 10 executes the computer program 21, it implements the aforementioned method for analyzing the insertion information of the transpose element, for example, implementing... Figure 1 The steps shown. Alternatively, when the processor 60 executes the computer program 62, it implements the functions of each module in the apparatus 800 for analyzing the insertion information of the transposon element, as described above, for example... Figure 3 The functions of modules 801, 802 and 803 are shown.

[0044] The device 100 for analyzing the insertion information of the transpose element can be a computing device such as a desktop computer, laptop, handheld computer, or cloud server. The device 100 for analyzing the insertion information of the transpose element may include, but is not limited to, a processor 10 and a memory 20. Those skilled in the art will understand that... Figure 4 This is merely an example of a device 100 for analyzing insertion information of transpose elements and does not constitute a limitation on the device 100 for analyzing insertion information of transpose elements. For example, it may include more or fewer components than illustrated, or combine certain components, or use different components. For example, the device 100 for analyzing insertion information of transpose elements may also include input / output devices, network access devices, buses, etc.

[0045] Processor 10 can be a Central Processing Unit (CPU), or other general-purpose processors, digital signal processors (DSPs), application-specific integrated circuits (ASICs), field-programmable gate arrays (FPGAs), or other programmable logic devices, discrete gate or transistor logic devices, discrete hardware components, etc. A general-purpose processor can be a microprocessor or any conventional processor.

[0046] The memory 20 can be an internal storage unit of the device 100 for analyzing the insertion information of the transpose element, such as a hard disk or memory. The memory 20 can also be an external storage device of the device 100 for analyzing the insertion information of the transpose element, such as a plug-in hard disk, smart media card (SMC), secure digital (SD) card, flash card, etc., equipped on the device 100. Furthermore, the memory 20 can include both internal storage units and external storage devices of the device 100 for analyzing the insertion information of the transpose element.

[0047] The memory 20 can be used to store the computer program 21, as well as other programs and data required by the device 100 for analyzing the insertion information of the transpose element. The memory 20 can also be used to temporarily store data that has been output or will be output.

[0048] Those skilled in the art will clearly understand that, for the sake of convenience and brevity, the above-described division of functional units and modules is merely an example. In practical applications, the above functions can be assigned to different functional units and modules as needed, that is, the internal structure of the device can be divided into different functional units or modules to complete all or part of the functions described above. The functional units and modules in the embodiments can be integrated into one processing unit, or each unit can exist physically separately, or two or more units can be integrated into one unit. The integrated unit can be implemented in hardware or as a software functional unit. Furthermore, the specific names of the functional units and modules are only for easy differentiation and are not intended to limit the scope of protection of this application. The specific working process of the units and modules in the above system can be referred to the corresponding process in the foregoing method embodiments, and will not be repeated here.

[0049] Those skilled in the art will recognize that the units and algorithm steps of the various examples described in conjunction with the embodiments disclosed herein can be implemented in electronic hardware, or a combination of computer software and electronic hardware. Whether these functions are implemented in hardware or software depends on the specific application and design constraints of the technical solution. Those skilled in the art can use different methods to implement the described functions for each specific application, but such implementation should not be considered beyond the scope of this application.

[0050] In the embodiments provided in this application, it should be understood that the disclosed devices / terminal equipment and methods can be implemented in other ways. For example, the device / terminal equipment embodiments described above are merely illustrative. For instance, the division of modules or units is only a logical functional division, and in actual implementation, there may be other division methods. For example, multiple units or components may be combined or integrated into another system, or some features may be ignored or not executed. Furthermore, the displayed or discussed mutual coupling or direct coupling or communication connection may be through some interfaces; the indirect coupling or communication connection between devices or units may be electrical, mechanical, or other forms.

[0051] The units described as separate components may or may not be physically separate. The components shown as units may or may not be physical units; that is, they may be located in one place or distributed across multiple network units. Some or all of the units can be selected to achieve the purpose of this embodiment according to actual needs.

[0052] Furthermore, the functional units in the various embodiments of this application can be integrated into one processing unit, or each unit can exist physically separately, or two or more units can be integrated into one unit. The integrated unit can be implemented in hardware or as a software functional unit.

[0053] If the integrated module / unit is implemented as a software functional unit and sold or used as an independent product, it can be stored in a computer-readable storage medium. Based on this understanding, all or part of the processes in the methods of the above embodiments can also be implemented by a computer program instructing related hardware. The computer program can be stored in a computer-readable storage medium, and when executed by a processor, it can implement the steps of the various method embodiments described above. The computer program includes computer program code, which can be in the form of source code, object code, executable files, or certain intermediate forms. The computer-readable medium can include: any entity or device capable of carrying the computer program code, recording media, USB flash drives, portable hard drives, magnetic disks, optical disks, computer memory, read-only memory (ROM), random access memory (RAM), electrical carrier signals, telecommunication signals, and software distribution media, etc. It should be noted that the content included in the computer-readable medium can be appropriately added or removed according to the requirements of legislation and patent practice in the jurisdiction. For example, in some jurisdictions, according to legislation and patent practice, computer-readable media do not include electrical carrier signals and telecommunication signals.

[0054] The present application will be further described in detail below through specific embodiments and comparative examples. The following embodiments are only for further illustration of the present application and should not be construed as limiting the present application. In the embodiments and comparative examples, unless otherwise specified, the instruments and software used are all commercially available, and the operating procedures are all performed in accordance with the product manuals and conventional operating procedures.

[0055] Example:

[0056] The NA18506 sample, from the Yoruba population in Africa, was selected from the 1000 Genomes Project. Whole-genome sequencing was performed on the CompleteGenomics platform. According to the literature (title: Alu repeat discovery and characterization within human genomes), researchers conducted PCR experiments to verify the transposon insertions detected in the sample. The 29 transposon insertions verified in the NA18506 sample are shown in Table 1 (coordinates are in hg19 version).

[0057] Download the BAM file of the sequencing data of sample NA18506 from ftp: / / ftp.1000genomes.ebi.ac.uk / vol1 / ftp / phase3 / data / for subsequent analysis.

[0058] Table 1. Insertion information of 29 transposon elements in NA18506 sample validated by PCR.

[0059] chromosome Starting coordinates Termination coordinates zygotic type chr20 15,035,661 15,035,666 INS chr8 18,661,749 18,661,756 INS chr5 141,738,823 141,738,857 INS chr7 153,596,976 153,596,978 INS + WT chr10 116,996,308 116,996,316 INS + WT chr4 185,888,665 185,888,669 INS + WT chr5 162,164,519 162,164,525 INS chr11 23,315,080 23,315,102 INS chr2 216,256,683 216,256,684 INS + WT chr3 7,108,386 7,108,389 INS chr3 57,600,576 57,600,582 INS + WT chr2 73,978,989 73,978,990 INS + WT chr10 36,507,312 36,507,338 INS chr11 119,619,971 119,619,980 INS + WT chr13 78,169,592 78,169,605 INS + WT chr14 26,595,992 26,595,995 INS + WT chr15 92,676,928 92,676,940 INS + WT chr5 50,821,401 50,821,434 INS + WT chr13 70,040,371 70,040,499 INS + WT chr6 17,312,778 17,312,928 INS + WT chr1 7,408,829 7,408,832 INS + WT chr1 44,126,080 44,126,086 INS + WT chr10 25,527,917 25,527,920 INS + WT chr11 86,025,802 86,025,815 INS chr13 37,339,114 37,339,116 INS + WT chr18 20,644,160 20,644,167 INS + WT chr2 83,197,699 83,197,714 INS + WT chr20 13,652,161 13,652,170 INS + WT chr3 72,176,431 72,176,450 INS + WT

[0060] In Table 1, INS (insertion) zygote type represents homozygote, and INS+WT (wild type) zygote type represents heterozygote.

[0061] 1. Obtain the reference genome and annotation files: Retrieve the corresponding transposon element annotation files from relevant databases and perform necessary organization and annotation as needed. Specifically, obtain the repetitive sequence (including all currently available transposon elements) files from the official website (https: / / hgdownload.soe.ucsc.edu / goldenPath / hg19 / database / rmsk.txt.gz), and select long scattered repetitive elements (LINE) and short scattered repetitive elements (SINE) from them, totaling 32,928,892.

[0062] 2. Calculate the mean (denoted as mean_s) and standard deviation (denoted as std, or S) of the inserted fragment size in correctly paired reads (FLAG value 0x2) in the BAM file. Specifically, use the pysam module in Python to calculate the mean and standard deviation of the inserted fragment size in correctly paired reads in the BAM file, obtaining mean_s as 151.6 and S as 2.3.

[0063] 3. Selecting candidate read pairs in the BAM file involves scanning the BAM file and removing any correctly matched read pairs (FLAG value 0x2), read pairs with inserted segment lengths shorter than the average calculated in step one, and read pairs where both reads align to transposable elements. Among the valid candidate read pairs, select one read pair that uniquely aligns to a non-transposable element location (denoted as the anchored read), and the other read pair that aligns to at least one location marked as a transposable element in the provided annotations (denoted as the transposable paired read). Further filtering removes read pairs with an alignment quality value less than 20 for the anchored read, resulting in the final set of candidate read pairs.

[0064] 4. Let the starting coordinate of the anchored read segment be A, the ending coordinate be B, and the length of the inserted segment of the read segment pair be L. Each candidate read segment pair can predict the interval where the transpose element insertion point is located (denoted as the insertion interval). The starting point of the interval is the starting point of the anchored read segment (forward read segment), or the ending point of the interval is the ending point of the anchored read segment (reverse read segment). The other coordinate of the interval is calculated as follows: when the anchored read segment is a forward read segment, the ending point of the insertion interval is: A + L + 2 × S; when the anchored read segment is a reverse read segment, the starting point of the insertion interval is: B - (L + 2 × S).

[0065] 5. Based on the predicted overlap of insertion intervals, cluster the anchor reads on the forward and reverse chains. If the predicted insertion intervals of the forward and reverse clusters overlap, pair them. The starting point of the final insertion interval is the ending point of the anchor read with the largest coordinate in the paired forward sequence cluster. The ending point of the final insertion interval is the starting point of the anchor read with the smallest coordinate in the paired reverse sequence cluster.

[0066] 6. Search for soft-sheared reads in the final insertion interval to calculate the precise insertion site. If three or more reads in the interval are soft-sheared at the same site, then that site is the precise insertion site. Otherwise, the insertion interval will be filtered out.

[0067] 7. Count the number of reads crossing the insertion site, denoted as M. Count the number of cut reads crossing the insertion site, denoted as N. The mutation frequency (VAF) of the transposable element insertion is then equal to N / M. If VAF equals 1, the mutation is homozygous; if it is less than 1, the mutation is heterozygous.

[0068] 8. Output the coordinates of all potential transposable element insertion sites, the insertion interval, the total number of reads, the number of forward reads, and the number of reverse reads within that interval, the total number of reads spanning the insertion site, the number of soft-shear reads, and the mutation frequency. (At this point, users can filter the insertion interval length, total number of reads, etc., according to their actual needs, and finally select the sites of interest for further experimental verification.)

[0069] Based on the above process, a total of 1730 transpose element insertions were analyzed, including 29 verified transpose element insertions, as shown in Table 2 below (only the 29 verified transpose element insertions are shown in the table):

[0070] Table 2. Insertion information of the transducer element obtained by the analysis method of this application.

[0071] chromosome Insertion site coordinates Insert the start of the interval Insert the endpoint of the interval Insertion interval length Total number of segments read Total number of reads across insertion sites Total number of soft-shear reads across the insertion site Mutation frequency (VAF) chr1 7,408,829 7408751 7408841 90 63 40 27 0.68 chr1 44,126,080 44126071 44126098 27 81 48 35 0.73 chr2 216,256,683 216256645 216256699 54 99 81 49 0.60 chr2 73,978,989 73978914 73978994 80 114 76 29 0.38 chr2 83,197,699 83197674 83197811 137 107 57 16 0.28 chr3 7,108,386 7108378 7108390 12 46 43 43 1.00 chr3 57,600,576 57600546 57600664 118 343 275 159 0.58 chr3 72,176,431 72176421 72176447 26 19 7 7 1.00 chr4 185,888,665 185888643 185888694 51 20 16 9 0.56 chr5 141,738,823 141738784 141738895 111 47 28 28 1.00 chr5 162,164,519 162164546 162164723 177 117 69 69 1.00 chr5 50,821,401 50821332 50821434 102 185 77 43 0.56 chr6 17,312,778 17312724 17312796 72 151 99 64 0.65 chr7 153,596,976 153596915 153596998 83 14 10 6 0.60 chr8 18661749 18661714 18661781 67 43 31 31 1.00 chr10 116996308 116996294 116996376 82 29 15 11 0.73 chr10 36,507,312 36507287 36507356 69 66 42 42 1.00 chr10 25,527,917 25527876 25527984 108 70 53 36 0.68 chr11 23,315,080 23315040 23315089 49 58 41 41 1.00 chr11 119,619,971 119619926 119619987 61 98 73 38 0.52 chr11 86,025,802 86025782 86025962 180 78 53 44 0.83 chr13 78,169,592 78169544 78169639 95 189 154 105 0.68 chr13 70,040,371 70040351 70040399 48 78 49 35 0.71 chr13 37,339,114 37339088 37339174 86 81 59 41 0.69 chr14 26,595,992 26595946 26595998 52 51 36 14 0.39 chr15 92,676,928 92676911 92676936 25 42 40 23 0.58 chr18 20,644,160 20644153 20644187 34 57 21 8 0.38 chr20 15,035,661 15035608 15035691 83 98 64 64 1.00 chr20 13,652,161 13652132 13652187 55 16 14 4 0.29

[0072] As shown in the table above, all transposon insertions verified by PCR experiments were detected by the analytical method of this application. Among them, only the transposon insertion at chr3:7108386 was judged as a homozygous mutation, and the transposon insertion at chr11:86025802 was judged as a heterozygous mutation, which is inconsistent with the verification results. The zygotic structures of the remaining mutations were consistent with the verification results. This fully demonstrates the effectiveness of the analytical method of this application for transposon insertion analysis.

[0073] The above description, in conjunction with specific embodiments, provides a further detailed explanation of this application and should not be construed as limiting the specific implementation of this application to these descriptions. Those skilled in the art to which this application pertains can make several simple deductions or substitutions without departing from the concept of this application.

Claims

1. A method for analyzing insertion information of a transpose element, characterized in that, include: Screening candidate read pairs: Obtain read pairs from paired-end sequencing data, each read pair including a forward read and a reverse read. From all read pairs, remove read pairs that cannot be used to analyze the insertion information of the transposable element to obtain the candidate read pairs. Selecting target read segment pairs: Selecting target read segment pairs from the candidate read segment pairs. The target read segment pair refers to a read segment pair in which one read segment is uniquely matched to the position of a non-rotating element and the other read segment is matched to the position of a rotating element. The read segment that is uniquely matched to the position of a non-rotating element is denoted as the anchored read segment. Predicting the insertion interval of the transposon element: If the anchored read is a forward read, the predicted starting position of the insertion interval of the transposon element is A, and the ending position is A + (L + n × S); if the anchored read is a reverse read, the predicted starting position of the insertion interval of the transposon element is B - (L + n × S), and the ending position is B; where A refers to the starting coordinate of the anchored read, B refers to the ending coordinate of the anchored read, L refers to the length of the inserted fragment between the read pairs corresponding to the anchored read, S refers to the standard deviation of the length of the inserted fragment of the read pairs correctly aligned to the genome in the paired-end sequencing data, and n is a natural number greater than 1; Determine the insertion interval of the transpose element: Based on the overlap between the insertion interval of the transpose element predicted by the forward reading segment and the insertion interval of the transpose element predicted by the reverse reading segment, the determined insertion interval of the transpose element is obtained. Determine the insertion position of the rotary element: If multiple reading segments undergo soft shearing at the same position within the determined insertion range of the rotary element, then the insertion position of the rotary element is the position where the rotary element is inserted.

2. The analytical method according to claim 1, characterized in that, The step of screening candidate read pairs includes: in the paired-end sequencing data, removing read pairs that meet at least one of the following conditions: both the forward and reverse reads of the read pair are aligned to the genome; both the forward and reverse reads of the read pair are aligned to transposable elements; and the length of the insert fragment corresponding to the read pair is less than the average length of the insert fragment of the read pair that is correctly aligned to the genome.

3. The analytical method according to claim 1, characterized in that, The step of determining the insertion interval of the transpose element includes: clustering the anchoring read segments according to the predicted overlap of the insertion intervals of the transpose element; pairing the forward and reverse read segments that overlap the predicted insertion intervals of the transpose element in the anchoring read segments; the starting position of the determined insertion interval of the transpose element is: the ending coordinate of the anchoring read segment with the largest coordinate in the paired forward read segment cluster; the ending position of the determined insertion interval of the transpose element is: the ending coordinate of the anchoring read segment with the smallest coordinate in the paired reverse read segment cluster.

4. The analytical method according to claim 1, characterized in that, The step of determining the insertion range of the transpose element includes: searching for soft-shear segments within the determined insertion range of the transpose element; if three or more segments within the determined insertion range of the transpose element undergo soft shearing at the same position, then that position is the insertion position of the transpose element.

5. The analytical method according to claim 1, characterized in that, It also includes determining the zygotic type of the transposable element insertion: based on the determined insertion position of the transposable element, the number of read segments crossing that position is counted, denoted as M, and the number of soft shear read segments crossing that position is counted, denoted as N. Then the insertion frequency VAF of the transposable element is N / M, and the zygotic type of the transposable element insertion is determined based on the VAF value.

6. The analytical method according to claim 5, characterized in that, If the VAF value is greater than or equal to 0.99, it is homozygous; if the VAF value is less than 0.99, it is heterozygous.

7. An apparatus for analyzing insertion information of a rotary element, characterized in that, include: Candidate read pair screening module: used to obtain read pairs in paired-end sequencing data, each read pair including a forward read and a reverse read, and remove read pairs that cannot be used to analyze the insertion information of the transposable element from all read pairs to obtain the candidate read pairs; Target read segment selection module: used to select target read segment pairs from the candidate read segment pairs. The target read segment pair refers to a read segment pair in which one read segment is uniquely matched to a non-rotor element position and the other read segment is matched to a rotor element position. The read segment that is uniquely matched to a non-rotor element position is denoted as the anchor read segment. Transposon element insertion interval prediction module: used to predict the insertion interval of transposon elements. If the anchored read is a forward read, the starting position of the insertion interval of the transposon element is A, and the ending position is A + (L + n × S); if the anchored read is a reverse read, the starting position of the insertion interval of the transposon element is B - (L + n × S), and the ending position is B; where A refers to the starting coordinate of the anchored read, B refers to the ending coordinate of the anchored read, L refers to the length of the inserted fragment between the read pairs corresponding to the anchored read, S refers to the standard deviation of the length of the inserted fragment of the read pairs correctly aligned to the genome in the paired-end sequencing data, and n is a natural number greater than 1. The module for determining the insertion interval of a transducer element is used to determine the insertion interval of the transducer element based on the overlap between the insertion interval of the transducer element predicted by the forward reading segment and the insertion interval of the transducer element predicted by the reverse reading segment. The module for determining the insertion position of a rotary element is used to determine the insertion position of the rotary element. If multiple reading segments are soft-cut at the same position within the determined insertion range of the rotary element, then the insertion position of the rotary element is the position where the rotary element is inserted.

8. The apparatus according to claim 7, characterized in that, It also includes a transposable element insertion mate type determination module: used to determine the mate type of the transposable element insertion, based on the determined insertion position of the transposable element, to count the number of read segments crossing the position, denoted as M, and to count the number of soft shear read segments crossing the position, denoted as N, then the insertion frequency VAF of the transposable element is N / M, and the mate type of the transposable element insertion is determined based on the VAF value.

9. A device for analyzing insertion information of a rotary element, characterized in that, It includes a memory and a processor, the memory being used to store a program, and the processor executing the program stored in the memory to implement the analysis method as described in any one of claims 1 to 6.

10. A computer-readable storage medium, characterized in that, The storage medium stores a program that can be executed by a processor to implement the analysis method as described in any one of claims 1 to 6.

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