CfDNA fragment analysis method and device based on sequencing data, storage medium and electronic equipment
By obtaining the sequencing reads and insertion fragment lengths from the comparative data files, the starting site and fragment length of the cfDNA fragment are determined, and the problem of large analysis errors in the prior art is solved and the analysis accuracy is improved.
Patent Information
- Application Number
- CN202310770650.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-06-27
- Publication Date
- 2024-12-27
AI Technical Summary
The existing cfDNA fragment oscillary analysis technology has poor results in the analysis of fragment length and terminal sites, with large errors and low analysis accuracy.
By obtaining the pair of sequencing reads corresponding to the target cfDNA fragment from the alignment data file, the sequencing reads are aligned to the start site and alignment matching results on the reference genome, and the alignment of the target cfDNA fragment on the reference genome Insert fragment length, determine the starting site, end site and fragment length of the target cfDNA fragment, and use this information for accurate analysis.
It improves the analysis accuracy of cfDNA fragment characteristics, reduces analysis errors, and can more accurately analyze the fragment length and terminal sites of cfDNA fragments.
Smart Images
Figure CN119207569A_ABST
Abstract
Description
Technical Field
[0001] The present application belongs to the field of biomedical technology, and in particular, relates to a cfDNA fragment analysis method, device, storage medium and electronic device based on sequencing data. Background Art
[0002] Different from traditional invasive detection methods, liquid biopsy uses non-invasive sampling to obtain tumor cell-related information. It is one of the most representative precision medical diagnostic technologies. Its principle is to detect the original tumor cells or DNA released into the blood. Among them, circulating cell-free DNA, namely cfDNA, contains all DNA information released into the blood.
[0003] The study of cfDNA fragmentation patterns, also known as "fragmentomics", is now an actively pursued area of biomarker research. Fragmentomics analysis based on cfDNA has become a biomarker for diagnosis, prognosis, and response prediction of various cancers. However, the existing fragmentomics analysis technology is not good at analyzing the fragment characteristics of cfDNA fragments, especially the fragment length and end sites, and there are certain errors and low analysis accuracy. Summary of the invention
[0004] The present application aims to solve at least one of the technical problems existing in the prior art. To this end, the present application proposes a cfDNA fragment analysis method, device, storage medium and electronic device based on sequencing data, which can accurately analyze the fragment length and end site of the cfDNA fragment with high analysis accuracy.
[0005] In a first aspect, the present application provides a cfDNA fragment analysis method based on sequencing data, comprising:
[0006] Obtaining from the alignment data file a pair of sequencing reads corresponding to the target cfDNA fragment, a start site and an alignment match result of each sequencing read in the pair of sequencing reads aligned to a reference genome, and an insert length of the target cfDNA fragment aligned to the reference genome, wherein the pair of sequencing reads are two sequencing reads obtained by performing double-end sequencing of the target cfDNA fragment;
[0007] Determining a target starting site and a target end site corresponding to the target cfDNA fragment according to the starting site and the length of the inserted fragment;
[0008] The fragment length of the target cfDNA fragment is determined based on the alignment matching result, the reference genome, the pair of sequencing reads and the length of the inserted fragment.
[0009] The cfDNA fragment analysis method based on sequencing data provided by the present application obtains a pair of sequencing reads corresponding to the target cfDNA fragment, the start site and the alignment matching result of each sequencing read in the pair of sequencing reads to the reference genome, and the length of the inserted fragment of the target cfDNA fragment to the reference genome from the comparison data file, wherein the pair of sequencing reads are two sequencing reads obtained by performing double-end sequencing on the target cfDNA fragment; according to the start site and the length of the inserted fragment, the target start site and the target end site corresponding to the target cfDNA fragment are determined; according to the alignment matching result, the reference genome, the pair of sequencing reads and the length of the inserted fragment, the fragment length of the target cfDNA fragment is determined, so that the comparison data file can be used to accurately analyze the fragment length and the end site of the target cfDNA fragment, thereby reducing the analysis error and improving the analysis effect of the fragment characteristics.
[0010] According to the method provided in the present application, determining the target starting site and the target end site corresponding to the target cfDNA fragment according to the starting site and the length of the inserted fragment includes:
[0011] Determine the sum value between the starting site of each sequencing read and the corresponding insert length in a pair of sequencing reads corresponding to the target cfDNA fragment, each target cfDNA fragment corresponds to two sum values; use the sum value with a larger value as the target end site of the corresponding target cfDNA fragment;
[0012] The starting site with a smaller value among the starting sites of a pair of the sequencing reads corresponding to the target cfDNA fragment is determined, and the starting site with the smaller value is used as the target starting site of the corresponding target cfDNA fragment.
[0013] According to the method provided in the present application, determining the fragment length of the target cfDNA fragment according to the alignment matching result, the reference genome, the pair of sequencing reads and the length of the inserted fragment includes:
[0014] When the alignment and matching results corresponding to the target cfDNA fragments all indicate that the corresponding sequencing reads can be matched to the reference genome and are completely matched, the length of the inserted fragment is used as the fragment length of the target cfDNA fragment;
[0015] When the alignment and matching results corresponding to the target cfDNA fragment include an alignment and matching result indicating that the corresponding sequencing read can be matched to the reference genome but is not completely matched, at least one incompletely matched sequence is extracted from the reference genome or the corresponding sequencing read; and the fragment length of the target cfDNA fragment is determined based on the at least one incompletely matched sequence and the length of the inserted fragment.
[0016] According to the method provided in the present application, determining the fragment length of the target cfDNA fragment according to the at least one incompletely matched sequence and the length of the inserted fragment includes:
[0017] Determine the sequence length of each of the incompletely matched sequences and the sequence type to which it belongs, wherein the sequence type includes a newly added sequence type and a missing sequence type;
[0018] The fragment length of the target cfDNA fragment is determined based on the sequence length, the inserted fragment length and the sequence type.
[0019] According to the method provided in the present application, determining the fragment length of the target cfDNA fragment according to the sequence length, the inserted fragment length and the sequence type includes:
[0020] When there is an incompletely matched sequence whose sequence type is the missing sequence class, subtracting the sequence length of the corresponding incompletely matched sequence from the length of the inserted fragment to update the length of the inserted fragment;
[0021] When there is an incompletely matched sequence whose sequence type is the newly added sequence class, adding the sequence length of the corresponding incompletely matched sequence to the length of the inserted fragment to update the length of the inserted fragment;
[0022] The updated insert fragment length is used as the fragment length of the target cfDNA fragment.
[0023] According to the method provided by the present application, the method further comprises:
[0024] Generate the comparison data file corresponding to the target library, the target library includes multiple cfDNA fragments, the comparison data file includes a pair of sequencing reads corresponding to each of the cfDNA fragments, and a comparison match result of each sequencing read being aligned to the reference genome;
[0025] According to the comparison and matching results, a target cfDNA fragment is screened out from the multiple cfDNA fragments.
[0026] According to the method provided in the present application, screening out a target cfDNA fragment from the multiple cfDNA fragments according to the comparison and matching results includes:
[0027] Obtaining the alignment and matching results of a pair of the sequencing reads corresponding to each of the cfDNA fragments to obtain two alignment and matching results;
[0028] When the two alignment matching results both indicate that the corresponding sequencing read can be matched to the reference genome, the corresponding cfDNA fragment is used as the target cfDNA fragment.
[0029] According to the method provided in the present application, before generating the comparison data file corresponding to the target library, it also includes:
[0030] Extracting cfDNA from the sample blood of the test subject to obtain the sample cfDNA;
[0031] constructing a methylation library based on the sample cfDNA;
[0032] The methylation library is hybridized, amplified and purified by a preset probe to obtain the target library, wherein the preset probe is used to capture a specific methylation region.
[0033] In a second aspect, the present application provides a cfDNA analysis device based on sequencing data, comprising:
[0034] An acquisition module is used to obtain from the alignment data file a pair of sequencing reads corresponding to the target cfDNA fragment, a start site and an alignment match result of each sequencing read in the pair of sequencing reads to the reference genome, and an insert length of the target cfDNA fragment aligned to the reference genome, wherein the pair of sequencing reads is two sequencing reads obtained by performing double-end sequencing on the target cfDNA fragment;
[0035] A first determination module is used to determine a target start site and a target end site corresponding to the target cfDNA fragment according to the start site and the length of the inserted fragment;
[0036] The second determination module is used to determine the fragment length of the target cfDNA fragment based on the comparison and matching results, the reference genome, the pair of sequencing reads and the length of the inserted fragment.
[0037] According to the device of the present application, the first determining module is specifically used for:
[0038] Determine the sum value between the starting site of each sequencing read and the corresponding insert length in a pair of sequencing reads corresponding to the target cfDNA fragment, each target cfDNA fragment corresponds to two sum values; use the sum value with a larger value as the target end site of the corresponding target cfDNA fragment;
[0039] The starting site with a smaller value among the starting sites of a pair of the sequencing reads corresponding to the target cfDNA fragment is determined, and the starting site with the smaller value is used as the target starting site of the corresponding target cfDNA fragment.
[0040] According to the device of the present application, the second determining module is specifically used for:
[0041] When the alignment and matching results corresponding to the target cfDNA fragments all indicate that the corresponding sequencing reads can be matched to the reference genome and are completely matched, the length of the inserted fragment is used as the fragment length of the target cfDNA fragment;
[0042] When the alignment and matching results corresponding to the target cfDNA fragment include an alignment and matching result indicating that the corresponding sequencing read can be matched to the reference genome but is not completely matched, at least one incompletely matched sequence is extracted from the reference genome or the corresponding sequencing read; and the fragment length of the target cfDNA fragment is determined based on the at least one incompletely matched sequence and the length of the inserted fragment.
[0043] According to the device of the present application, the second determining module is specifically used for:
[0044] Determine the sequence length of each of the incompletely matched sequences and the sequence type to which it belongs, wherein the sequence type includes a newly added sequence type and a missing sequence type;
[0045] The fragment length of the target cfDNA fragment is determined based on the sequence length, the inserted fragment length and the sequence type.
[0046] According to the device of the present application, the second determining module is specifically used for:
[0047] When there is an incompletely matched sequence whose sequence type is the missing sequence class, subtracting the sequence length of the corresponding incompletely matched sequence from the length of the inserted fragment to update the length of the inserted fragment;
[0048] When there is an incompletely matched sequence whose sequence type is the newly added sequence class, adding the sequence length of the corresponding incompletely matched sequence to the length of the inserted fragment to update the length of the inserted fragment;
[0049] The updated insert fragment length is used as the fragment length of the target cfDNA fragment.
[0050] In a third aspect, the present application provides a non-transitory computer-readable storage medium having a computer program stored thereon, which, when executed by a processor, implements any of the above-mentioned methods for analyzing cfDNA fragmentation based on sequencing data.
[0051] In a fourth aspect, the present application provides an electronic device, comprising a memory, a processor, and a computer program stored in the memory and executable on the processor, wherein when the processor executes the program, the cfDNA fragmentation analysis method based on sequencing data as described above is implemented. BRIEF DESCRIPTION OF THE DRAWINGS
[0052] The above and / or additional aspects and advantages of the present application will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:
[0053] Figure 1 It is a schematic diagram of the process of the cfDNA fragmentation analysis method based on sequencing data provided in an embodiment of the present application;
[0054] Figure 2 It is another schematic diagram of the process of the cfDNA fragmentation analysis method based on sequencing data provided in an embodiment of the present application;
[0055] Figure 3 It is a schematic diagram showing the process of adding a linker to sample cfDNA provided in an embodiment of the present application;
[0056] Figure 4 It is a schematic diagram of the structure of a cfDNA fragmentation analysis device based on sequencing data provided in an embodiment of the present application;
[0057] Figure 5 is a schematic diagram of the structure of an electronic device provided in an embodiment of the present application;
[0058] Figure 6 It is a schematic diagram of the hardware structure of the electronic device provided in the embodiment of the present application. DETAILED DESCRIPTION
[0059] The embodiments of the present application are described in detail below, and examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present application, and cannot be understood as limiting the present application.
[0060] The embodiments of the present application provide a cfDNA fragment analysis method, device, storage medium and electronic device based on sequencing data.
[0061] See also Figure 1 , Figure 1 101 is a flow chart of a cfDNA fragment analysis method based on sequencing data provided in an embodiment of the present application. The cfDNA fragment analysis method includes the following steps 101-103, wherein:
[0062] 101. Obtain from the comparison data file a pair of sequencing reads corresponding to the target cfDNA fragment, the start site and the comparison match result of each sequencing read in the pair of sequencing reads compared to the reference genome, and the length of the inserted fragment of the target cfDNA fragment compared to the reference genome, wherein the pair of sequencing reads is two sequencing reads obtained by performing double-end sequencing on the target cfDNA fragment;
[0063] 102. Determine the target start site and target end site corresponding to the target cfDNA fragment according to the start site and the length of the inserted fragment;
[0064] 103. Determine the fragment length of the target cfDNA fragment based on the alignment result, the reference genome, the pair of sequencing reads and the length of the inserted fragment.
[0065] It should be understood that the steps shown in the above production method are not exclusive, and other steps may be performed before, after or between any of the steps shown. The size of the step number does not represent the actual execution order of the steps, but is only an example of an execution order. For example, the above step 102 and step 103 may be performed simultaneously or sequentially, such as first performing step 102 and then performing step 103, or first performing step 103 and then performing step 102.
[0066] The following is a detailed description of the cfDNA fragment analysis method based on sequencing data in the embodiments of the present application.
[0067] 101. Obtain from the alignment data file a pair of sequencing reads corresponding to the target cfDNA fragment, the starting site and alignment matching result of each sequencing read in the pair of sequencing reads aligned to the reference genome, and the length of the inserted fragment of the target cfDNA fragment aligned to the reference genome, wherein the pair of sequencing reads are two sequencing reads obtained by performing double-end sequencing on the target cfDNA fragment.
[0068] Among them, the comparison data file is a file obtained after comparing the sequencing data with the reference genome. It can be a bam file in binary format and can be used for annotation of gene sequences, variation detection, differential expression and other analyses. The sequencing data is obtained after double-end sequencing of all cfDNA fragments in the target library, and these cfDNA fragments include the above-mentioned target cfDNA fragments. Double-end sequencing refers to reading a sequence of tens to hundreds of bp (the number of base pairs) from the beginning and end of the gene sequence to be tested (such as the above-mentioned target cfDNA fragment) to obtain a pair of sequencing reads. That is, if the gene sequence to be tested is the above-mentioned target cfDNA fragment, a pair of reads are short sequences read from the beginning and end of the target cfDNA fragment, respectively. The sequence length of a single read is generally less than the length of the target cfDNA fragment to be tested. By aligning a large number of reads (all reads corresponding to the same sequencing object) to the reference genome, the complete DNA of the test object can be obtained.
[0069] These cfDNA fragments in the target library can be obtained after the library is established based on cfDNA extracted from the blood of the same test subject. cfDNA is a free DNA fragment in the blood. When extracting cfDNA from the test subject's blood, the extracted cfDNA may include both human cfDNA fragments and microbial cfDNA fragments. The target cfDNA fragments are DNA fragments obtained based on human cfDNA fragments, that is, the target library includes cfDNA fragments related to the test subject (such as the above-mentioned target cfDNA fragments), as well as other cfDNA fragments that may be generated due to reasons such as the library construction environment or the extraction environment.
[0070] In the comparison data file of the present application, the reads of all cfDNA fragments in the target library, the comparison information of each read and the reference genome, and the length of the inserted fragment on the reference genome after each cfDNA fragment is compared to the reference genome are stored. These can be directly obtained using existing sequencing comparison technology, and because it is double-end sequencing, each cfDNA fragment corresponds to a pair of reads. The comparison information mainly includes the start site of the read comparison to the reference genome and the comparison matching result. The comparison matching result reflects the matching situation of the read and the reference genome, which roughly includes three matching situations: the read can be matched to the reference genome but not completely matched, the read can be matched to the reference genome and completely matched, and the read cannot be matched to the reference genome. Among them, the read can be matched to the reference genome and completely matched, which means that the gene sequence corresponding to the read can be found on the reference genome, and the read and the gene sequence are completely consistent. The read can be matched to the reference genome but not completely matched, which means that the gene sequence corresponding to the read can be found on the reference genome, but there are some differences between the read and the gene sequence, such as more or less part of the sequence. The read cannot be matched to the reference genome, which means that the gene sequence corresponding to the read cannot be found on the reference genome.
[0071] It should be pointed out that although the target library is constructed based on the cfDNA of the test object, since there are some cfDNA fragments that are not related to the test object, it is necessary to first identify which cfDNA fragments in the target library are related to the test object and which are not. In this way, only the fragment characteristics of those cfDNA fragments related to the test object need to be analyzed subsequently, reducing the data processing volume and processing difficulty of subsequent fragment analysis.
[0072] That is, in some embodiments, see Figure 2 Before executing the above step 101, the cfDNA fragment analysis method based on sequencing data may further include:
[0073] 1041. Generate a comparison data file corresponding to the target library, the target library includes multiple cfDNA fragments, and the comparison data file includes a pair of sequencing reads corresponding to each cfDNA fragment, and a comparison matching result of each sequencing read aligned to the reference genome.
[0074] Among them, the next-generation sequencing (NGS) instrument can be used to perform double-end sequencing on all cfDNA fragments in the target library to obtain sequencing data. After that, the sequencing data is preprocessed, connected, filtered, and quality controlled. The sequencing data is then aligned to the reference genome to obtain the alignment data file corresponding to the entire target library.
[0075] 1042. Based on the comparison and matching results, the target cfDNA fragment is screened out from the multiple cfDNA fragments.
[0076] In some embodiments, the above step 1042 may specifically include:
[0077] Obtaining the alignment and matching results of a pair of sequencing reads corresponding to each cfDNA fragment, and obtaining two alignment and matching results;
[0078] When the two alignment matching results both indicate that the corresponding sequencing read can match the reference genome, the corresponding cfDNA fragment is used as the target cfDNA fragment.
[0079] There are two situations in which the corresponding sequencing read can be matched to the reference genome: the corresponding sequencing read can be matched to the reference genome and is completely matched, or the corresponding sequencing read can be matched to the reference genome but is not completely matched.
[0080] In some embodiments, before the above step 1041, the cfDNA fragment analysis method based on sequencing data may further include:
[0081] Extracting cfDNA from the sample blood of the test subject to obtain the sample cfDNA;
[0082] A methylation library was constructed based on the cfDNA of the sample;
[0083] The methylation library is hybridized, amplified and purified by a preset probe to obtain the target library, and the preset probe is used to capture a specific methylation region.
[0084] The steps of establishing a methylation library can generally include: end repair, A-tail ligation, adapter ligation, purification, elution and other operation steps. After a series of experimental operations, such as Figure 3 , Figure 3 1 is a schematic diagram showing the process of adding a linker to sample cfDNA provided in the embodiment of the present application. The target library is a target-targeted capture methylation sequencing library.
[0085] 102. Based on the starting site and the length of the inserted fragment, determine the target starting site and target end site corresponding to the target cfDNA fragment.
[0086] Among them, since the insert fragment length calculated by the existing second-generation sequencer is calculated by aligning the cfDNA fragment (such as the target cfDNA fragment) to the reference genome and calculating its length on the reference genome, if there is a mutated gene part in the cfDNA fragment, such as sequence insertion or sequence deletion in the cfDNA fragment relative to the reference genome, there will be a certain error between the insert fragment length calculated by the second-generation sequencer and the actual fragment length of the cfDNA fragment. At the same time, there is also an analytical error in the position of the end site. In fragment omics analysis, fragment characteristics such as fragment length and end site are extremely important research information. Therefore, it is urgent to develop a method that can accurately extract the actual end site and actual fragment length of cfDNA fragments.
[0087] This application proposes to further combine the read site information to determine the actual end site and actual fragment length of the cfDNA fragment based on the length of the inserted fragment. For details, please continue to refer to Figure 2 , the above step 102 may include:
[0088] Determine the sum value between the start site of each sequencing read and the corresponding length of the insert fragment in a pair of sequencing reads corresponding to the target cfDNA fragment, and each target cfDNA fragment corresponds to two sum values; use the sum value with a larger value as the target end site of the corresponding target cfDNA fragment;
[0089] The starting site with a smaller value among the starting sites of a pair of sequencing reads corresponding to the target cfDNA fragment is determined, and the starting site with the smaller value is used as the target starting site of the corresponding target cfDNA fragment.
[0090] For example, the above steps can be expressed as the following formula:
[0091]
[0092]
[0093] Where i is the number of detection objects, xi is the i-th detection object, and the second-generation sequencer may sequence the libraries of multiple detection objects at the same time. The comparison data file may contain sequencing comparison data of multiple libraries. start_site (xi) is an array consisting of all target cfDNA fragments and their corresponding target starting sites corresponding to the i-th detection object, that is, including each target cfDNA fragment and its target starting site. end_site(xi) is an array consisting of all target cfDNA fragments and their target end sites corresponding to the i-th detection object, that is, it includes each target cfDNA fragment and its target end site.
[0094] R1 and R2 are a pair of reads obtained by double-end sequencing, N is the number of reads of all target cfDNA fragments corresponding to the i-th detection object, and R is one of the reads of all target cfDNA fragments corresponding to the i-th detection object. ins_len is the insert length of the target cfDNA fragment corresponding to R. k 1 start_site is the starting point of the kth R1, is the starting point of the kth R2.
[0095] From the above formula, it can be seen that for the reads of all target cfDNA fragments corresponding to any detection object, the start site with a more forward position among the two start sites corresponding to a pair of reads can be used as the target start site of the corresponding target cfDNA fragment, and the sum of the two start sites and the length of the inserted fragment of the corresponding target cfDNA fragment is calculated respectively, each sum value indicates an end site, and then the end site with a more backward position is used as the target end site of the corresponding target cfDNA fragment.
[0096] 103. Determine the fragment length of the target cfDNA fragment based on the alignment result, the reference genome, the pair of sequencing reads and the length of the inserted fragment.
[0097] Among them, since the target cfDNA fragment is a cfDNA fragment related to the test object, the alignment and matching results of its read usually only include two matching situations: the read can match the reference genome but not completely match, and the read can match the reference genome and completely match. For different matching situations, different methods are used to determine the fragment length.
[0098] In some embodiments, please see Figure 2 , the above step 103 may specifically include:
[0099] When the alignment matching results corresponding to the target cfDNA fragment all indicate that the corresponding sequencing read can match the reference genome and completely match, the length of the inserted fragment is used as the fragment length of the target cfDNA fragment;
[0100] When the alignment and matching results corresponding to the target cfDNA fragment include an alignment and matching result indicating that the corresponding sequencing read can be matched to the reference genome but is not completely matched, at least one incompletely matched sequence is extracted from the reference genome or the corresponding sequencing read; and the fragment length of the target cfDNA fragment is determined based on the at least one incompletely matched sequence and the length of the inserted fragment.
[0101] Among them, when the pair of reads corresponding to the target cfDNA fragment can completely match the reference genome, the length of the inserted fragment can be directly used as the actual fragment length of the target cfDNA fragment. When there are incompletely matched reads in the pair of reads corresponding to the target cfDNA fragment, such as one or two reads that are not completely matched, it is necessary to correct the length of the inserted fragment according to the incompletely matched sequence to obtain the actual fragment length. The incompletely matched sequence can be the part of the sequence that is extra to the reference genome relative to the read, or the part of the sequence that is extra to the reference genome relative to the read.
[0102] In some embodiments, the above step of "determining the fragment length of the target cfDNA fragment according to the at least one incompletely matched sequence and the length of the inserted fragment" may specifically include:
[0103] Determine the sequence length of each incompletely matched sequence and the sequence type to which it belongs, where the sequence type includes a newly added sequence type and a missing sequence type;
[0104] The fragment length of the target cfDNA fragment is determined based on the sequence length, the inserted fragment length and the sequence type.
[0105] If the incompletely matched sequence belongs to the category of newly added sequences, it means that it is the part of the sequence that is extra in the read relative to the reference genome. The incompletely matched sequence is usually extracted from the read and can also be called an added fragment. If the incompletely matched sequence belongs to the category of deleted sequences, it means that it is the part of the sequence that is extra in the reference genome relative to the read. The incompletely matched sequence is usually extracted from the reference genome and can also be called a deleted fragment.
[0106] Furthermore, the above step of “determining the fragment length of the target cfDNA fragment according to the sequence length, the inserted fragment length and the sequence type” may specifically include:
[0107] When there is an incompletely matched sequence whose sequence type is the missing sequence class, the length of the corresponding incompletely matched sequence is subtracted from the length of the inserted fragment to update the length of the inserted fragment;
[0108] When there is an incompletely matched sequence whose sequence type is the newly added sequence class, the length of the corresponding incompletely matched sequence is added to the length of the inserted fragment to update the length of the inserted fragment;
[0109] The updated inserted fragment length is used as the fragment length of the target cfDNA fragment.
[0110] Among them, when calculating the actual fragment length of the target cfDNA fragment, the sequence length of the newly added fragment can be added to the length of the inserted fragment, and / or the sequence length of the deleted fragment can be subtracted. For example, if a pair of reads of the target cfDNA fragment corresponds to n incompletely matched sequences, then the length of its inserted fragment needs to perform n corresponding addition and subtraction operations, and the final inserted fragment length will be used as the actual fragment length, thereby effectively eliminating the problem of deviation between the inserted fragment length obtained by existing sequencing technology and the actual fragment length due to gene insertion or deletion of the target cfDNA fragment.
[0111] In addition, after analyzing the two fragment features of the target cfDNA fragment, the fragment length and the target end position, these two fragment features can be further used alone or in combination with other fragment omics information to jointly construct a machine learning model to achieve cancer prediction, screening and other functions.
[0112] As can be seen from the above, the cfDNA fragment analysis method based on sequencing data provided in the embodiment of the present application obtains a pair of sequencing reads corresponding to the target cfDNA fragment, the starting site and the alignment matching result of each sequencing read in the pair of sequencing reads to the reference genome, and the length of the inserted fragment of the target cfDNA fragment aligned to the reference genome from the comparison data file. The pair of sequencing reads are two sequencing reads obtained by performing double-end sequencing on the target cfDNA fragment; according to the starting site and the length of the inserted fragment, the target starting site and the target end site corresponding to the target cfDNA fragment are determined; according to the alignment matching result, the reference genome, the pair of sequencing reads and the length of the inserted fragment, the fragment length of the target cfDNA fragment is determined, so that the comparison data file can be used to accurately analyze the fragment length of the target cfDNA fragment and the fragment characteristics of the end site, thereby reducing the analysis error and improving the analysis effect of the fragment characteristics.
[0113] According to the method described in the above embodiment, the present application embodiment also provides a cfDNA fragment analysis device based on sequencing data. Figure 4 , Figure 42 is a schematic diagram of a cfDNA fragment analysis device 200 based on sequencing data provided in an embodiment of the present application. The cfDNA fragment analysis device 200 based on sequencing data includes an acquisition module 201, a first determination module 202, and a second determination module 203, wherein:
[0114] An acquisition module 201 is used to acquire, from the alignment data file, a pair of sequencing reads corresponding to the target cfDNA fragment, a start site and an alignment match result of each sequencing read in the pair of sequencing reads aligned to a reference genome, and an insert length of the target cfDNA fragment aligned to the reference genome, wherein the pair of sequencing reads are two sequencing reads obtained by performing double-end sequencing on the target cfDNA fragment;
[0115] A first determination module 202 is used to determine a target start site and a target end site corresponding to the target cfDNA fragment according to the start site and the length of the inserted fragment;
[0116] The second determination module 203 is used to determine the fragment length of the target cfDNA fragment according to the alignment result, the reference genome, the pair of sequencing reads and the length of the inserted fragment.
[0117] In some embodiments, the first determining module 202 is specifically configured to:
[0118] Determine the sum value between the start site of each sequencing read and the corresponding length of the insert fragment in a pair of sequencing reads corresponding to the target cfDNA fragment, and each target cfDNA fragment corresponds to two sum values; use the sum value with a larger value as the target end site of the corresponding target cfDNA fragment;
[0119] The starting site with a smaller value among the starting sites of a pair of sequencing reads corresponding to the target cfDNA fragment is determined, and the starting site with the smaller value is used as the target starting site of the corresponding target cfDNA fragment.
[0120] In some embodiments, the second determining module 203 is specifically configured to:
[0121] When the alignment matching results corresponding to the target cfDNA fragment all indicate that the corresponding sequencing read can match the reference genome and completely match, the length of the inserted fragment is used as the fragment length of the target cfDNA fragment;
[0122] When the alignment and matching results corresponding to the target cfDNA fragment include an alignment and matching result indicating that the corresponding sequencing read can be matched to the reference genome but is not completely matched, at least one incompletely matched sequence is extracted from the reference genome or the corresponding sequencing read; and the fragment length of the target cfDNA fragment is determined based on the at least one incompletely matched sequence and the length of the inserted fragment.
[0123] In some embodiments, the second determining module 203 is specifically configured to:
[0124] Determine the sequence length of each incompletely matched sequence and the sequence type to which it belongs, where the sequence type includes a newly added sequence type and a missing sequence type;
[0125] The fragment length of the target cfDNA fragment is determined based on the sequence length, the inserted fragment length and the sequence type.
[0126] In some embodiments, the second determining module 203 is specifically configured to:
[0127] When there is an incompletely matched sequence whose sequence type is the missing sequence class, the length of the corresponding incompletely matched sequence is subtracted from the length of the inserted fragment to update the length of the inserted fragment;
[0128] When there is an incompletely matched sequence whose sequence type is the newly added sequence class, the length of the corresponding incompletely matched sequence is added to the length of the inserted fragment to update the length of the inserted fragment;
[0129] The updated inserted fragment length is used as the fragment length of the target cfDNA fragment.
[0130] In some embodiments, the cfDNA fragment analysis device 200 based on sequencing data further includes a screening module for:
[0131] Generate a comparison data file corresponding to a target library, the target library including multiple cfDNA fragments, the comparison data file including a pair of sequencing reads corresponding to each cfDNA fragment, and a comparison match result of each sequencing read being aligned to the reference genome;
[0132] According to the comparison and matching results, the target cfDNA fragment is screened out from the multiple cfDNA fragments.
[0133] In some embodiments, the cfDNA fragment analysis device 200 based on sequencing data further includes a library building module for:
[0134] Before generating the comparison data file corresponding to the target library, cfDNA is extracted from the sample blood of the test subject to obtain the sample cfDNA;
[0135] A methylation library was constructed based on the cfDNA of the sample;
[0136] The methylation library is hybridized, amplified and purified by a preset probe to obtain the target library, and the preset probe is used to capture a specific methylation region.
[0137] It should be noted that the specific details of each module unit in the above-mentioned cfDNA fragment analysis device 200 based on sequencing data have been described in detail in the above-mentioned corresponding display compensation method, and will not be repeated here.
[0138] The cfDNA fragment analysis device based on sequencing data in the embodiment of the present application can be an electronic device, or a component in an electronic device, such as an integrated circuit or a chip. The electronic device can be a terminal, or other devices other than a terminal. Exemplary, the electronic device can be a mobile phone, a tablet computer, a laptop computer, a PDA, a car electronic device, a mobile Internet device (Mobile Internet Device, MID), an augmented reality (augmented reality, AR) / virtual reality (virtual reality, VR) device, a robot, a wearable device, an ultra-mobile personal computer (ultra-mobile personal computer, UMPC), a netbook or a personal digital assistant (personal digital assistant, PDA), etc., and can also be a server, a network attached storage (Network Attached Storage, NAS), a personal computer (personal computer, PC), a television (television, TV), a teller machine or a self-service machine, etc., and the embodiment of the present application is not specifically limited.
[0139] In some embodiments, Figure 5 As shown, the embodiment of the present application also provides an electronic device 300, including a processor 301, a memory 302, and a computer program stored in the memory 302 and executable on the processor 301. When the program is executed by the processor 301, each process of the embodiment of the cfDNA fragment analysis method based on sequencing data is implemented, and the same technical effect can be achieved. To avoid repetition, it will not be described here.
[0140] It should be noted that the electronic devices in the embodiments of the present application include the above-mentioned mobile electronic devices and non-mobile electronic devices.
[0141] Figure 6 A schematic diagram of the hardware structure of an electronic device to implement an embodiment of the present application.
[0142] The electronic device 400 includes but is not limited to: a radio frequency unit 401, a network module 402, an audio output unit 403, an input unit 404, a sensor 405, a display unit 406, a user input unit 407, an interface unit 408, a memory 409, a processor 410 and other components.
[0143] Those skilled in the art will appreciate that the electronic device 400 may also include a power source (such as a battery) for supplying power to each component, and the power source may be logically connected to the processor 410 through a power management system, thereby implementing functions such as managing charging, discharging, and power consumption management through the power management system. Figure 6 The electronic device structure shown in the figure does not constitute a limitation on the electronic device. The electronic device may include more or fewer components than shown in the figure, or combine certain components, or arrange the components differently, which will not be described in detail here.
[0144] It should be understood that in the embodiment of the present application, the input unit 404 may include a graphics processor (Graphics Processing Unit, GPU) 4041 and a microphone 4042, and the graphics processor 4041 processes the image data of the static picture or video obtained by the image capture device (such as a camera) in the video capture mode or the image capture mode. The display unit 406 may include a display panel 4061, and the display panel 4061 may be configured in the form of a liquid crystal display, an organic light emitting diode, etc. The user input unit 407 includes a touch panel 4071 and at least one of other input devices 4072. The touch panel 4071 is also called a touch screen. The touch panel 4071 may include two parts: a touch detection device and a touch controller. Other input devices 4072 may include, but are not limited to, a physical keyboard, function keys (such as volume control keys, switch keys, etc.), a trackball, a mouse, and a joystick, which will not be repeated here.
[0145] The memory 409 can be used to store software programs and various data. The memory 409 may mainly include a first storage area for storing programs or instructions and a second storage area for storing data, wherein the first storage area may store an operating system, an application program or instructions required for at least one function (such as a sound playback function, an image playback function, etc.), etc. In addition, the memory 409 may include a volatile memory or a non-volatile memory, or the memory 409 may include both volatile and non-volatile memories. Among them, the non-volatile memory may be a read-only memory (ROM), a programmable read-only memory (PROM), an erasable programmable read-only memory (EPROM), an electrically erasable programmable read-only memory (EEPROM), or a flash memory. The volatile memory may be a random access memory (RAM), a static random access memory (SRAM), a dynamic random access memory (DRAM), a synchronous dynamic random access memory (SDRAM), a double data rate synchronous dynamic random access memory (DDRSDRAM), an enhanced synchronous dynamic random access memory (ESDRAM), a synchronous link dynamic random access memory (SLDRAM) and a direct memory bus random access memory (DRRAM). The memory 409 in the embodiment of the present application includes but is not limited to these and any other suitable types of memory.
[0146] The processor 410 may include one or more processing units; the processor 410 integrates an application processor and a modem processor, wherein the application processor mainly processes operations related to an operating system, a user interface, and application programs, and the modem processor mainly processes wireless communication signals, such as a baseband processor. It is understandable that the modem processor may not be integrated into the processor 410.
[0147] An embodiment of the present application also provides a non-transitory computer-readable storage medium, on which a computer program is stored. When the computer program is executed by a processor, the various processes of the above-mentioned cfDNA fragment analysis method embodiment based on sequencing data are implemented, and the same technical effect can be achieved. To avoid repetition, it will not be repeated here.
[0148] The processor is the processor in the electronic device in the above embodiment. The readable storage medium includes a computer readable storage medium, such as a computer read-only memory ROM, a random access memory RAM, a magnetic disk or an optical disk.
[0149] An embodiment of the present application also provides a computer program product, including a computer program, which, when executed by a processor, implements the above-mentioned cfDNA fragment analysis method based on sequencing data.
[0150] It should be noted that, in this article, the term "comprises", "includes" or any other variant thereof is intended to cover non-exclusive inclusion, so that the process, method, article or device including a series of elements includes not only those elements, but also includes other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "including one..." do not exclude the presence of other identical elements in the process, method, article or device including the element. In addition, it should be noted that the scope of the method and device in the embodiment of the present application is not limited to performing functions in the order shown or discussed, and may also include performing functions in a substantially simultaneous manner or in reverse order according to the functions involved, for example, the described method may be performed in an order different from that described, and various steps may also be added, omitted, or combined. In addition, the features described with reference to certain examples may be combined in other examples.
[0151] Through the description of the above embodiments, those skilled in the art can clearly understand that the above embodiment methods can be implemented by means of software plus a necessary general hardware platform, and of course by hardware, but in many cases the former is a better embodiment. Based on such an understanding, the technical solution of the present application, or the part that contributes to the prior art, can be embodied in the form of a computer software product, which is stored in a storage medium (such as ROM / RAM, a magnetic disk, or an optical disk), and includes a number of instructions for enabling a terminal (which can be a mobile phone, a computer, a server, or a network device, etc.) to execute the methods described in each embodiment of the present application.
[0152] The embodiments of the present application are described above in conjunction with the accompanying drawings, but the present application is not limited to the above-mentioned specific embodiments. The above-mentioned specific embodiments are merely illustrative and not restrictive. Under the guidance of the present application, ordinary technicians in this field can also make many forms without departing from the purpose of the present application and the scope of protection of the claims, all of which are within the protection of the present application.
[0153] The terms "first", "second", etc. in the specification and claims of the present application are used to distinguish similar objects, and are not used to describe a specific order or sequence. It should be understood that the data used in this way can be interchangeable under appropriate circumstances, so that the embodiments of the present application can be implemented in an order other than those illustrated or described here, and the objects distinguished by "first", "second", etc. are generally of one type, and the number of objects is not limited. For example, the first object can be one or more. In addition, "and / or" in the specification and claims represents at least one of the connected objects, and the character " / " generally indicates that the objects associated with each other are in an "or" relationship.
[0154] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials, or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.
[0155] Although the embodiments of the present application have been shown and described, those skilled in the art will appreciate that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present application, and that the scope of the present application is defined by the claims and their equivalents.
Claims
1. A cfDNA fragment analysis method based on sequencing data, characterized in that: include: Obtaining from the alignment data file a pair of sequencing reads corresponding to the target cfDNA fragment, a start site and an alignment match result of each sequencing read in the pair of sequencing reads aligned to a reference genome, and an insert length of the target cfDNA fragment aligned to the reference genome, wherein the pair of sequencing reads are two sequencing reads obtained by performing double-end sequencing of the target cfDNA fragment; Determining a target starting site and a target end site corresponding to the target cfDNA fragment according to the starting site and the length of the inserted fragment; The fragment length of the target cfDNA fragment is determined based on the alignment matching result, the reference genome, the pair of sequencing reads and the length of the inserted fragment.
2. The method according to claim 1, characterized in that Determining the target starting site and the target end site corresponding to the target cfDNA fragment according to the starting site and the length of the inserted fragment includes: Determine the sum value between the starting site of each sequencing read and the corresponding insert length in a pair of sequencing reads corresponding to the target cfDNA fragment, each target cfDNA fragment corresponds to two sum values; use the sum value with a larger value as the target end site of the corresponding target cfDNA fragment; The starting site with a smaller value among the starting sites of a pair of the sequencing reads corresponding to the target cfDNA fragment is determined, and the starting site with the smaller value is used as the target starting site of the corresponding target cfDNA fragment.
3. The method according to claim 1, characterized in that The determining the fragment length of the target cfDNA fragment according to the alignment and matching result, the reference genome, the pair of sequencing reads and the length of the inserted fragment comprises: When the alignment and matching results corresponding to the target cfDNA fragments all indicate that the corresponding sequencing reads can be matched to the reference genome and are completely matched, the length of the inserted fragment is used as the fragment length of the target cfDNA fragment; When the alignment and matching results corresponding to the target cfDNA fragment include an alignment and matching result indicating that the corresponding sequencing read can be matched to the reference genome but is not completely matched, at least one incompletely matched sequence is extracted from the reference genome or the corresponding sequencing read; and the fragment length of the target cfDNA fragment is determined based on the at least one incompletely matched sequence and the length of the inserted fragment.
4. The method according to claim 3, characterized in that The determining the fragment length of the target cfDNA fragment according to the at least one incompletely matched sequence and the length of the inserted fragment comprises: Determine the sequence length of each of the incompletely matched sequences and the sequence type to which it belongs, wherein the sequence type includes a newly added sequence type and a missing sequence type; The fragment length of the target cfDNA fragment is determined based on the sequence length, the inserted fragment length and the sequence type.
5. The method according to claim 4, characterized in that The determining the fragment length of the target cfDNA fragment according to the sequence length, the inserted fragment length and the sequence type comprises: When there is an incompletely matched sequence whose sequence type is the missing sequence class, subtracting the sequence length of the corresponding incompletely matched sequence from the length of the inserted fragment to update the length of the inserted fragment; When there is an incompletely matched sequence whose sequence type is the newly added sequence class, adding the sequence length of the corresponding incompletely matched sequence to the length of the inserted fragment to update the length of the inserted fragment; The updated insert fragment length is used as the fragment length of the target cfDNA fragment.
6. The method according to claim 1, characterized in that The method further comprises: Generate the comparison data file corresponding to the target library, the target library includes multiple cfDNA fragments, the comparison data file includes a pair of sequencing reads corresponding to each of the cfDNA fragments, and a comparison match result of each sequencing read being aligned to the reference genome; According to the comparison and matching results, a target cfDNA fragment is screened out from the multiple cfDNA fragments.
7. The method according to claim 6, characterized in that The step of screening out a target cfDNA fragment from the plurality of cfDNA fragments according to the comparison and matching results includes: Obtaining the alignment and matching results of a pair of the sequencing reads corresponding to each of the cfDNA fragments to obtain two alignment and matching results; When the two alignment matching results both indicate that the corresponding sequencing read can be matched to the reference genome, the corresponding cfDNA fragment is used as the target cfDNA fragment.
8. The method according to claim 6, characterized in that Before generating the comparison data file corresponding to the target library, the method further includes: Extracting cfDNA from the sample blood of the test subject to obtain the sample cfDNA; constructing a methylation library based on the sample cfDNA; The methylation library is hybridized, amplified and purified by a preset probe to obtain the target library, wherein the preset probe is used to capture a specific methylation region.
9. A cfDNA fragmentation analysis device based on sequencing data, characterized in that: include: An acquisition module is used to obtain from the alignment data file a pair of sequencing reads corresponding to the target cfDNA fragment, a start site and an alignment match result of each sequencing read in the pair of sequencing reads to the reference genome, and an insert length of the target cfDNA fragment aligned to the reference genome, wherein the pair of sequencing reads is two sequencing reads obtained by performing double-end sequencing on the target cfDNA fragment; A first determination module is used to determine a target start site and a target end site corresponding to the target cfDNA fragment according to the start site and the length of the inserted fragment; The second determination module is used to determine the fragment length of the target cfDNA fragment based on the comparison and matching results, the reference genome, the pair of sequencing reads and the length of the inserted fragment.
10. The device according to claim 9, characterized in that The first determining module is specifically used for: Determine the sum value between the starting site of each sequencing read and the corresponding insert length in a pair of sequencing reads corresponding to the target cfDNA fragment, each target cfDNA fragment corresponds to two sum values; use the sum value with a larger value as the target end site of the corresponding target cfDNA fragment; The starting site with a smaller value among the starting sites of a pair of the sequencing reads corresponding to the target cfDNA fragment is determined, and the starting site with the smaller value is used as the target starting site of the corresponding target cfDNA fragment.
11. The device according to claim 9, characterized in that The second determining module is specifically used for: When the alignment and matching results corresponding to the target cfDNA fragments all indicate that the corresponding sequencing reads can be matched to the reference genome and are completely matched, the length of the inserted fragment is used as the fragment length of the target cfDNA fragment; When the alignment and matching results corresponding to the target cfDNA fragment include an alignment and matching result indicating that the corresponding sequencing read can be matched to the reference genome but is not completely matched, at least one incompletely matched sequence is extracted from the reference genome or the corresponding sequencing read; and the fragment length of the target cfDNA fragment is determined based on the at least one incompletely matched sequence and the length of the inserted fragment.
12. The device according to claim 11, characterized in that The second determining module is specifically used for: Determine the sequence length of each of the incompletely matched sequences and the sequence type to which it belongs, wherein the sequence type includes a newly added sequence type and a missing sequence type; The fragment length of the target cfDNA fragment is determined based on the sequence length, the inserted fragment length and the sequence type.
13. The device according to claim 12, characterized in that The second determining module is specifically used for: When there is an incompletely matched sequence whose sequence type is the missing sequence class, subtracting the sequence length of the corresponding incompletely matched sequence from the length of the inserted fragment to update the length of the inserted fragment; When there is an incompletely matched sequence whose sequence type is the newly added sequence class, adding the sequence length of the corresponding incompletely matched sequence to the length of the inserted fragment to update the length of the inserted fragment; The updated insert fragment length is used as the fragment length of the target cfDNA fragment.
14. A non-transitory computer-readable storage medium having a computer program stored thereon, characterized in that: When the computer program is executed by a processor, the cfDNA fragmentation analysis method based on sequencing data is implemented as described in any one of claims 1-8.
15. An electronic device comprising a memory, a processor, and a computer program stored in the memory and executable on the processor, characterized in that: When the processor executes the program, it implements the cfDNA fragmentation analysis method based on sequencing data as described in any one of claims 1-8.