HLA typing method, system, device and medium based on third-generation full-length transcript sequencing data

Abstract

The present invention relates to the field of bioinformatics technology and discloses an HLA typing method, system, device, and medium based on third-generation full-length transcript sequencing data. By leveraging the technical advantages of third-generation sequencing's single-molecule long reads, the complete transcript sequence of the HLA gene is directly obtained, enabling full-length HLA typing without the need for PCR amplification and short-read splicing. This method effectively addresses the challenges of balancing cost, efficiency, and accuracy in traditional HLA typing techniques. It not only avoids PCR amplification bias and short-read assembly errors, significantly improving typing accuracy, but also reduces analytical complexity through optimized bioinformatics workflows. The present invention can also provide an efficient, cost-effective, and reliable solution for applications such as clinical transplant matching and disease association studies, possessing significant clinical application value and broad market prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of bioinformatics, and in particular to an HLA typing method, system, device and medium based on third-generation full-length transcript sequencing data. Background Art

[0002] HLA typing is a method for determining an individual's HLA genotype by analyzing the genetic polymorphism of human leukocyte antigen (HLA) genes. Existing HLA typing is primarily based on technologies such as PCR, sequencing, or gene chips, which determine specific allele types by detecting highly polymorphic regions of the HLA gene. However, these methods face the challenge of balancing cost, efficiency, and accuracy. For example, PCR methods (such as SSP / SSOP) are low-cost but have limited resolution and are prone to misidentification due to primer cross-reactions. Sanger sequencing offers high accuracy but low throughput, is time-consuming, and is expensive, making it difficult to promote clinically. While NGS sequencing can achieve ultra-high resolution, it requires large equipment investments, complex data analysis, and relies on database integrity. Gene chips offer high throughput but can only detect known loci, cannot identify novel alleles, and are subject to interference from probe hybridization.

[0003] Therefore, a new HLA typing method is urgently needed to improve the efficiency and accuracy of HLA typing while reducing costs. Summary of the Invention

[0004] The present invention provides an HLA typing method, system, device and medium based on third-generation full-length transcript sequencing data, aiming to solve the core problem of the difficulty in balancing cost, efficiency and accuracy in existing HLA typing methods.

[0005] The present invention provides an HLA typing method based on third-generation full-length transcript sequencing data, comprising:

[0006] S1. Obtain the third-generation full-length transcript sequencing data of the sample to be tested;

[0007] S2. Obtain the fasta file based on the third-generation full-length transcript sequencing data of the sample to be tested;

[0008] S3. Map the fasta file to the HLA reference gene data, and based on the mapping results, filter out the transcripts in the third-generation full-length transcript sequencing data that cannot be mapped to the HLA reference gene data;

[0009] S4. Correct the transcripts retained in the third-generation full-length transcript sequencing data;

[0010] S5. For each corrected transcript, translate it and obtain the corresponding transcript-peptide chain, and search the HLA amino acid database for an HLA second-level typing set that meets the first preset requirements;

[0011] S6. For each corrected transcript, compare it with the HLA third-level typing included in the corresponding HLA second-level typing set to obtain an HLA third-level typing set that meets the second preset requirement;

[0012] S7. For each corrected transcript, based on its corresponding HLA level 3 typing set and according to the first preset rule, obtain a unique HLA level 3 typing;

[0013] S8. Integrate all corrected transcripts and their corresponding unique HLA third-level typing, and obtain the HLA typing result of the sample to be tested according to the second preset rule.

[0014] According to an HLA typing method based on third-generation full-length transcript sequencing data provided by the present invention, the correction process includes deleting inserted base fragments and / or supplementing deleted base fragments.

[0015] According to an HLA typing method based on third-generation full-length transcript sequencing data provided by the present invention, HLA second-level typing refers to polymorphisms retained in the second field coding region, such as HLA-C*07:02.

[0016] According to an HLA typing method based on third-generation full-length transcript sequencing data provided by the present invention, the first preset requirement is: the same as the transcript-peptide chain length and the amino acid difference value is within a first preset range, wherein the amino acid difference value refers to the number of different amino acids.

[0017] According to an HLA typing method based on third-generation full-length transcript sequencing data provided by the present invention, HLA third-level typing refers to variations retained in the third field non-coding region, such as HLA-C*07:02:01.

[0018] According to an HLA typing method based on third-generation full-length transcript sequencing data provided by the present invention, the second preset requirement is: the base difference value is within a second preset range, wherein the base difference value refers to the number of different bases.

[0019] According to an HLA typing method based on third-generation full-length transcript sequencing data provided by the present invention, step S7 includes:

[0020] S701, for each corrected transcript corresponding to the HLA level 3 typing set, pairwise compare transcript sets corresponding to different HLA level 3 typings;

[0021] S702: If the transcript set corresponding to one HLA level 3 typing is a subset of the transcript set corresponding to another HLA level 3 typing, discard the HLA level 3 typing;

[0022] S703. If the transcript sets corresponding to the two HLA level 3 typings are the same, compare the sum of base difference values ​​of the transcript sets corresponding to the two HLA level 3 typings, and retain the HLA level 3 typing with the smaller sum of base difference values.

[0023] S704. For each corrected transcript, the HLA level 3 typing obtained after steps S701-S703 is counted. If a transcript still corresponds to multiple HLA level 3 typings, the base difference between the different HLA level 3 typings and the transcript is compared, and the HLA level 3 typing with the smallest difference is selected as the unique HLA level 3 typing for the corrected transcript.

[0024] According to an HLA typing method based on third-generation full-length transcript sequencing data provided by the present invention, step S8 comprises:

[0025] S801. Obtaining the coverage of HLA third-level typing based on the differences between HLA third-level typing and different transcripts;

[0026] S802. For multiple HLA level 3 typings under the same HLA level 2 typing, obtain a unique HLA level 3 typing based on the coverage of the HLA level 3 typing and the differences between the HLA level 3 typing and different transcripts;

[0027] S803: Count the HLA third-level typing set obtained after step S802, and obtain the HLA typing result of the sample to be tested based on the coverage of each HLA third-level typing in the set.

[0028] According to an HLA typing method based on third-generation full-length transcript sequencing data provided by the present invention, the coverage expression is:

[0029] ,

[0030] In the expression of coverage, Indicates HLA third level typing The coverage, Indicates HLA third level typing and transcripts The difference between Represents the difference parameter, indicating function Indicates that if the condition in the brackets is met, it is equal to 1, otherwise it is equal to 0. Indicates the total number of transcripts.

[0031] According to an HLA typing method based on third-generation full-length transcript sequencing data provided by the present invention, step S802 includes:

[0032] If there are multiple HLA third-level typings under the same HLA second-level typing, priority will be given to coverage, and the HLA third-level typing with the highest coverage will be retained;

[0033] If multiple HLA level 3 typings have the same coverage, priority will be given to the number of transcripts with a difference of 0 from each HLA level 3 typing, and the HLA level 3 typing with the largest number of transcripts with a difference of 0 from the HLA level 3 typing will be retained;

[0034] If there are multiple HLA level 3 typings with the same number of transcripts having a degree of difference of 0 from the HLA level 3 typing, priority is given to the HLA level 3 typing with the smaller sum of the degrees of difference from the individual transcripts.

[0035] According to an HLA typing method based on third-generation full-length transcript sequencing data provided by the present invention, step S803 includes:

[0036] The HLA level 3 typing set obtained after step S802 is counted. Based on the coverage of each HLA level 3 typing in the set, the two HLA level 3 typings with the highest coverage are retained as the HLA typing result of the sample to be tested. If there is only one HLA level 3 typing in the HLA level 3 typing set obtained after step S802, the HLA typing result of the sample to be tested is determined to be homozygous.

[0037] According to the HLA typing method based on third-generation full-length transcript sequencing data provided by the present invention, step S803 further includes:

[0038] Output the HLA second-level typing results of the sample to be tested.

[0039] The present invention also provides an HLA typing system based on third-generation full-length transcript sequencing data, comprising:

[0040] The data acquisition module is used to obtain the third-generation full-length transcript sequencing data of the sample to be tested;

[0041] The data processing module is used to obtain the fasta file based on the third-generation full-length transcript sequencing data of the sample to be tested;

[0042] The mapping module is used to map the fasta file to the HLA reference gene data and, based on the mapping results, filter out transcripts in the third-generation full-length transcript sequencing data that cannot be mapped to the HLA reference gene data;

[0043] The correction module is used to correct the transcripts retained in the third-generation full-length transcript sequencing data;

[0044] The HLA second-level typing module is configured to: translate each corrected transcript and obtain a corresponding transcript-peptide chain, and search the HLA amino acid database for an HLA second-level typing set that meets the first preset requirements;

[0045] An HLA third-level typing module is configured to compare each corrected transcript with the HLA third-level typing included in the corresponding HLA second-level typing set to obtain an HLA third-level typing set that meets a second preset requirement;

[0046] A screening module is configured to: for each corrected transcript, obtain a unique HLA level 3 typing based on its corresponding HLA level 3 typing set according to a first preset rule;

[0047] The HLA typing module is used to integrate all corrected transcripts and their corresponding unique HLA third-level typing, and obtain the HLA typing result of the sample to be tested according to the second preset rule.

[0048] The present invention also provides an electronic device comprising a processor and a memory storing a computer program, wherein when the processor executes the computer program, any of the above-mentioned HLA typing methods based on third-generation full-length transcript sequencing data is implemented.

[0049] The present invention also 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 HLA typing methods based on third-generation full-length transcript sequencing data.

[0050] The present invention also provides a computer program product, which includes a computer program. The computer program can be stored on a non-transitory computer-readable storage medium. When the computer program is executed by a processor, the computer can execute any of the above-mentioned HLA typing methods based on third-generation full-length transcript sequencing data.

[0051] The present invention provides an HLA typing method, system, device and medium based on third-generation full-length transcript sequencing data. By leveraging the technical advantages of third-generation sequencing single-molecule long reads, the complete transcript sequence of the HLA gene is directly obtained, achieving full-length HLA typing without the need for PCR amplification and short-read splicing, effectively solving the problem of balancing cost, efficiency and accuracy in traditional HLA typing methods.

[0052] Compared with traditional methods, the present invention has at least the following outstanding advantages:

[0053] Improved detection efficiency: High-resolution typing can be completed with only third-generation sequencing raw data, avoiding the cost waste caused by repeated sequencing due to typing needs;

[0054] Accuracy breakthrough: Through single-molecule mapping of full-length transcripts, PCR amplification bias and short-read assembly errors are completely eliminated, significantly improving the detection rate of rare alleles;

[0055] Clinical application value: It can provide economical and reliable solutions for scenarios such as organ transplant matching and autoimmune disease research, and has important clinical application value and broad market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] In order to more clearly illustrate the technical solutions in the present invention or the prior art, a brief introduction will be given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0057] Figure 1 A schematic diagram of the process of an HLA typing method based on third-generation full-length transcript sequencing data provided by the present invention.

[0058] Figure 2 This is a schematic diagram of the structure of an HLA typing system based on third-generation full-length transcript sequencing data provided by the present invention.

[0059] Figure 3 This is a schematic structural diagram of the electronic device provided by the present invention. DETAILED DESCRIPTION

[0060] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the drawings in the present invention. Obviously, the embodiments described are part of the embodiments of the present invention, not all of the embodiments, and they should not be understood as limitations on the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. In the description of the present invention, it should be understood that the terms used are only for descriptive purposes and cannot be understood as indicating or implying relative importance.

[0061] HLA genes play a key role in the immune system and are closely related to organ transplant matching, susceptibility to autoimmune diseases, and adverse drug reactions. Determining the HLA typing of a sample can provide accurate matching for organ transplantation and reduce the risk of rejection. It also facilitates disease risk assessment, personalized medicine, and research into the mechanisms of immune-related diseases.

[0062] Figure 1 This is a schematic diagram of a process flow for an HLA typing method based on third-generation full-length transcript sequencing data provided by the present invention. The method can be performed by any applicable terminal-side or network-side device, such as an HLA typing device based on third-generation full-length transcript sequencing data.

[0063] See also Figure 1 The present invention provides an HLA typing method based on third-generation full-length transcript sequencing data, which may include:

[0064] S1. Obtain the third-generation full-length transcript sequencing data of the sample to be tested.

[0065] In one embodiment, researchers may first use PacBio's third-generation sequencing technology to sequence the sample to be tested to obtain third-generation full-length transcript sequencing data of the sample to be tested. The sample to be tested may be a biological tissue sample, such as a human tumor sample.

[0066] PacBio's third-generation sequencing technology, also known as single-molecule real-time sequencing (SMRT), is a high-throughput sequencing method based on single-molecule sequencing. Its core is the real-time detection of the fluorescent signal released by DNA polymerase as it synthesizes a complementary strand on a single DNA template using zero-mode waveguides (ZMWs).

[0067] PacBio technology utilizes Iso-Seq (Isoform Sequencing) for full-length transcript sequencing. Iso-Seq directly sequences intact mRNA molecules without fragmentation, resulting in full-length transcript sequences. This method is particularly well-suited for studying the complexities of gene expression, including alternative splicing and transcript isoforms.

[0068] S2. Obtain a fasta file based on the third-generation full-length transcript sequencing data of the sample to be tested. The fasta file may include information such as the transcript temporary number, transcript full-field coverage, and transcript base sequence.

[0069] In one embodiment, step S2 can use sequencing data processing software (such as isoseq3 (version 3.3.0)) to process the third-generation full-length transcript sequencing data of the sample to be tested to obtain a fasta file.

[0070] S3. Map the fasta file to the HLA reference gene data, and based on the mapping results, filter out the transcripts in the third-generation full-length transcript sequencing data that cannot be mapped to the HLA reference gene data.

[0071] In one embodiment, step S3 may map the fasta file to HLA reference gene data using mapping software (eg, Minimap2 (version 3.3.0)), and the HLA reference gene data may be data from the IPD-IMGT / HLA database.

[0072] S4. Perform correction processing on the transcripts retained in the third-generation full-length transcript sequencing data, wherein the correction processing includes deleting the inserted base fragments and supplementing the missing base fragments.

[0073] In one embodiment, step S4 may perform correction processing on the retained transcripts according to the CIGAR information in the Minimap2 mapping result.

[0074] S5. For each corrected transcript, translate it and obtain a corresponding transcript-peptide chain. Using the HLA amino acid database (in this embodiment, the IPD-IMGT / HLA database) as a reference, search the database to obtain an HLA second-level typing set that meets the first preset requirement. HLA second-level typing refers to polymorphisms retained in the coding region of the second field, for example, HLA-C*07:02. The first preset requirement is that the length is the same as the transcript-peptide chain and the amino acid difference value is within a first preset range (in this embodiment, the amino acid difference value is less than or equal to 5). The amino acid difference value refers to the number of different amino acids.

[0075] S6. For each corrected transcript, compare it with the HLA level-3 typing included in the corresponding HLA level-2 typing set to obtain an HLA level-3 typing set that meets a second preset requirement. The HLA level-3 typing refers to variants retained in the non-coding region of the third field, for example, HLA-C*07:02:01. The second preset requirement is that the base difference value be within a second preset range (in this embodiment, a base difference value less than or equal to 5), where the base difference value refers to the number of base differences.

[0076] S7. For each corrected transcript, based on its corresponding HLA level 3 typing set and according to the first preset rule, a unique HLA level 3 typing is obtained.

[0077] In one embodiment, step S7 includes:

[0078] S701, for each corrected transcript corresponding to the HLA level 3 typing set, cyclically aligning the transcript sets corresponding to every two different HLA level 3 typing sets;

[0079] S702: If the transcript set corresponding to one HLA level 3 typing is a subset of the transcript set corresponding to another HLA level 3 typing, discard the HLA level 3 typing;

[0080] S703. If the transcript sets corresponding to the two HLA level 3 typings are the same, compare the sum of base difference values ​​of the transcript sets corresponding to the two HLA level 3 typings, and retain the HLA level 3 typing with the smaller sum of base difference values.

[0081] S704. For each corrected transcript, the HLA level 3 typing obtained after steps S701-S703 is counted. If a transcript still corresponds to multiple HLA level 3 typings, the base difference between the different HLA level 3 typings and the transcript is compared, and the HLA level 3 typing with the smallest difference is selected as the unique HLA level 3 typing for the corrected transcript.

[0082] S8. Integrate all corrected transcripts and their corresponding unique HLA third-level typing, and obtain the HLA typing result of the sample to be tested according to the second preset rule.

[0083] In one embodiment, step S8 includes:

[0084] S801. Obtain coverage of HLA level 3 typing based on the differences between the HLA level 3 typing and different transcripts, where the expression for coverage is:

[0085] ,

[0086] In the expression of coverage, Indicates HLA third level typing The coverage, Indicates HLA third level typing and transcripts The difference between Indicates the difference parameter (in this embodiment The value is 5), indicating the function Indicates that if the condition in the brackets is met, it is equal to 1, otherwise it is equal to 0. represents the total number of transcripts;

[0087] S802. For multiple HLA level 3 typings under the same HLA level 2 typing, obtain a unique HLA level 3 typing based on the coverage of the HLA level 3 typing and the differences between the HLA level 3 typing and different transcripts;

[0088] In one embodiment, step S802 includes:

[0089] If there are multiple HLA third-level typings under the same HLA second-level typing, priority will be given to coverage, and the HLA third-level typing with the highest coverage will be retained;

[0090] If multiple HLA level 3 typings have the same coverage, priority will be given to the number of transcripts with a difference of 0 from each HLA level 3 typing, and the HLA level 3 typing with the largest number of transcripts with a difference of 0 from the HLA level 3 typing will be retained;

[0091] If there are multiple HLA level 3 typings with the same number of transcripts having a degree of difference of 0 from the HLA level 3 typing, priority is given to the HLA level 3 typing with the smaller sum of the degrees of difference from the individual transcripts.

[0092] S803: Count the HLA third-level typing set obtained after step S802, and obtain the HLA typing result of the sample to be tested based on the coverage of each HLA third-level typing in the set.

[0093] In one embodiment, step S803 includes:

[0094] The HLA level 3 typing set obtained after step S802 is counted. Based on the coverage of each HLA level 3 typing in the set, the two HLA level 3 typings with the highest coverage are retained as the HLA typing result of the sample to be tested. If there is only one HLA level 3 typing in the HLA level 3 typing set obtained after step S802, the HLA typing result of the sample to be tested is determined to be homozygous.

[0095] In one embodiment, step S803 further includes the step of outputting the HLA second-level typing result of the sample to be tested for use in immune and other research.

[0096] After obtaining the HLA typing results of the sample to be tested, relevant immunological research can be carried out, such as antigen prediction, autoimmune disease research, and susceptibility gene screening for some diseases; it can also be used as a genetic marker to assist genetic research, which is of great significance.

[0097] In this example, an HLA typing method based on third-generation full-length transcript sequencing data was used to perform HLA typing on eight samples to be tested (named sample1, sample2...sample8). The results are shown in Table 1.

[0098]

[0099] For the eight samples to be tested, this example also used Pacbio's proprietary HiFiHLA method for HLA typing analysis. Comparison revealed that the HLA typing method based on third-generation full-length transcript sequencing data provided in this example achieved superior accuracy compared to the HiFiHLA method. Furthermore, the HLA typing method based on third-generation full-length transcript sequencing data provided in this example has higher data utilization, can save samples, avoid multiple sequencing runs, and effectively reduce time and economic costs.

[0100] The present invention provides an HLA typing method, system, device, and storage medium based on third-generation full-length transcript sequencing data. By leveraging the technical advantages of third-generation sequencing single-molecule long reads, the complete transcript sequence of the HLA gene is directly obtained, achieving full-length HLA typing without PCR amplification and short-read splicing, thus filling the gap in the detection of HLA typing using third-generation full-field transcripts. This method effectively solves the problem of balancing cost, efficiency, and accuracy in traditional HLA typing technology. It not only avoids PCR amplification bias and short-read assembly errors, significantly improves typing accuracy, but also reduces analysis complexity through optimized bioinformatics processes. The present invention can also provide an efficient solution that is both economical and reliable for application scenarios such as clinical transplant matching and disease association studies, and has important clinical application value and broad market prospects.

[0101] The HLA typing system based on third-generation full-length transcript sequencing data provided by the present invention is described below. The HLA typing system based on third-generation full-length transcript sequencing data described below and the HLA typing method based on third-generation full-length transcript sequencing data described above can be referenced to each other.

[0102] The present invention provides an HLA typing system based on third-generation full-length transcript sequencing data, which may include:

[0103] The data acquisition module is used to obtain the third-generation full-length transcript sequencing data of the sample to be tested;

[0104] The data processing module is used to obtain the fasta file based on the third-generation full-length transcript sequencing data of the sample to be tested;

[0105] The mapping module is used to map the fasta file to the HLA reference gene data and, based on the mapping results, filter out transcripts in the third-generation full-length transcript sequencing data that cannot be mapped to the HLA reference gene data;

[0106] The correction module is used to correct the transcripts retained in the third-generation full-length transcript sequencing data;

[0107] The HLA second-level typing module is configured to: translate each corrected transcript and obtain a corresponding transcript-peptide chain, and search the HLA amino acid database for an HLA second-level typing set that meets the first preset requirements;

[0108] An HLA third-level typing module is configured to compare each corrected transcript with the HLA third-level typing included in the corresponding HLA second-level typing set to obtain an HLA third-level typing set that meets a second preset requirement;

[0109] A screening module is configured to: for each corrected transcript, obtain a unique HLA level 3 typing based on its corresponding HLA level 3 typing set according to a first preset rule;

[0110] The HLA typing module is used to integrate all corrected transcripts and their corresponding unique HLA third-level typing, and obtain the HLA typing result of the sample to be tested according to the second preset rule.

[0111] Figure 3 An example of a physical structure diagram of an electronic device is shown below. Figure 3As shown, the electronic device may include: a processor 810, a communication interface 820, a memory 830, and a communication bus 840, wherein the processor 810, the communication interface 820, and the memory 830 communicate with each other via the communication bus 840. The processor 810 may call the logic instructions in the memory 830 to perform the following steps:

[0112] S1. Obtain the third-generation full-length transcript sequencing data of the sample to be tested;

[0113] S2. Obtain the fasta file based on the third-generation full-length transcript sequencing data of the sample to be tested;

[0114] S3. Map the fasta file to the HLA reference gene data, and based on the mapping results, filter out the transcripts in the third-generation full-length transcript sequencing data that cannot be mapped to the HLA reference gene data;

[0115] S4. Correct the transcripts retained in the third-generation full-length transcript sequencing data;

[0116] S5. For each corrected transcript, translate it and obtain the corresponding transcript-peptide chain, and search the HLA amino acid database for an HLA second-level typing set that meets the first preset requirements;

[0117] S6. For each corrected transcript, compare it with the HLA third-level typing included in the corresponding HLA second-level typing set to obtain an HLA third-level typing set that meets the second preset requirement;

[0118] S7. For each corrected transcript, based on its corresponding HLA level 3 typing set and according to the first preset rule, obtain a unique HLA level 3 typing;

[0119] S8. Integrate all corrected transcripts and their corresponding unique HLA third-level typing, and obtain the HLA typing result of the sample to be tested according to the second preset rule.

[0120] Furthermore, the logic instructions in the aforementioned memory 830 can be implemented as software functional units and, when sold or used as independent products, can be stored in a computer-readable storage medium. Based on this understanding, the technical solution of the present invention, or the portion that contributes to the prior art, or a portion of the technical solution, can be embodied in the form of a software product. This computer software product, stored in a storage medium, includes instructions for enabling a computer device (which can be a personal computer, server, or network device, etc.) to perform all or part of the steps of the methods described in various embodiments of the present invention. The aforementioned storage medium includes various media capable of storing program code, such as USB flash drives, mobile hard drives, read-only memories (ROMs), random access memories (RAMs), magnetic disks, or optical disks.

[0121] In another aspect, the present invention further provides a computer program product, comprising a computer program, which may be stored on a non-transitory computer-readable storage medium, and wherein when the computer program is executed by a processor, the computer is capable of performing the following steps:

[0122] S1. Obtain the third-generation full-length transcript sequencing data of the sample to be tested;

[0123] S2. Obtain the fasta file based on the third-generation full-length transcript sequencing data of the sample to be tested;

[0124] S3. Map the fasta file to the HLA reference gene data, and based on the mapping results, filter out the transcripts in the third-generation full-length transcript sequencing data that cannot be mapped to the HLA reference gene data;

[0125] S4. Correct the transcripts retained in the third-generation full-length transcript sequencing data;

[0126] S5. For each corrected transcript, translate it and obtain the corresponding transcript-peptide chain, and search the HLA amino acid database for an HLA second-level typing set that meets the first preset requirements;

[0127] S6. For each corrected transcript, compare it with the HLA third-level typing included in the corresponding HLA second-level typing set to obtain an HLA third-level typing set that meets the second preset requirement;

[0128] S7. For each corrected transcript, based on its corresponding HLA level 3 typing set and according to the first preset rule, obtain a unique HLA level 3 typing;

[0129] S8. Integrate all corrected transcripts and their corresponding unique HLA third-level typing, and obtain the HLA typing result of the sample to be tested according to the second preset rule.

[0130] In another aspect, the present invention further provides a non-transitory computer-readable storage medium having a computer program stored thereon, which is configured to execute the following steps when executed by a processor:

[0131] S1. Obtain the third-generation full-length transcript sequencing data of the sample to be tested;

[0132] S2. Obtain the fasta file based on the third-generation full-length transcript sequencing data of the sample to be tested;

[0133] S3. Map the fasta file to the HLA reference gene data, and based on the mapping results, filter out the transcripts in the third-generation full-length transcript sequencing data that cannot be mapped to the HLA reference gene data;

[0134] S4. Correct the transcripts retained in the third-generation full-length transcript sequencing data;

[0135] S5. For each corrected transcript, translate it and obtain the corresponding transcript-peptide chain, and search the HLA amino acid database for an HLA second-level typing set that meets the first preset requirements;

[0136] S6. For each corrected transcript, compare it with the HLA third-level typing included in the corresponding HLA second-level typing set to obtain an HLA third-level typing set that meets the second preset requirement;

[0137] S7. For each corrected transcript, based on its corresponding HLA level 3 typing set and according to the first preset rule, obtain a unique HLA level 3 typing;

[0138] S8. Integrate all corrected transcripts and their corresponding unique HLA third-level typing, and obtain the HLA typing result of the sample to be tested according to the second preset rule.

[0139] The device embodiments described above are merely illustrative. The units described as separate components may or may not be physically separate, and the components shown as units may or may not be physical units, i.e., they may be located in one location or distributed across multiple network units. Some or all of the modules may be selected based on actual needs to achieve the objectives of the present embodiment. Persons of ordinary skill in the art will be able to understand and implement the present invention without inventive effort.

[0140] Through the above description of the embodiments, those skilled in the art will clearly understand that each embodiment can be implemented using software plus a necessary general-purpose hardware platform, or of course, hardware. Based on this understanding, the essence of the above technical solution, or the portion that contributes to the prior art, can be embodied in the form of a software product. This computer software product can be stored in a computer-readable storage medium, such as ROM / RAM, a magnetic disk, or an optical disk, and includes a number of instructions for causing a computer device (such as a personal computer, server, or network device) to execute the methods described in each embodiment or certain portions of the embodiments.

[0141] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. An HLA typing method based on third-generation full-length transcript sequencing data, characterized in that: include: S1. Obtain the third-generation full-length transcript sequencing data of the sample to be tested; S2. Obtain the fasta file based on the third-generation full-length transcript sequencing data of the sample to be tested; S3. Map the fasta file to the HLA reference gene data, and based on the mapping results, filter out the transcripts in the third-generation full-length transcript sequencing data that cannot be mapped to the HLA reference gene data; S4. Correct the transcripts retained in the third-generation full-length transcript sequencing data; S5. For each corrected transcript, translate it and obtain the corresponding transcript-peptide chain, and search the HLA amino acid database for an HLA second-level typing set that meets the first preset requirements; S6. For each corrected transcript, compare it with the HLA third-level typing included in the corresponding HLA second-level typing set to obtain an HLA third-level typing set that meets the second preset requirement; S7. For each corrected transcript, based on its corresponding HLA level 3 typing set and according to the first preset rule, obtain a unique HLA level 3 typing; S8. Integrate all corrected transcripts and their corresponding unique HLA level 3 typing, and obtain the HLA typing result of the sample to be tested according to the second preset rule; Among them, HLA second-level typing refers to the polymorphism retained in the second field coding region. The first preset requirement is: the same as the transcript-peptide chain length and the amino acid difference value is within the first preset range, where the amino acid difference value refers to the number of different amino acids; HLA level 3 typing refers to variants retained in the non-coding region of the third field. The second preset requirement is that the base difference value is within the second preset range, where the base difference value refers to the number of different bases. Wherein, step S7 includes: S701, for each corrected transcript corresponding to the HLA level 3 typing set, pairwise compare transcript sets corresponding to different HLA level 3 typings; S702: If the transcript set corresponding to one HLA level 3 typing is a subset of the transcript set corresponding to another HLA level 3 typing, discard the HLA level 3 typing; S703. If the transcript sets corresponding to the two HLA level 3 typings are the same, compare the sum of base difference values ​​of the transcript sets corresponding to the two HLA level 3 typings, and retain the HLA level 3 typing with the smaller sum of base difference values. S704. For each corrected transcript, the HLA level 3 typing obtained after steps S701-S703 is counted. If a transcript still corresponds to multiple HLA level 3 typings, the HLA level 3 typing with the smallest base difference is compared with the transcript, and the HLA level 3 typing with the smallest base difference is used as the unique HLA level 3 typing of the corrected transcript. Wherein, step S8 includes: S801. Obtaining the coverage of HLA third-level typing based on the differences between HLA third-level typing and different transcripts; S802. For multiple HLA level 3 typings under the same HLA level 2 typing, obtain a unique HLA level 3 typing based on the coverage of the HLA level 3 typing and the differences between the HLA level 3 typing and different transcripts; S803: Count the HLA third-level typing set obtained after step S802, and obtain the HLA typing result of the sample to be tested based on the coverage of each HLA third-level typing in the set; The expression of coverage is: ， In the expression of coverage, Indicates HLA third level typing The coverage, Indicates HLA third level typing and transcripts The difference between Represents the difference parameter, indicating function Indicates that if the condition in the brackets is met, it is equal to 1, otherwise it is equal to 0. represents the total number of transcripts; Wherein, step S802 includes: If there are multiple HLA third-level typings under the same HLA second-level typing, priority will be given to coverage, and the HLA third-level typing with the highest coverage will be retained; If multiple HLA level 3 typings have the same coverage, priority will be given to the number of transcripts with a difference of 0 from each HLA level 3 typing, and the HLA level 3 typing with the largest number of transcripts with a difference of 0 from the HLA level 3 typing will be retained; If there are multiple HLA level 3 typings with the same number of transcripts that are 0 different from the HLA level 3 typing, the HLA level 3 typing with the smaller sum of differences from each transcript will be given priority; Wherein, step S803 includes: The HLA level 3 typing set obtained after step S802 is counted. Based on the coverage of each HLA level 3 typing in the set, the two HLA level 3 typings with the highest coverage are retained as the HLA typing result of the sample to be tested. If there is only one HLA level 3 typing in the HLA level 3 typing set obtained after step S802, the HLA typing result of the sample to be tested is determined to be homozygous.

2. An HLA typing system based on the HLA typing method based on third-generation full-length transcript sequencing data according to claim 1, characterized in that: include: The data acquisition module is used to obtain the third-generation full-length transcript sequencing data of the sample to be tested; The data processing module is used to obtain the fasta file based on the third-generation full-length transcript sequencing data of the sample to be tested; The mapping module is used to map the fasta file to the HLA reference gene data and, based on the mapping results, filter out transcripts in the third-generation full-length transcript sequencing data that cannot be mapped to the HLA reference gene data; The correction module is used to correct the transcripts retained in the third-generation full-length transcript sequencing data; The HLA second-level typing module is configured to: translate each corrected transcript and obtain a corresponding transcript-peptide chain, and search the HLA amino acid database for an HLA second-level typing set that meets the first preset requirements; An HLA third-level typing module is configured to compare each corrected transcript with the HLA third-level typing included in the corresponding HLA second-level typing set to obtain an HLA third-level typing set that meets a second preset requirement; A screening module is configured to: for each corrected transcript, obtain a unique HLA level 3 typing based on its corresponding HLA level 3 typing set according to a first preset rule; The HLA typing module is used to integrate all corrected transcripts and their corresponding unique HLA third-level typing, and obtain the HLA typing result of the sample to be tested according to the second preset rule.

3. 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 HLA typing method based on third-generation full-length transcript sequencing data as claimed in claim 1 is implemented.

4. 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 HLA typing method based on third-generation full-length transcript sequencing data as claimed in claim 1 is implemented.

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