HK1240371B - Method and device for determining v/j gene sequences before rearrangement - Google Patents

Description

Method and device for determining V/J gene sequence before rearrangement

Technical Field

The present invention belongs to the field of bioinformatics. Specifically, the present invention relates to a method and device for determining a V/J gene sequence before rearrangement.

Background Art

Germline cells contain a cluster of V genes and a cluster of J genes, sometimes with a cluster of D genes between them. The genes in a cluster are separated by introns and arranged in tandem on the same chromosome, with very high homology between them [Animal Immunology [M]. China Agricultural University Press, 1996]. Typically, a cluster contains dozens of genes, and each gene can vary between individuals. For example, the V gene cluster encoding the heavy chain (IGH) of antibodies in humans has 40 genes, the D gene cluster has 25 genes, and the J gene cluster has 6 genes. The 40 V genes have a total of 425 alleles.

During the maturation of lymphocytes, V, J, or D genes undergo intergenic rearrangement [Parkin J, Cohen B. An overview of the immune system [J]. The Lancet, 2001, 357(9270): 1777-1789.], forming genes encoding T cell receptors (TCRs), B cell receptors (BCRs), or antibodies (Ig). The collection of these B cell receptors/antibodies or T cell receptors that make up the body's immune system forms the immune repertoire.

The constant regions (C regions) of immunoglobulins TCR and BCR are relatively conserved and relatively easy to sequence. The C regions of many animals are known. However, the diversity of the V, D, and J gene regions is high [Yu Jiang, Yao Xinsheng. High-throughput sequencing analysis of the characteristics of the T cell receptor β chain CDR3 repertoire in autoimmune diseases [J]. Guizhou Medicine, 2015, 3: 037.]; moreover, except for humans and mice, the genes in this region of other species have not been found or only a part of them has been identified; this has hindered the progress of immunological research to a certain extent. For example, monkeys are a usable animal model for vaccine evaluation and antibody and are widely used. However, only a small number of monkey IgH sequences [Link J M, Hellinger MA, Schroeder H W. The Rhesus monkey immunoglobulin IGHD and IGHJ germline repertoire [J]. Immunogenetics, 2002, 54(4): 240-250.] have been discovered, which is far from meeting the requirements of analysis. Therefore, studying the germline sequence of species is a basic problem that needs to be solved urgently.

Currently, there are several methods that attempt to explore germline sequences. The traditional method is to use a PCR cloning strategy, using human genomic DNA sequences as primers to PCR amplify the species' germline. This method can be used to detect partial germline sequences in camels [Nguyen V K, Hamers R, Wyns L, et al. Camel heavy-chain antibodies: diverse germline VHH and specific mechanisms enlarge the antigen-binding repertoire[J]. The EMBO journal, 2000, 19(5): 921-930.] and monkeys [Diaz O L, Daubenberger C A, Rodriguez R, et al. Immunoglobulin kappa light-chain V, J, and C gene sequences of the owl monkey Aotus nancymaae[J]. Immunogenetics, 2000, 51(3): 212-218.]. This is the most direct method for obtaining sequences, but it is only applicable to species homologous to humans and requires the design of multiple paired primers, which is time-consuming.

Recently, applying bioinformatics methods to assemble species genomes from reference sequences has become an important area of research. However, these bioinformatics strategies rely on known species genomes and germline sequences. Accurately correcting the assembly of highly repetitive regions within a species' germline region is challenging, hindering germline inference. Furthermore, no software or tools exist to date for inferring germline sequences.

Summary of the Invention

The present invention aims to solve at least one of the above problems or to provide a commercial alternative. To this end, the inventors provide a de novo method to infer the V/J germline sequence, that is, to infer the V/J gene sequence before rearrangement.

According to one aspect of the present invention, the present invention provides a method for determining the V and/or J gene sequence before rearrangement, the method comprising: (1) obtaining sequencing data of an RNA sample to be tested, the sequencing data comprising a plurality of reads from the variable region of TCR, BCR and/or Ig, the length of the read being L, L ≥ 100 bp; (2) based on the sequencing data, determining the portion of the read from the V gene segment and/or the J gene segment according to the arrangement relationship between the V gene segment and the J gene segment and the C gene segment in the variable region, and obtaining a plurality of V region portions and/or a plurality of J region portions; (3) extracting at least one sequence from each of the V region portions and/or the J region portions as a seed sequence, and obtaining a sequence comprising a plurality of seed sequences. (4) clustering the V region portion and/or the J region portion according to the difference in the number of supports of the V region portion and/or the J region portion of each seed sequence in the seed sequence set to obtain a plurality of V region portion clusters and/or a plurality of J region portion clusters; (5) extending the seed sequence supported by each V region portion cluster and/or the J region portion cluster to obtain a plurality of candidate pre-rearrangement V gene sequences and/or a plurality of candidate pre-rearrangement J gene sequences; (6) filtering the support of the candidate pre-rearrangement V gene sequence and/or the candidate pre-rearrangement J gene sequence using the read segments in the sequencing data to obtain the V and/or J gene sequence before the rearrangement.

According to another aspect of the present invention, a computer-readable medium is provided for storing a computer-executable program. Execution of the program includes performing the method for determining the pre-rearrangement V and/or J gene sequence according to one aspect of the present invention. Those skilled in the art will appreciate that, when executing the computer-executable program, the associated hardware can perform all or part of the steps of the above-described method by instructing the hardware. The storage medium may include a read-only memory, a random access memory, a magnetic disk, or an optical disk.

According to another aspect of the present invention, the present invention provides a device for determining the V and/or J gene sequence before rearrangement, the device comprising: a data input unit for inputting data; a data output unit for outputting data; a storage unit for storing data, comprising a computer executable program; a processor connected to the data input unit, the data output unit and the storage unit, for executing the computer executable program, wherein executing the program comprises completing the method for determining the V and/or J gene sequence before rearrangement according to the above-mentioned aspect of the present invention.

According to another aspect of the present invention, a system for determining V and/or J gene sequences before rearrangement is provided, the system comprising: a data acquisition device for acquiring sequencing data of an RNA sample to be tested, the sequencing data comprising multiple reads from the variable regions of TCR, BCR and/or Ig, the length of the reads being L, L ≥ 100 bp; a V/J region portion determination device for determining, based on the sequencing data and the arrangement relationship between the V gene segments and the J gene segments and the C gene segments in the variable regions, the portions of the reads from the V gene segments and/or the J gene segments, thereby obtaining multiple V region segments and/or multiple J region segments; and a seed sequence set acquisition device for extracting at least one sequence from each of the V region segments and/or the J region segments as a seed sequence, thereby obtaining a seed sequence set comprising multiple seed sequences. a set, wherein the length of the seed sequence is K; a V/J region partial cluster determining device, for clustering the V region parts and/or J region parts according to the difference in the number of supports of the V region parts and/or the J region parts of each seed sequence in the seed sequence set, to obtain multiple V region partial clusters and/or multiple J region partial clusters; a candidate pre-rearrangement V/J gene sequence acquiring device, for extending the seed sequence supported by each V region partial cluster and/or the J region partial cluster, to obtain multiple candidate pre-rearrangement V gene sequences and/or multiple candidate pre-rearrangement J gene sequences; a pre-rearrangement V/J gene sequence determining device, for filtering the support status of the candidate pre-rearrangement V gene sequence and/or the candidate pre-rearrangement J gene sequence using reads in the sequencing data, to obtain the V and/or J gene sequence before the rearrangement.

The methods, devices, and/or systems of the present invention, based on sequencing data obtained from high-throughput sequencing of immune repertoires, can accurately deduce V/J germline sequences using only information analysis methods. The methods of the present invention can determine germline sequences for many species where V/J germlines have not been identified, facilitating further research on species' T cell receptors, B cell receptors, or antibodies. Compared to traditional and currently available methods, the methods of the present invention significantly reduce difficulty, time, and cost.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and/or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments with reference to the following drawings, in which:

FIG1 is a schematic diagram of the steps of a method for determining the V and/or J gene sequence before rearrangement in one embodiment of the present invention.

FIG2 is a schematic diagram of the structure of an apparatus for determining the V and/or J gene sequence before rearrangement in one embodiment of the present invention.

FIG3 is a schematic diagram of the structure of a system for determining the V and/or J gene sequence before rearrangement in one embodiment of the present invention.

FIG4 is a flow chart of a method for determining V and/or J gene sequences before rearrangement in one embodiment of the present invention.

FIG5 is a schematic diagram showing the coverage of the combined TRB-J gene of three samples in the human JGermline gene region determined in one embodiment of the present invention.

FIG6 is a schematic diagram showing the coverage of the combined TRB-V gene of three samples in the human VGermline gene region determined in one embodiment of the present invention.

DETAILED DESCRIPTION

Embodiments of the present invention are described in detail below, examples of which 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 intended only to explain the present invention, and are not to be construed as limiting the present invention. It should be noted that the terms "first," "second," "first category," "second category," or "first portion" used herein are for convenience of description only and are not to be construed as indicating or implying relative importance, nor as implying a sequential order. In the description of the present invention, unless otherwise specified, "plurality" means two or more.

In this document, unless otherwise clearly specified or limited, terms such as "connected" and "connection" should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium, or it can be the internal connection of two elements.

As shown in FIG1 , a method for determining the V and/or J gene sequence before rearrangement is provided according to one embodiment of the present invention. The method comprises the following steps:

S10 obtains sequencing data of the RNA sample to be tested.

The obtained sequencing data of the RNA sample to be tested includes multiple reads from the variable regions of TCR, BCR and/or Ig, and the length of the reads is L, where L is ≥ 100 bp.

The so-called RNA sample is derived from RNA or free RNA in cells in which V and/or J gene rearrangement occurs, generally from specific immune cells, such as T lymphocytes and/or B lymphocytes.

The so-called sequencing data is obtained by preparing a sequencing library for the nucleic acid sequence of the RNA sample to be tested and sequencing the library on a machine. According to an embodiment of the present invention, obtaining the sequencing data includes: obtaining nucleic acid from the sample to be tested, preparing a sequencing library of the nucleic acid, and sequencing the sequencing library. The preparation method of the sequencing library is carried out according to the requirements of the selected sequencing method. The sequencing method can be selected from, but not limited to, Illumina's Hisq2000/2500 sequencing platform, Life Technologies' Ion Torrent platform, and a single-molecule sequencing platform based on the selected sequencing platform. The sequencing method can be selected from single-end sequencing or double-end sequencing. The data obtained off the machine are fragments read out, called reads.

According to one embodiment of the present invention, the sequencing data has been preprocessed. The preprocessing includes at least one of filtering out reads containing adapter sequences, trimming bases with a sequence quality score less than 10 at the ends of reads, and trimming adapter sequences at the ends of reads. This preprocessing improves the overall quality of the sequencing data, facilitating subsequent accurate analysis and estimation of V/J germline sequences.

According to one embodiment of the present invention, the sequencing data is obtained using paired-end sequencing, i.e., the sequencing data comprises multiple pairs of paired reads. The paired reads are then spliced into a spliced sequence using the overlapping portions between the reads. The following steps are then performed using the spliced sequence instead of the paired reads. This results in longer sequence fragments, which facilitate subsequent accurate analysis and estimation of the sequence before rearrangement.

S20 acquires a plurality of V region portions and/or a plurality of J region portions.

Based on the sequencing data, according to the arrangement relationship between the V gene segments and the J gene segments and the C gene segments in the variable region, the portion of the read segment from the V gene segment and/or the J gene segment is determined to obtain multiple V region portions and/or multiple J region portions.

According to one embodiment of the present invention, S20 includes: determining the portion of the read segment that is derived from the C gene segment, for example, by using local alignment to determine the portion of the read segment that is derived from the C gene segment; cutting off the portion of the read segment that is derived from the C gene segment to obtain a cut portion; extracting a sequence of no less than 60 bp from the 3' end to the 5' end of the cut portion to obtain the J region portion; and/or cutting off 40 bp from the 3' end to the 5' end of the cut portion to obtain the remaining portion as the multiple V region portions. This example preliminarily determines the V region portion and J region portion of the read segment that are derived from the V gene segment and the J gene segment based on the arrangement relationship between the variable region V gene segment and the constant region C gene segment in Ig or TRB, as well as the size of the target gene segment.

According to a preferred embodiment of the present invention, S20 further includes filtering out J region portions and/or V region portions less than 40 bp in length. In this way, based on the size of the target gene fragments, fragments not derived from the target gene or short target fragments are removed, facilitating subsequent simple and accurate data processing.

S30 obtains a seed sequence set.

At least one sequence is taken from each of the V region parts and/or the J region parts as a seed sequence to obtain a seed sequence set comprising a plurality of seed sequences, wherein the length of the seed sequence is K.

Considering that the length of the J region ranges from 40 to 60 bp, according to one embodiment of the present invention, K is set to no more than 40 bp. This facilitates the conversion of each V region portion or J region portion into multiple seed sequences.

According to one embodiment of the present invention, S30 includes: performing a sliding cut on each of the V region portions and/or the J region portions at a length of 1 bp to convert the V region portion and/or the J region portion into a seed sequence subset, wherein the seed sequence subset includes (L-K+1) seed sequences, and the plurality of seed sequence subsets constitute the seed sequence set. Thus, the V region portion or the J region portion is converted into a corresponding seed sequence subset, i.e., into a Kmer set. This conversion, on the one hand, ensures that there is an overlap of (K-1) bp between two Kmers with a 1 bp sliding distance. This overlap relationship does not need to be obtained through alignment, thus saving alignment time. On the other hand, each V region portion or the J region portion is equivalent to a Kmer group, and the linear directional relationship of this Kmer group is determined. This facilitates subsequent seed sequence-based extension and facilitates the inference of the V/J gene sequence before rearrangement.

S40: Obtain a plurality of V region partial clusters and/or a plurality of J region partial clusters.

According to the difference in the support number of the V region portion and/or the J region portion of each seed sequence in the seed sequence set, the V region portion and/or the J region portion are clustered to obtain multiple V region portion clusters and/or multiple J region portion clusters.

According to one embodiment of the present invention, S40 includes repeating the following (i) and (ii) until no seed sequences remain: (i) determining the seed sequence that receives the most support from the V region portions and/or J region portions, and grouping all V region portions and/or J region portions that support the seed sequence into one category, thereby obtaining a corresponding V region portion cluster and/or a corresponding J region portion cluster; and (ii) removing the seed sequence in (i) and all V region portions and/or J region portions that support the seed sequence. This cycle is repeated until no seed sequences remain.

S50 obtains a candidate pre-rearrangement V gene sequence and/or a candidate pre-rearrangement J gene sequence.

Each of the V region partial clusters and/or the J region partial clusters is used to extend the seed sequence supported by it to obtain multiple candidate pre-rearrangement V gene sequences and/or multiple candidate pre-rearrangement J gene sequences. The so-called extension is performed based on the overlapping relationship between the V region portions or J region portions. For example, the J region portions in the same J region partial cluster are aligned to the seed sequence supported by them, that is, these J region portions are mapped, and the extension is performed based on the overlapping relationship between the mapped J region partial sequences.

The term "aligned" is synonymous with matching. The specific alignment can be performed using known alignment software, such as SOAP, BWA, and TeraMap, and is not limited in this embodiment. During the alignment process, depending on the alignment parameter settings, a pair or sequence is allowed to have a maximum of n base mismatches. For example, if n is set to 1 or 2, if more than n base mismatches occur in the sequence, the sequence/pair is considered to be unable to be aligned to the reference sequence.

When the match is a perfect match, for example, when a certain site in the aligned sequence is identical to that site in the reference sequence, the sequence is said to be a sequence supporting the site.

Since both V and D genes have multiple copies, the random combination of each fragment is a variety of rearrangement modes, which is displayed as a variety of bases at the same position in the V/J region after positioning. When extending, it is necessary to set a trust condition to determine the base type at the position. According to one embodiment of the present invention, S50 includes: using the V region partial cluster and/or the J region partial cluster, extending the seed sequence supported by the V region partial cluster and/or the J region partial cluster to obtain a plurality of the candidate pre-rearrangement V gene sequences and/or a plurality of the candidate pre-rearrangement J gene sequences, including performing at least one of the following: (a) for the seed sequence supported by the J region partial cluster, using the J region partial cluster to extend the 3' end and/or 5' end of the seed sequence by one base, the following conditions need to be met at the same time: the number of J region parts supporting the base accounts for more than 3% of the total number of J region parts contained in the J region partial cluster, and the number of types of J region parts supporting the base accounts for more than 1% of the total number of J region parts contained in the J region partial cluster. The proportion of the total number of types included is greater than 5%; (b) For a seed sequence supported by a V region partial cluster, using the V region partial cluster to extend the 3' end of the seed sequence by one base must simultaneously meet the following conditions: the number of V region portions supporting the base accounts for greater than 3% of the total number of V region portions included in the V region partial cluster, and the number of types of V region portions supporting the base accounts for greater than 5% of the total number of types of V region portions included in the V region partial cluster; (c) For a seed sequence supported by a V region partial cluster, using the V region partial cluster to extend the 5' end of the seed sequence by one base must simultaneously meet the following conditions: the number of V region portions supporting the base is greater than 100, and the number of types of V region portions supporting the base is greater than 2. The type of J region portion supporting a base refers to the J region portion that has the same base at that position but not completely identical bases at other positions. The type of V region portion supporting a base refers to the V region portion that has the same base at that position but not completely identical bases at other positions.

To obtain a candidate pre-rearrangement V gene sequence, according to one embodiment of the present invention, S50 includes performing steps (b) and (c) above, and splicing the sequences obtained after steps (b) and (c) to obtain a candidate pre-rearrangement V gene sequence. This embodiment takes into account that after the V region gene is interrupted, the fragment lengths vary, which is more complex than the J region. Therefore, the left and right ends are extended separately and different filtering conditions are used to facilitate the acquisition of highly accurate candidate V gene sequences.

S60 filtering was used to obtain the V and/or J gene sequences before rearrangement.

The support status of the candidate pre-rearrangement V gene sequence and/or the candidate pre-rearrangement J gene sequence is filtered using the reads in the sequencing data to obtain the pre-rearrangement V and/or J gene sequence.

According to one embodiment of the present invention, before performing S60, candidate pre-rearrangement V gene sequences with a sequence similarity of not less than 95% are merged, and/or candidate pre-rearrangement J gene sequences with a sequence similarity of not less than 95% are merged. This avoids repeated analysis of the same data and reduces computational complexity.

According to one embodiment of the present invention, S60 includes performing the following (d) and/or (e): (d) starting from the first base at the 3' end of the candidate pre-rearrangement V gene sequence, a sequence of the length of the seed sequence is cut off in the 5' direction as the first fragment, starting from the P-th base at the 3' end of the candidate pre-rearrangement V gene sequence, a sequence of the length of the seed sequence is cut off in the 5' direction as the second fragment, and filtering the candidate pre-rearrangement V gene sequence based on the degree of difference between the read support number of the first fragment and the read support number of the second fragment; (e) starting from the first base at the 5' end of the candidate pre-rearrangement J gene sequence, a sequence of the length of the seed sequence is cut off in the 3' direction as the third fragment, starting from the P-th base at the 5' end of the candidate pre-rearrangement J gene sequence, a sequence of the length of the seed sequence is cut off in the 3' direction as the fourth fragment, and filtering the candidate pre-rearrangement J gene sequence based on the degree of difference between the read support number of the third fragment and the read support number of the fourth fragment.

According to one embodiment of the present invention, step (d) in S60 includes retaining candidate pre-rearrangement V gene sequences that simultaneously meet the following two conditions: the read support number of the second segment/the read support number of the first segment>1.5, and the read support number of the first segment/the average read support number of the first segment>5%; and/or step (e) in S60 includes retaining candidate pre-rearrangement J gene sequences that simultaneously meet the following two conditions: the read support number of the fourth segment/the read support number of the third segment>1.5, and the read support number of the third segment/the average read support number of the third segment>5%. The above embodiment screens candidate V/J gene sequences based on the obtained read support numbers, which helps to ultimately retain reliable pre-rearrangement sequences.

This method of the present invention accurately derives V/J germline sequences using only information analysis techniques. This method can determine germline sequences for many species where V/J germlines are not known, enabling further research on T cell receptors, B cell receptors, or antibodies in any species. Compared to traditional and currently available methods, the method of the present invention significantly reduces difficulty, time, and cost.

Those skilled in the art will appreciate that all or part of the steps of the above-mentioned method for determining the V and/or J gene sequence before rearrangement can be written into a program in a machine-readable language and stored in a storage medium. According to another embodiment of the present invention, a computer-readable medium is provided, which is used to store a computer-executable program, and executing the program includes completing the method for determining the V and/or J gene sequence before rearrangement in any of the above-mentioned embodiments. Those skilled in the art will appreciate that when executing the computer-executable program, all or part of the steps of any of the above-mentioned methods for determining the V and/or J gene sequence before rearrangement can be completed by instructing the relevant hardware. The so-called storage medium may include: read-only memory, random access memory, magnetic disk or optical disk, etc.

As shown in Figure 2, according to another embodiment of the present invention, a device for determining the V and/or J gene sequence before rearrangement is provided. The device 100 includes: a data input unit 110, for inputting data; a data output unit 120, for outputting data; a storage unit 130, for storing data, including a computer-executable program; a processor 140, connected to the data input unit 110, the data output unit 120 and the storage unit 130, for executing the computer-executable program, and executing the program includes completing the method for determining the V and/or J gene sequence before rearrangement in any of the above embodiments.

As shown in FIG3 , a system for determining the V and/or J gene sequence before rearrangement is provided according to another embodiment of the present invention. The system can be used to implement the method for determining the V and/or J gene sequence before rearrangement in any of the embodiments of the present invention described above. The system 1000 includes: a data acquisition device 1010 for acquiring sequencing data of an RNA sample to be tested, wherein the sequencing data includes multiple reads from the variable region of TCR and/or Ig, wherein the length of the read is L, and L ≥ 100bp; a V/J region portion determination device 1020 for determining the portion from the V gene segment and/or J gene segment on the read based on the sequencing data and the arrangement relationship between the V gene segment and the J gene segment and the C gene segment in the variable region, thereby obtaining multiple V region portions and/or multiple J region portions; a seed sequence set acquisition device 1030 for extracting at least one sequence from each of the V region portions and/or the J region portions as a seed sequence, thereby obtaining a seed sequence set comprising multiple seed sequences, wherein the length of the seed sequence is K; a V/J region portion The cluster determination device 1040 is used to cluster the V region portions and/or J region portions based on the difference in the number of supports for the V region portions and/or J region portions of each seed sequence in the seed sequence set to obtain multiple V region portion clusters and/or multiple J region portion clusters; the candidate pre-rearrangement V/J gene sequence acquisition device 1050 is used to extend the seed sequence supported by each V region portion cluster and/or the J region portion cluster to obtain multiple candidate pre-rearrangement V gene sequences and/or multiple candidate pre-rearrangement J gene sequences; the pre-rearrangement V/J gene sequence determination device 1060 is used to filter the support status of the candidate pre-rearrangement V gene sequence and/or the candidate pre-rearrangement J gene sequence using the reads in the sequencing data to obtain the pre-rearrangement V and/or J gene sequence. The above description of the technical features and advantages of the method for determining the pre-rearrangement V and/or J gene sequence of the present invention is also applicable to this system and will not be repeated here.

According to an embodiment of the present invention, the system of the present invention may further have at least one of the following additional technical features:

According to one embodiment of the present invention, the sequencing data in the data acquisition device 1010 is preprocessed sequencing data, and the preprocessing includes at least one of the following: filtering out reads containing adapter sequences, cutting off the end sequence portion of the end sequence of the read segment with a quality value less than 10, and cutting off the end sequence portion of the end sequence of the read segment containing the adapter sequence.

According to one embodiment of the present invention, the V/J region portion determination apparatus performs the following steps: determining the portion of the read segment originating from the C gene segment, cutting off the portion of the read segment originating from the C gene segment to obtain a cut portion, extracting a sequence of no less than 60 bp from the 3' end to the 5' end of the cut portion to obtain the J region portion; and/or cutting off 40 bp from the 3' end to the 5' end of the cut portion to obtain the remaining portion as the multiple V region portions. According to one embodiment of the present invention, the portion of the read segment originating from the C gene segment is determined using local alignment.

According to one embodiment of the present invention, the V/J region portion determination device is further used to filter out the J region portion with a length less than 40 bp and/or the V region portion with a length less than 40 bp.

Taking into account the length of the target sequence, according to one embodiment of the present invention, K is set to be no greater than 40 bp.

According to one embodiment of the present invention, the seed sequence set acquisition device is used to perform the following: sliding cutting is performed on each of the V region portions and/or the J region portions at a length of 1 bp to convert one of the V region portions and/or the J region portions into a seed sequence subset, wherein one of the seed sequence subsets includes (L-K+1) seed sequences, and multiple seed sequence subsets constitute the seed sequence set.

According to one embodiment of the present invention, the V/J region partial cluster determination device is used to repeatedly perform the following (i) and (ii) until no seed sequence remains: determining a seed sequence that obtains the largest number of V region portions and/or J region portions supporting it, grouping all V region portions and/or J region portions supporting the seed sequence into one category, and correspondingly obtaining a V region partial cluster and/or a J region partial cluster; (ii) removing the seed sequence in (i) and all V region portions and/or J region portions supporting the seed sequence.

According to one embodiment of the present invention, the candidate pre-rearrangement V/J gene sequence acquisition device is used to perform the following: using the V region partial cluster and/or the J region partial cluster, the seed sequence supported by the V region partial cluster and/or the J region partial cluster is extended to obtain multiple candidate pre-rearrangement V gene sequences and/or multiple candidate pre-rearrangement J gene sequences, including performing at least one of the following: (a) for the seed sequence supported by the J region partial cluster, using the J region partial cluster to extend the 3' end and/or 5' end of the seed sequence by one base, which needs to meet the following conditions at the same time: the proportion of the number of J region parts supporting the base to the total number of J region parts contained in the J region partial cluster is greater than 3%, and the type of the J region part supporting the base is greater than 1%. (b) for a seed sequence supported by a V region partial cluster, extending the 3' end of the seed sequence by one base using the V region partial cluster must simultaneously meet the following conditions: the number of V region portions supporting the base must simultaneously meet the following conditions: the number of V region portions supporting the base must simultaneously meet the following conditions: the number of V region portions supporting the base must simultaneously meet the following conditions: the number of V region portions supporting the base must simultaneously meet the following conditions: the number of V region portions supporting the base must simultaneously meet the following conditions: the number of V region portions supporting the base must simultaneously meet the following conditions: the number of V region portions supporting the base must simultaneously meet the following conditions: the number of V region portions supporting the base must simultaneously meet the following conditions: the number of V region portions supporting the base must simultaneously meet the following conditions: the number of V region portions supporting the base must simultaneously meet the following conditions: the number of V region portions supporting the base must simultaneously meet the following conditions: the number of V region portions supporting the base must simultaneously meet the following conditions: the number of V region portions supporting the base must simultaneously meet the following conditions: the number of V region portions supporting the base must simultaneously meet the following conditions: the number of V region portions supporting the base must simultaneously meet the following conditions: the number of V region portions supporting the base must simultaneously meet the following conditions:

According to one embodiment of the present invention, before obtaining the V and/or J gene sequence before rearrangement using the pre-rearrangement V/J gene sequence determination device, the candidate pre-rearrangement V gene sequences with a sequence similarity of not less than 95% are merged, and/or the candidate pre-rearrangement J gene sequences with a sequence similarity of not less than 95% are merged.

According to one embodiment of the present invention, the apparatus for determining the pre-rearrangement V/J gene sequence is used to perform the following (d) and/or (e): (d) starting from the first base at the 3' end of the candidate pre-rearrangement V gene sequence, a sequence of the length of the seed sequence is intercepted in the 5' direction as the first fragment, starting from the P-th base at the 3' end of the candidate pre-rearrangement V gene sequence, a sequence of the length of the seed sequence is intercepted in the 5' direction as the second fragment, and based on the difference in the read support number of the first fragment and the read support number of the second fragment, (e) starting from the first base at the 5' end of the candidate J gene sequence before rearrangement, a sequence with the length of the seed sequence is intercepted in the 3' direction as the third segment, starting from the P'th base at the 5' end of the candidate J gene sequence before rearrangement, a sequence with the length of the seed sequence is intercepted in the 3' direction as the fourth segment, and filtering the candidate J gene sequence before rearrangement based on the difference between the read support number of the third segment and the read support number of the fourth segment.

According to one embodiment of the present invention, the device for determining the V/J gene sequence before rearrangement is used to perform (d) including retaining candidate pre-rearrangement V gene sequences that simultaneously meet the following two conditions: the read support number of the second fragment/the read support number of the first fragment>1.5, and the read support number of the first fragment/the average read support number of the first fragment>5%, and/or the device for determining the V/J gene sequence before rearrangement is used to perform (e) including retaining candidate pre-rearrangement J gene sequences that simultaneously meet the following two conditions: the read support number of the fourth fragment/the read support number of the third fragment>1.5, and the read support number of the third fragment/the average read support number of the third fragment>5%.

To further clarify the technical solutions and advantages of the present invention, the following detailed description of the method, apparatus, and/or system for determining the pre-rearrangement V and/or J gene sequence of the present invention is provided in conjunction with specific examples. It should be understood that the following examples are provided to illustrate the present invention and are not intended to limit the present invention.

Unless otherwise stated, the reagents, sequences (linkers, tags, and primers), software, and instruments not specifically stated in the following examples are all conventional commercially available products or open source, such as Illumina sequencing library construction kits.

Example 1

The general method comprises the following steps:

For RNA samples, a set of universal primers optimized by the inventors can be used to amplify the variable regions of TCR, BCR or Ig via 5’race:

The variable region is formed by the rearrangement of the V, D, and J gene segments of a TCR or Ig. During the rearrangement process, nucleotide insertions and deletions occur at the junctions between the gene segments. This region reflects the diversity of surface receptors in adaptive immune molecules. The C region is the constant region. Primers can be designed within the C region to amplify the variable region. The 5' race method can then be used to amplify the variable region resulting from the rearrangement of the V and J regions of different subfamilies.

(2) Library preparation

Step 1: Synthesize one strand of cDNA using the reverse transcription primer in the C region and Superscript II. Then, digest the RNA in the cDNA with RNasemix, add C to the 5' end, and finally amplify by PCR using the Abridged Anchor primer in the 5'race kit and the biotin-labeled C region primer.

In step 2, the amplified product was fragmented to about 250 bp, and the biotinylated DNA was enriched using Dynabeads M-270 streptavidin magnetic beads. The DNA was then digested with the restriction endonuclease PacⅠ and collected.

Step three: Library construction: DNA is end-repaired using enzymes such as T4 DNA Polymerase, Klenow Fragment, and T4 Polynucleotide Kinase with dNTP as the substrate to form blunt-ended phosphorylated DNA fragments. If the subsequent TA sticky end connection is to be performed, Klenow Fragment (3’-5’exo-) polymerase and dATP can be used to add an “A” base to the 3’ end of the blunt-ended sequence. It is then connected to the adapter under the action of T4 DNA Ligase. In order to facilitate the mixed sequencing of RNA libraries prepared from different samples and to distinguish them after sequencing, a label sequence can be introduced into the adapter to distinguish libraries prepared from different samples. If it is necessary to enrich the fragments connected to the adapter, a PCR step with a common primer can be added.

The sequencing library was purified by magnetic beads throughout the process, and the library was detected by Agilent 2100 and quantified by Q-PCR.

(3) High-throughput sequencing

The prepared library is sequenced on a high-throughput sequencing platform. The high-throughput sequencing platform can be selected from at least one of the Illumina Hiseq and Miseq sequencing platforms, the Roche 454 sequencing platform, and the Life Technologies SOLiD and IonTorrent sequencing platforms.

(4) Data Analysis

As shown in Figure 4, the main steps include:

Step 1: Preliminary data processing

Data filtering: Check the sequence for contamination by sequencing adapters. If adapter sequences are present and located at the end (last 50 bp), the contaminating portion is trimmed. Otherwise, the entire sequence is filtered out. Bases with low sequencing quality (<Q10) are trimmed at the end of the sequence. Read concatenation: For paired-end sequencing, two reads are concatenated through the overlapping region to form a single sequence. The concatenation requires that the overlap region be >10 bp in length and that the mismatch ratio be <=10%.

Step 2: Identify the C region and divide the sequence into V and J parts

1) Identify the constant region (C region): Perform a local alignment (e.g., BLAST) of the filtered sequence against a reference sequence for the C region. Determine the C region through alignment, cut off the C region, and convert the antisense strand to the sense strand.

2) Extract the V/J parts separately: Because the D region is short and insertions/deletions make it impossible to determine the junction between the J region and the D region, and because the length of the J region ranges from 40 to 60 bp, a certain read length (such as 70 bp) is extracted from the start of the C region to the J region as the J region; similarly, 40 bp is cut off from the start of the C region toward the 5' end, and the remaining sequence is the V region.

Step 3: Seed-based clustering

For the V and J regions, separate clustering is performed. Sequences of a certain length (e.g., 40 bp) are used as seeds. Sequences are read, and the sequence support count for each seed is recorded. The seed with the largest support count is selected first, and all sequences supporting this seed are output as one class. The remaining seeds and their sequence support counts are recounted, and the largest seed is selected and its supported sequences are output as another class. The remaining sequences are recounted again, and the largest class is output. This cycle repeats until the remaining sequences are zero.

Step 4: Seed extension

J-region seed extension: For each type of sequence, the sequence is extended one base to the left and right according to the seed. During each extension, if the following conditions are met simultaneously: (1) the number of sequence supports accounts for >3% of the sequence type, and (2) the number of sequence supports accounts for >5% of the sequence type; then the extension continues. If a branch occurs during the extension (i.e., multiple bases meet the same conditions at the same position), multiple sequences are generated based on the branch. When the extension stops, the extended sequence is considered a candidate germline.

V region seed extension: For all seed cluster subsets of the V region, since the fragment lengths are different after the V region is interrupted, the situation is more complicated than that of the J region. We extend the left and right ends separately with different filtering conditions. When extending to the 3' end, the retention conditions are similar to those of the J region; but when extending to the 5' end, the filtering conditions are: (1) sequence support number > 100, (2) sequence support number type > 2; finally, the two extended parts are spliced together.

Step 5: Merge candidate germlines

After each seed cluster is extended, duplicate germlines may appear between different subsets. The merging process removes duplicate sequences from candidate germlines. Candidate germlines are then compared pairwise. If the similarity exceeds 95%, the two sequences are merged into a single sequence.

Step 6: Filter

At the 3' end of the candidate V germline or the 5' end of the candidate J germline, 40 bp forward from the end was taken as fragment 1, and 40 bp forward from the 5th base from the end was taken as fragment 2. Fragments 1 and 2 were searched in the original data set (after preliminary data processing) and their sequence support numbers were counted. If the following conditions were met at the same time: (1) the sequence support number of fragment 2 / the sequence support number of fragment 1 > 1.5; (2) the sequence support number of fragment 1 / the average support number of fragment 1 > 5%, the sequence was retained; otherwise, it was filtered out.

Example 2

(1) Experimental process

(1) 5’ RACE enrichment of target fragments

Peripheral blood was drawn from three healthy individuals, and peripheral blood mononuclear cells (PBMCs) were isolated and RNA was extracted. Three RNA samples were obtained, designated Sample 1 (HRB), Sample 2 (HXY), and Sample 3 (XHS). The RNA was reverse transcribed into cDNA using primers specific for the TCR constant region C. The following system uses the reaction number for one sample as an example.

1.1 cDNA 1st synthesis

1) Prepare according to the following system (1 sample)

TCRC region primer: TTGATGGCTCAAACACAGCGA (SEQ ID NO: 1)

2) Incubate at 70°C for 10 min, place on ice for 1 min, add the following system, and incubate at 42°C for 1 min.

3) Add 1 μL Superscript II and react at 42°C for 50 min and then at 70°C for 15 min.

4) Add 1 μL of RNase mix and incubate at 37°C for 30 min.

1. Purify cDNA using magnetic beads 21.5 times and redissolve in 18ul nuclease-free water.

1.3TdT Tailing cDNA

1) Prepare according to the following system

2) Incubate at 94°C for 2-3 minutes and cool on ice for 1 minute.

3) Add 1 μL of TdT, mix well, and incubate at 37°C for 10 min and then at 65°C for 10 min.

1.4 PCR of dC-tailed cDNA

1) Prepare according to the following system

2) Place in a PCR instrument and react according to the following procedure.

a. 94℃ 2min

b.94℃ 15s

c.60℃ 30s

d.72℃ 30s

e. Repeat steps b-d 29 times (30 cycles in total)

f.72℃ 5min

g.12℃ Hold

3) Purify with 1x magnetic beads and re-dissolve in 20 μL nuclease-free water.

(2) Covaris interruption sample

Take out 3 μL of sample for electrophoresis to detect the disruption effect.

(3) Washing and elution of interrupted sequences

Open the water bath in advance and adjust the temperature to 47°C and equilibrate it to heat the Washing Buffer.

3.1 Prepare washing solution

Prepare various wash buffer reagents in advance and prepare two wash buffer reagents (1×Binding and Wash Buffer, 2×Binding and Wash Buffer) according to the proportion.

3.2 Preparation of Streptavidin Magnetic Beads M-270

3.3 Bind the fragmented DNA to streptavidin magnetic beads and wash

(4) Restriction enzyme endonuclease

1) Prepare according to the following system

2) Place the tube in a PCR instrument and follow the following reaction schedule. Place the tube on a magnetic rack and aspirate the supernatant, which is the desired product.

a.37℃ 2h

b.65℃ 20min

(5) Sequencing adapters are introduced by ligase and sequencing libraries are prepared according to the standard library preparation process developed by each sequencing platform.

(6) Library detection

The size and content of the library inserts were detected by Bioanalyzer analysis system (Agilent, Santa Clara, USA); the concentration of the library was accurately quantified by Q-PCR.

(7) Sequencing

After the library is qualified, it is sequenced on the corresponding sequencing platform and sequenced on the HiSeq2000 sequencer according to the double-end read length of 151 bases.

(2) Data Analysis

1. Data Preprocessing

Data filtering: Check the sequence for contamination by sequencing adapters. If there is a contamination adapter at the end (last 50 bp), the contamination is removed. Otherwise, the entire sequence is filtered out. Bases with low sequencing quality (<Q10) at the end of the sequence are removed.

Splicing reads: For paired-end sequencing, two reads are spliced together through the overlapping portion in the middle to form a single sequence. (Overlapping region, length > 10bp, mismatch <= 10%)

According to the filtering conditions, the filtering results of the three samples are as follows: Sample 1 (HRB) filtered out 14,695,238 sequences, with a filtering rate of 97.97%; Sample 2 (HXY) filtered out 17,459,894 sequences, with a data filtering rate of 98.14%; Sample 3 (XHS) filtered out 16,515,129 sequences, with a filtering rate of 96.01%.

2. Identify the C region and divide the sequence into V and J parts

Identify the constant region (C region): Perform a local alignment (e.g., BLAST) of the filtered sequence against a reference sequence for the C region. Determine the C region by alignment, cut off the C region, and convert the antisense strand to the sense strand.

Separate extraction of the V and J regions: Because the D region is short and insertions/deletions prevent the J-D region junction from being identified, and because the J region ranges from 40 to 60 bp in length, a 70-bp segment extending from the start of the C region toward the J region is extracted as the J region. Similarly, 40 bp from the start of the C region toward the 5' end is trimmed, and the remaining sequence is considered the V region. V and J sequences shorter than 40 bp are filtered out. Table 1 shows the number and proportion of successfully extracted V and J region sequences from the three samples.

Table 1

3. Seed-based clustering and extension

Sequence Clustering

Cluster the V and J regions separately, taking a 40-bp seed and reading the sequence. Record the sequence support for each seed. First, select the seed with the highest support, and output all sequences supporting it as one cluster. Then, recount the seed and its sequence support for the remaining sequences, select the seed with the highest support, and output the sequences it supports as another cluster. Recount the remaining sequences again, outputting the highest cluster, and so on. Repeat this cycle until the remaining sequences are zero.

J zone seed extension

For each type of sequence, the sequence is extended one base to the left and right based on the seed. Each time the extension is performed, if the following conditions are met simultaneously: (1) the number of sequence supports accounts for >3% of the sequence type, and (2) the number of sequence types supports accounts for >5% of the sequence types; the extension is continued. If a branch occurs during the extension (i.e., multiple bases meet the same conditions at the same position), multiple sequences are generated based on the branch. When the extension is finally stopped, the extended sequence is considered a candidate germline.

V zone seed extension

For all seed cluster subsets in the V region, since the fragment lengths are different after the V region is interrupted, the situation is more complicated than that of the J region. We extend the left and right ends separately with different filtering conditions. When extending to the 3' end, the retention conditions are similar to those of the J region; but when extending to the 5' end, the filtering conditions are: (1) sequence support number > 100, (2) sequence support number type > 2; finally, the two extended parts are spliced together.

4. Merge candidate germlines

After each seed cluster is extended, duplicate germlines may appear between different subsets. The merging process removes duplicate sequences from candidate germlines. Candidate germlines are then compared pairwise. If the similarity exceeds 95%, the two sequences are merged into a single sequence.

5. Filter and get reference Germline

At the 3' end of the candidate V germline or the 5' end of the candidate J germline, 40 bp forward from the end was taken as fragment 1, and 40 bp forward from the 5th base from the end was taken as fragment 2. Fragments 1 and 2 were searched in the original data set (after preliminary data processing) and their sequence support numbers were counted. If the following conditions were met at the same time: (1) Fragment 2 sequence support number / Fragment 1 sequence support number > 1.5; (2) Fragment 1 sequence support number / average Fragment 1 support number > 5%, the sequence was retained; otherwise, it was filtered out.

After analysis, we identified 11 candidate germline lines for all three samples of TRB-J. For the TRB-V germline, we deduced 34 lines for sample 1, 30 for sample 2, and 36 for sample 3. The following section analyzes the accuracy and coverage of the germline results.

6. Verify the credibility of Germline

6.1 Collecting TRB-J Germline Alignment Information

Table 2 shows the predicted TRB-J genes of the germline of the three samples and their matching with the known human TRB-J genes.

Because there are many V/J genes and their diversity in genes encoding immune cell receptor proteins, the similarity in the table refers to the comparison of a gene segment of TRB-V and TRB-J predicted by the present method with a currently known human V/J gene. A similarity of 100% indicates a 100% match between the V/J genes.

Table 2

6.2 Predicted Germline TRB-J (TRB J gene sequence before rearrangement) Distribution

Figure 5 shows the combined TRB-J gene coverage of the human germline region for the three samples. As shown in Figure 5, after the statistical analysis above, the number of TRB-J region genes in samples 1-3 was 11, with an average length of 50 bp. The overall similarity was >= 90%, base deletions <= 5 bp, base insertions <= 5 bp, and mismatch rate <= 2. The inferred coverage distribution of each J gene shows complete coverage of the entire TRB-J region, demonstrating that this method has high predictive and accuracy for both the number and accuracy of TRB-J region genes and can be used for inferring J region genes.

6.3 Statistics of TRB-V Germline Alignment Information

Table 3 below shows the alignment of the V Germline sequences deduced from the three samples with the known human TRB-V Germline.

Table 3

6.4 Predicted Germline TRB-V Distribution

Figure 6 shows the coverage of the TRB-V gene in the human germline gene region of the three samples combined.

The statistical analysis results in Table 2 above show that the number of TRB-V region genes imputed for samples 1-3 was 34, 30, and 36, respectively; the overall similarity was >= 90%, the base deletions were <= 5 bp, the base insertions were <= 5 bp, and the mismatch rate was <= 3. The coverage distribution of individual genes in the inferred V region in Figure 6 shows that the coverage of the entire TRB-V region exceeds 80%. Three genes were not included in the inferred germline, and the results were consistent with the TRB-J region genes, but the overall coverage was slightly lower.

Throughout this specification, references to terms such as "one embodiment," "some embodiments," "examples," "specific examples," "implementation," or "some examples" indicate that the specific features, structures, materials, or characteristics described in conjunction with that embodiment or example are included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to the embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the claims and their equivalents.

Claims (27)

1. A method for determining the V and/or J gene sequences before rearrangement, characterized by comprising the following steps: (1) Obtain sequencing data of the RNA sample to be tested, wherein the sequencing data includes multiple reads from the variable regions of TCR, BCR and/or Ig, wherein the length of the reads is L, and L≥100bp; (2) Based on the sequencing data, according to the arrangement relationship between the V gene fragment, the J gene fragment and the C gene fragment in the variable region, determine the part of the read from the V gene fragment and/or the J gene fragment, and obtain multiple V region parts and/or multiple J region parts; (3) Take at least one sequence from each of the V region portions and/or the J region portions as a seed sequence to obtain a seed sequence set containing multiple seed sequences, wherein the length of the seed sequence is K; (4) Based on the difference in the number of support for each seed sequence in the seed sequence set, the V region and/or J region are clustered to obtain multiple V region clusters and/or multiple J region clusters. (5) Extend the seed sequence supported by each V region partial cluster and/or J region partial cluster to obtain multiple candidate pre-rearrangement V gene sequences and/or multiple candidate pre-rearrangement J gene sequences. (6) Filter the reads in the sequencing data to determine the support for the candidate pre-rearrangement V gene sequence and/or the candidate pre-rearrangement J gene sequence, so as to obtain the pre-rearrangement V and/or J gene sequences. 2. The method as described in claim 1, wherein the length K of the seed sequence is no greater than 40 bp. 3. The method as described in claim 1, wherein the sequencing data is preprocessed sequencing data, and the preprocessing includes at least one of the following: filtering out reads containing adapter sequences, removing bases with a quality value of less than 10 from the end sequences of reads, and removing adapter sequences from the ends of reads. 4. The method as described in claim 1, wherein step (2) comprises: Identify the portion of the read sequence originating from the C gene fragment, and cut off this portion to obtain the cut portion. Extract a sequence of at least 60 bp from the 3' end to the 5' end of the cut portion to obtain the J region portion, and/or Cut off 40bp from the 3' end to the 5' end of the cut portion, and the remaining portion is the plurality of V-region portions. 5. The method of claim 4, wherein the portion of the read from the C gene fragment is determined by local alignment. 6. The method as described in claim 4, wherein step (2) further comprises: filtering out the J region portion with a length less than 40 bp and/or the V region portion with a length less than 40 bp. 7. The method as described in claim 1, wherein step (3) comprises: Each V region portion and/or J region portion is slidably cut by a length of 1 bp to transform one V region portion and/or J region portion into a seed sequence subset. A seed sequence subset includes (L-K+1) seed sequences, and multiple seed sequence subsets constitute the seed sequence set. 8. The method as claimed in claim 1, wherein step (4) comprises repeating steps (i) and (ii) until no seed sequence remains: (i) Identify the seed sequence that receives the most support from the V region and/or J region portions, and group all V region portions and/or J region portions that support the seed sequence into one class, thereby obtaining a corresponding V region portion cluster and/or a J region portion cluster. (ii) Remove the seed sequence from (i) and all V region portions and/or J region portions that support the seed sequence. 9. The method as described in claim 1, wherein step (5) comprises: Extending the seed sequences supported by the partial clusters of region V and/or region J to obtain multiple candidate pre-rearrangement V gene sequences and/or multiple candidate pre-rearrangement J gene sequences, including performing at least one of the following steps: (a) For a seed sequence supported by a partial cluster in region J, extending the 3' end and/or 5' end of the seed sequence by one base using this partial cluster in region J requires the following conditions to be met simultaneously: The number of J-regions supporting this base accounts for more than 3% of the total number of J-regions contained in this J-region cluster. The number of species supporting this base in the J region accounts for more than 5% of the total number of species contained in the J region cluster. (b) For a seed sequence supported by a partial cluster in region V, extending the 3' end of the seed sequence by one base using this partial cluster in region V requires the following conditions to be met simultaneously: The number of V-regions supporting this base accounts for more than 3% of the total number of V-regions contained in this V-region cluster. The number of species supporting this base in the V region accounts for more than 5% of the total number of species in the V region of the V region cluster. (c) For a seed sequence supported by a partial cluster in region V, extending the 5' end of the seed sequence by one base using this partial cluster in region V requires the following conditions to be met simultaneously: The number of V-regions supporting this base is greater than 100. The number of types of V region regions supporting this base is greater than 2; if steps (b) and (c) are performed simultaneously, the sequences obtained after steps (b) and (c) are spliced together. 10. The method as described in claim 1, characterized in that, before performing step (6), candidate pre-rearrangement V gene sequences with a sequence similarity of not less than 95% are merged, and/or Candidate J gene sequences with a sequence similarity of not less than 95% were merged before rearrangement. 11. The method as claimed in claim 1, wherein step (6) comprises performing step (d) and/or step (e): (d) Starting from the first base at the 3' end of the candidate pre-rearranged V gene sequence, extract a sequence of the seed sequence length towards the 5' end as the first fragment. Starting from the P-th base at the 3' end of the candidate pre-rearranged V gene sequence, a sequence of the seed sequence length is truncated towards the 5' end as the second fragment. Based on the degree of difference between the read support number of the first fragment and the read support number of the second fragment, the candidate pre-rearrangement V gene sequences are filtered. (e) Starting from the first base at the 5' end of the candidate pre-rearranged J gene sequence, a sequence of the seed sequence length is truncated towards the 3' end as the third segment. Starting from the P' base at the 5' end of the candidate rearranged J gene sequence, a sequence of the seed sequence length is truncated towards the 3' end as the fourth segment. Based on the degree of difference between the read support number of the third fragment and the read support number of the fourth fragment, the candidate pre-rearrangement J gene sequences are filtered. 12. The method of claim 11, wherein step (d) in step (6) includes retaining candidate pre-rearrangement V gene sequences that simultaneously satisfy the following two conditions: The ratio of the number of read segments supporting the second segment to the number of read segments supporting the first segment is greater than 1.5. The ratio of the number of read segments supported in the first segment to the average number of read segments supported in the first segment is >5%; and/or Step (e) in step (6) includes retaining candidate pre-rearrangement J gene sequences that simultaneously meet the following two conditions: The ratio of the number of read segments supporting the fourth segment to the number of read segments supporting the third segment is greater than 1.5. The ratio of the number of reads supporting the third segment to the average number of reads supporting the third segment is greater than 5%. 13. The method according to any one of claims 1-12, wherein the sequencing data comprises multiple pairs of paired reads, and a pair of paired reads is spliced into a spliced sequence by utilizing the overlapping portion between the reads, and the spliced sequence replaces the paired reads. 14. An apparatus for determining the V and/or J gene sequences before rearrangement, characterized in that it comprises: The data input unit is used to input data. The data output unit is used to output data. Storage unit, used to store data, including computer-executable programs; A processor, connected to the data input unit, the data output unit, and the storage unit, is configured to execute the computer-executable program, wherein executing the program includes performing any one of the methods of claims 1-13. 15. A system for determining the V and/or J gene sequences before rearrangement, characterized in that it comprises: A data acquisition device is used to acquire sequencing data of a RNA sample to be tested. The sequencing data includes multiple reads from the variable regions of the TCR, BCR and/or Ig, and the length of each read is L, where L ≥ 100 bp. V/J region partial determination device, used to determine the portion of the read from the V gene fragment and/or the J gene fragment based on the sequencing data and according to the arrangement relationship between the V gene fragment and the J gene fragment and the C gene fragment in the variable region, to obtain multiple V region portions and/or multiple J region portions; A seed sequence set acquisition device is used to extract at least one sequence from each of the V region portions and/or the J region portions as a seed sequence to obtain a seed sequence set containing multiple seed sequences, wherein the length of the seed sequence is K; V/J region partial cluster determination device, used to cluster the V region and/or J region based on the difference in the number of support for each seed sequence in the seed sequence set, to obtain multiple V region partial clusters and/or multiple J region partial clusters; A candidate pre-rearrangement V/J gene sequence acquisition device is used to extend the seed sequence supported by each V region partial cluster and/or J region partial cluster to obtain multiple candidate pre-rearrangement V gene sequences and/or multiple candidate pre-rearrangement J gene sequences. A device for determining the pre-rearrangement V/J gene sequence is used to filter the support of the candidate pre-rearrangement V gene sequence and/or the candidate pre-rearrangement J gene sequence using reads in the sequencing data, so as to obtain the pre-rearrangement V and/or J gene sequences. 16. The system of claim 15, wherein the sequencing data is preprocessed sequencing data, the preprocessing comprising at least one of the following: filtering out reads containing adapter sequences, removing bases with a quality value of less than 10 at the end of the reads, and removing adapter sequences at the ends of the reads. 17. The system as claimed in claim 15, characterized in that the V/J region partial determination device performs the following: Identify the portion of the read from the C gene fragment. The portion of the read segment originating from the C gene fragment is cut off to obtain the cut portion. Extract a sequence of at least 60 bp from the 3' end to the 5' end of the cut portion to obtain the J region portion; and/or Cut off 40bp from the 3' end to the 5' end of the cut portion, and the remaining portion is the plurality of V-region portions. 18. The system of claim 17, wherein a portion of the read from the C gene fragment is determined by local alignment. 19. The system of claim 17, characterized in that the V/J region portion determination device is further used to: filter out the J region portion with a length less than 40 bp and/or the V region portion with a length less than 40 bp. 20. The system as described in claim 15, wherein K is no greater than 40bp. 21. The system as claimed in claim 15, characterized in that the seed sequence set acquisition device performs the following: Each V region portion and/or J region portion is slidably cut by a length of 1 bp to transform one V region portion and/or J region portion into a seed sequence subset. A seed sequence subset includes (L-K+1) seed sequences, and multiple seed sequence subsets constitute the seed sequence set. 22. The system of claim 15, characterized in that the steps (i) and (ii) are repeated using the V/J region partial cluster determination device until no seed sequence remains: (i) Identify the seed sequence that receives the most support from the V region and/or J region portions, and group all V region portions and/or J region portions that support the seed sequence into one class, thereby obtaining a corresponding V region portion cluster and/or a J region portion cluster. (ii) Remove the seed sequence from (i) and all V region portions and/or J region portions that support the seed sequence. 23. The system as described in claim 15, characterized in that the following is performed using the candidate rearrangement pre-V/J gene sequence acquisition device: Extending the seed sequences supported by the partial clusters of region V and/or region J to obtain multiple candidate pre-rearrangement V gene sequences and/or multiple candidate pre-rearrangement J gene sequences, including performing at least one of the following steps: (a) For a seed sequence supported by a partial cluster in region J, extending the 3' end and/or 5' end of the seed sequence by one base using this partial cluster in region J requires the following conditions to be met simultaneously: The number of J-regions supporting this base accounts for more than 3% of the total number of J-regions contained in this J-region cluster. The number of species supporting this base in the J region accounts for more than 5% of the total number of species contained in the J region cluster. (b) For a seed sequence supported by a partial cluster in region V, extending the 3' end of the seed sequence by one base using this partial cluster in region V requires the following conditions to be met simultaneously: The number of V-regions supporting this base accounts for more than 3% of the total number of V-regions contained in this V-region cluster. The number of species supporting this base in the V region accounts for more than 5% of the total number of species in the V region of the V region cluster. (c) For a seed sequence supported by a partial cluster in region V, extending the 5' end of the seed sequence by one base using this partial cluster in region V requires the following conditions to be met simultaneously: The number of V-regions supporting this base is greater than 100. The number of species supporting the V region of this base is greater than 2; If steps (b) and (c) are performed simultaneously using the partial clusters of region V and/or the partial clusters of region J, the sequences obtained after performing steps (b) and (c) are spliced together. 24. The system as claimed in claim 15, characterized in that, before obtaining the pre-rearrangement V and/or J gene sequences using the pre-rearrangement V/J gene sequence determination device, candidate pre-rearrangement V gene sequences with a sequence similarity of not less than 95% are merged, and/or Candidate J gene sequences with a sequence similarity of not less than 95% were merged before rearrangement. 25. The system as claimed in claim 15, characterized in that the following steps (d) and/or (e) are performed using the pre-rearrangement V/J gene sequence determination device. (d) Starting from the first base at the 3' end of the candidate pre-rearranged V gene sequence, extract a sequence of the seed sequence length towards the 5' end as the first fragment. Starting from the P-th base at the 3' end of the candidate pre-rearranged V gene sequence, a sequence of the seed sequence length is truncated towards the 5' end as the second fragment. Based on the degree of difference between the read support counts of the first fragment and the read support counts of the second fragment, the candidate pre-rearrangement V gene sequences are filtered. (e) Starting from the first base at the 5' end of the candidate pre-rearranged J gene sequence, a sequence of the seed sequence length is truncated towards the 3' end as the third segment. Starting from the P' base at the 5' end of the candidate rearranged J gene sequence, a sequence of the seed sequence length is truncated towards the 3' end as the fourth segment. Based on the degree of difference between the read support number of the third fragment and the read support number of the fourth fragment, the candidate pre-rearrangement J gene sequences are filtered. 26. The system of claim 25, wherein step (d) using the pre-rearrangement V/J gene sequence determination device includes retaining candidate pre-rearrangement V gene sequences that simultaneously satisfy the following two conditions: The ratio of the number of read segments supporting the second segment to the number of read segments supporting the first segment is greater than 1.5. The ratio of the number of read segments supporting the first segment to the average number of read segments supporting the first segment is >5%, and/or Step (e) using the pre-rearrangement V/J gene sequence determination device includes retaining candidate pre-rearrangement J gene sequences that simultaneously meet the following two conditions: The ratio of the number of read segments supporting the fourth segment to the number of read segments supporting the third segment is greater than 1.5, and the ratio of the number of read segments supporting the third segment to the average number of read segments supporting the third segment is greater than 5%. 27. The system of claim 15, wherein the data acquisition device performs the following: the sequencing data includes multiple pairs of reads, and a pair of reads is spliced into a spliced sequence using the overlapping portion between the reads, and the spliced sequence replaces the pair of reads.