CN108388773A - A kind of identification method of tumor neogenetic antigen - Google Patents
A kind of identification method of tumor neogenetic antigen Download PDFInfo
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Abstract
The present invention provides a kind of methods for the tumour specific antigen that individual specimen can be analyzed from NGS data；The present invention can utilize NGS data, the tumour specific antigen for fast and accurately analyzing individual specimen provides reference frame for the diagnosis and analysis of doctor, and can synchronize the comparison for carrying out normal cell and tumour cell and mankind's reference gene group, analysis time is short, and analysis efficiency is high.
Description
Technical field
The present invention relates to a kind of technical fields for identifying tumor neogenetic antigen using bioinformatics method.
Background technology
In immunotherapy of tumors, the neoantigen (neoantigen) that identification tumor tissue cell generates is to determine that downstream is faced
The committed step of bed treatment.Normal cell is in Carcinogenesis, since inhereditary material changes, leads to its DNA sequence dna and its
His normal cell is variant, therefore will produce tumor associated antigen (expression of tumour cell height) or tumour specific antigen and (only exist
It is expressed in tumour cell).These antigens can theoretically be resisted due to the specific epitope with mark tumour cell
Then original is combined, and then activate T cell in human leukocyte antigen (HLA) identification on delivery cell with TCR, start immune anti-
It answers, is the potential target spot of immunotherapy of tumors.Pass through the analysis to patient's tumor tissues and normal structure NGS data, Ke Yijian
Make somatic mutation of the tissue, such as point mutation, insertion and deletion mutation, structure variation, Gene Fusion etc..From these mutation
Event can predict the variation that tumour cell occurs in downstream transcription and expression process, and then deduce that may be present new anti-
Original provides foundation for clinical treatment.
Invention content
The purpose of the present invention is to provide a kind of tumour specific antigens that individual specimen can be analyzed from NGS data
Method.
The technical solution adopted by the present invention to solve the technical problems is：
A kind of identification method of tumor neogenetic antigen, includes the following steps：
S1：Obtain the normal cell DNA sequencing result and DNA of tumor cell sequencing result of tumor patient；
S2：Respectively by the short read sequence of normal cell sequencing and the short read sequence alignment of tumour cell sequencing to the mankind
Reference gene group obtains the comparison result of the comparison result and tumour cell of normal cell respectively；
S3：All believable mutation are detected from the comparison result of short read and carry out functional annotation, and mutation includes embryonal system
Mutation and somatic mutation,
S4：According to the annotation of mutation as a result, being directed to the corresponding transcript sequence of each catastrophic event editor, a packet is obtained
Polypeptide sequence is combined the length needed to intercept into small peptide section by the polypeptide sequence containing all mutation according to HLA partings；
S5：Identify that the HLA partings of HLA (human leukocyte antigen) partings and tumour cell of normal cell, prediction S4 obtain
HLA partings, the binding ability of the HLA partings of small peptide and tumour cell of the small peptide and normal cell that obtain,；
S6：It is preferred that going out and there is candidate data of the small peptide of binding ability as tumour antigen with tumour cell HLA partings.
Further, after step S2 obtains comparison result, Quality Control is carried out to comparison result, the content of Quality Control includes：Entirely catch
Obtain the coverage in region, average sequencing depth, comparison rate, the proportion of repetitive sequence and the proportion for uniquely comparing short read；If than
Quality Control requirement is met to result, then enters S3；If comparison result does not meet Quality Control requirement, sample is reacquired, repeats step
S1 and S2.
Further, the step S2 comparison results obtained are optimized, optimization includes being directed to insertion and deletion region again
It compares and the amendment of target area base quality, the optimization for obtaining the optimization comparison result and normal cell of tumour cell compares knot
Fruit.
Further, in step S2, the short read sequence alignment of normal cell sequencing result to mankind's reference gene group and swells
The short read sequence alignment of oncocyte sequencing result is synchronized to mankind's reference gene group and is carried out.
Further, the range of step S3 functional annotations includes sudden change region, gene, transcript, base variation, amino acid change
Change, clinvar, thousand human genomes, esp6500, dbsnp, cosmic, polyphen, sift, Membrane protein conformation, cancer is related
Gene.
Further, the method that step S3 detects all believable mutation is：
S3.1, the optimization comparison result for optimizing comparison result and normal cell for obtaining tumour cell, detect tumour cell
With the germline mutation site of normal cell；
S3.2, the believable mutation of acquisition is filtered to obtained germline mutation site and somatic mutation site；
S3.3, the somatic mutation filtered out and germline mutation are annotated.
Further, step S4 includes that the method for the polypeptide sequence of all mutation is：
S4.1, normal nucleotide sequence is found out from ensembl databases according to transcript number, according to the work(of mutation
It can annotate and the nucleotide of corresponding position on nucleotide sequence is made into modification, obtain a nucleotides sequence for including all mutation
Row；That provide such as functional annotation is G100A, then the 100th bases G for splicing nucleotide sequence is revised as A.
S4.2, the transcript nucleotide sequence comprising all mutation is translated into polypeptide sequence；
S4.3, sequence obtain sporting in polypeptide sequence and work as premutation, and centered on being currently mutated, interception n is a forward
Amino acid and backward m amino acid of interception obtain polypeptide sequence, the maximum length that n can present for HLA partings, and m is HLA partings
The maximum length or m that can be presented are from when premutation to the length of first terminator codon；
S4.4, in the small peptide that polypeptide sequence successively intercepted length is N, N is that HLA partings combine the length needed；To include
When N small peptide of premutation is used for step S6.Such as：It is 8, N 8 that HLA partings, which combine the length needed,；Since mutated site
7 amino acid are intercepted forward, form the small peptide that a length is 8 amino acid with the mutated site；Since mutated site to
6 amino acid of preceding interception intercept 1 amino acid backward, and it is 8 amino that this 7 amino acid form the 2nd article of length with mutated site
The small peptide of acid, and so on, 8 small peptides containing the mutation can be obtained altogether.
Further, the method for formation nucleotide sequence is in step S4.1：It is found out from database according to transcript number
CDS sequences, the sequence that upper 3 ' UTR region is spliced after CDS sequences form nucleotide sequence.
Further, the rule of the interception of step S4.3 is：
A, for point mutation：Centered on the position of mutation, n amino acid is intercepted forward, intercepts m amino backward
Acid, the longest peptide fragment that n=m=HLA partings can present, if when leading portion or back segment curtailment, how many cuts how many；If
Point mutation belongs to stop loss, then m is from when premutation to the length of first terminator codon；
B, for non-frameshift mutation：Non- frameshit insertion will intercept n amino forward from first amino acid of insetion sequence
Acid；M amino acid, the longest peptide fragment that n=m=HLA partings can present are intercepted backward from the last one amino acid of insetion sequence；
Centered on insetion sequence, m amino acid after n amino acid and insetion sequence before insetion sequence, insetion sequence and
Collectively constitute polypeptide sequence；
Non- frameshift deletion then centered on deletion segment, respectively forwardly intercepts n amino acid, intercepts m amino backward
Acid, the longest peptide fragment that n=m=HLA partings can present；
C, for frameshift mutation：Centered on first amino acid for starting frameshift mutation, n amino acid, n are intercepted forward
The longest peptide fragment that=HLA partings can present；M amino acid is intercepted backward, and m is from when premutation to first terminator codon
Length, i.e., intercept backward to first terminator codon.
The advantage of the invention is that：
1. entire analytic process, since fastq files, user is without preparing other input files.
2. all analytical procedures have corresponding Quality Control step, the accuracy of result is improved.
3. the identification for mutational site is more comprehensive, not only the case where consideration individual cells, it is also considered that monoblock tissue
Mutation distribution.
4. there is multigroup confirmation for learning analysis result.
5. committed step is all carried out at the same time there are many algorithm, it can both be mutually authenticated, and also reduce the false negative of result.
6. having carried out algorithm optimization and parallel processing for taking longer step, the speed of single sample analysis is accelerated
Degree.
Description of the drawings
Fig. 1 is the flow chart of the present invention.
Specific implementation mode
A kind of identification method of tumor neogenetic antigen, includes the following steps：
S1：Obtain the normal cell DNA sequencing result and DNA of tumor cell sequencing result of tumor patient；
S2：Respectively by the short read sequence ratio of the short read sequence of normal cell sequencing result and tumour cell sequencing result
To arriving mankind's reference gene group, the comparison result of the comparison result and tumour cell of normal cell is obtained respectively；
S3：All believable mutation are detected from the comparison result of short read and carry out functional annotation, and mutation includes embryonal system
Mutation and somatic mutation, the embryo of tumour cell and normal cell is detected using the haplotypecaller functions of GATK respectively
It is mutational site；Detect the somatic mutation position of tumour cell and normal cell respectively using tools such as Mutect (1.1.7)
Point；.
The range of functional annotation include sudden change region, gene, transcript, base variation, amino acid variation, clinvar,
Thousand human genomes, esp6500, dbsnp, cosmic, polyphen, sift, Membrane protein conformation, cancer related gene；
S4：According to mutation annotation as a result, obtain a polypeptide sequence for including all mutation, by polypeptide sequence according to
HLA partings combine the length needed to intercept into small peptide section；
S4.1, it is numbered from ensembl databases according to transcript find out corresponding CDS Region Nucleotides sequence first, so
The sequence for splicing upper 3 ' UTR region afterwards generates the transcript sequence of wild type.According to the functional annotation of mutation by nucleotide sequence
The nucleotide of upper corresponding position makes modification, obtains a nucleotide sequence for including all mutation.As functional annotation provides
It is G100A, then the 100th bases G for splicing nucleotide sequence is revised as A.
S4.2, the nucleotide sequence comprising all mutation is translated into polypeptide sequence；
S4.3, sequence obtain sporting in polypeptide sequence and work as premutation, and centered on being currently mutated, interception n is a forward
Amino acid and backward m amino acid of interception obtain polypeptide sequence, the maximum length that n can present for HLA partings, and m is HLA partings
The maximum length or m that can be presented are from when premutation to the length of first terminator codon；
S4.4, in the small peptide that polypeptide sequence successively intercepted length is N, N is that HLA partings combine the length needed；To include
When the small peptide of premutation is used for step S5.By taking N=11 as an example, 11 will be obtained and include the small peptide when premutation.If polypeptide sequence
Also include other mutation, and have certain small peptides simultaneously comprising current in N small peptide in row except when other than premutation
Mutation and other mutation, then become the small peptide with mutation combination.
S5：Identify that the HLA partings of HLA (human leukocyte antigen) partings and tumour cell of normal cell, prediction S4 obtain
HLA partings, the binding ability of the HLA partings of small peptide and tumour cell of the small peptide and normal cell that obtain.
The HLA of tumour cell is likely to occur change, compares the difference of normal cell HLA partings and tumour cell HLA partings
It is different, compare wild type peptide fragment (normal cell expression) and HLA binding abilities and saltant type peptide fragment (tumor cells expression) and HLA
The difference of binding ability knows the difference of tumour cell and normal cell, is the preferred accumulation foundation of follow-up neoantigen.
The HLA partings of normal structure sample and tumor tissues sample are predicted using tools such as SOAP-HLA, obtain HLA_I types
(A, B, C) and HLA_II types (DR, DQ, DP).
S6：It is preferred that go out has the small peptide of binding ability as the candidate as tumour antigen with tumour cell HLA partings
Data.
Affinity prediction is carried out using multiple HLA partings affinity forecasting softwares respectively, is such as directed to HLA I types
(netMHC4.0 etc.), for HLA II types (netMHCII 2.2 etc.), all parameters are acquiescence.Prediction result can provide often
The affinity of one HLA partings and each nascent polypeptide filters out the relatively strong (NM of wherein affinity<500) polypeptide.
In one embodiment, after step S2 obtains comparison result, Quality Control, the content packet of Quality Control are carried out to comparison result
Contain：The coverage of entire capture region, average sequencing depth, comparison rate, the proportion of repetitive sequence and unique ratio for comparing reads
Weight；If comparison result meets Quality Control requirement, enter S3；If comparison result does not meet Quality Control requirement, sample is reacquired, weight
Multiple step S1 and S2.
In general we require Q30>80%, i.e. sequencing error rate is more than that 80%, GC contains less than millesimal reads
Amount 50% or so, chain skewed popularity are less than 70%, then it is assumed that normal cell sequencing result and tumour cell the sequencing knot currently obtained
The analysis of step after fruit can be used in.
In one embodiment, the step S2 comparison results obtained are optimized, optimization includes being directed to insertion and deletion area
Domain again compare and target area base quality amendment, obtain tumour cell optimization comparison result and normal cell it is excellent
Change comparison result.
In step S2, the short read sequence alignment of normal cell sequencing result is surveyed to mankind's reference gene group and tumour cell
The short read sequence alignment of sequence result is synchronized to mankind's reference gene group and is carried out.
The method that step S3 detects all believable mutation is：
S3.1, the optimization comparison result for optimizing comparison result and normal cell for obtaining tumour cell, detect tumour cell
With the germline mutation site of normal cell；
S3.2, the believable mutation of acquisition is filtered to obtained germline mutation site and somatic mutation site；
S3.3, the somatic mutation filtered out and germline mutation are annotated.
In one embodiment, when mutation type is point mutation, the rule of the interception of step S4.3 is：With mutation
Centered on position, n amino acid is intercepted forward, intercepts m amino acid, the longest peptide that n=m=HLA partings can present backward
Section, if when leading portion or back segment curtailment, how many cuts how many；If point mutation, which belongs to stop loss, (loses termination codon
Son), then m is from when premutation to the length of first terminator codon.
In this way, for each mutation, it can simulate and be possible to the small peptide containing this mutation in tumour cell,
It is predicted finally by with the binding force of patient's HLA partings, filters out the small peptide of binding ability, the candidate as tumour antigen
Data.
Currently, it is that HLA II types combine 16 amino acid needed, therefore, n=that HLA partings, which combine the maximum length needed,
M=16.
Combine the length needed for for 8, N=8 with HLA partings；Intercept 7 amino forward since current point mutation
Acid is mutated the small peptide to form that a length is 8 sites with current point；Intercept 6 amino acid forward since current point mutation,
1 amino acid is intercepted backward, this 7 amino acid are mutated the small peptide to form that the 2nd article of length is 8 sites with current point, with such
It pushes away, 8 small peptides for containing current point mutation can be obtained altogether.
In one embodiment, when mutation type is non-frameshift mutation, the rule of the interception of step S4.3 is：Non- frameshit is inserted
N amino acid will be intercepted forward from first amino acid of insetion sequence by entering；Backward from the last one amino acid of insetion sequence
Intercept m amino acid, the longest peptide fragment that n=m=HLA partings can present；Centered on insetion sequence, insetion sequence is inserted into sequence
M amino acid after n amino acid and insetion sequence before row and collectively constitute polypeptide sequence.
Non- frameshift deletion then centered on deletion segment, respectively forwardly intercepts n amino acid, intercepts m amino backward
Acid, the longest peptide fragment that n=m=HLA partings can present.
Currently, it is that HLA II types combine 16 amino acid needed, therefore, n=that HLA partings, which combine the maximum length needed,
M=16.
In one embodiment, when mutation type is frameshift mutation, the rule of the interception of step S4.3 is：From beginning frameshit
Centered on first amino acid of mutation, n amino acid, the longest peptide fragment that n=HLA partings can present are intercepted forward；Backward
Intercept m amino acid, m is from when premutation is to the length of first terminator codon, i.e., intercepts close to first termination backward
Numeral.
Currently, it is that HLA II types combine 16 amino acid needed, therefore, n=that HLA partings, which combine the maximum length needed,
16。
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
Member, without departing from the inventive concept of the premise, can also make several improvements and modifications, these improvements and modifications also should be regarded as
In the scope of the present invention.
Claims (9)
1. a kind of identification method of tumor neogenetic antigen, which is characterized in that include the following steps：
S1：Obtain the normal cell DNA sequencing result and DNA of tumor cell sequencing result of tumor patient；
S2：The short read sequence alignment of the short read sequence of normal cell sequencing and tumour cell sequencing is referred to the mankind respectively
Genome obtains the comparison result of the comparison result and tumour cell of normal cell respectively；
S3：All believable mutation are detected from the comparison result of short read and carry out functional annotation, and mutation includes germline mutation
And somatic mutation,
S4：According to the annotation of mutation as a result, being directed to the corresponding transcript sequence of each catastrophic event editor, it includes institute to obtain one
Polypeptide sequence is combined the length needed to intercept into small peptide section by the polypeptide sequence for having mutation according to HLA partings；
S5：Identify that the HLA partings of HLA (human leukocyte antigen) partings and tumour cell of normal cell, prediction S4 obtain
The binding ability of the HLA partings of small peptide and normal cell, the HLA partings of small peptide and tumour cell,；
S6：It is preferred that going out and there is candidate data of the small peptide of binding ability as tumour antigen with tumour cell HLA partings.
2. a kind of identification method of tumor neogenetic antigen according to claim 1, it is characterised in that：Step S2 obtains ratio
After result, Quality Control is carried out to comparison result, the content of Quality Control includes：The coverage of entire capture region, average sequencing depth,
Comparison rate, the proportion of repetitive sequence and the proportion for uniquely comparing short read；If comparison result meets Quality Control requirement, enter S3；
If comparison result does not meet Quality Control requirement, sample is reacquired, repeats step S1 and S2.
3. a kind of identification method of tumor neogenetic antigen according to claim 2, it is characterised in that：Step S2 is obtained
Comparison result optimize, optimization include for insertion and deletion region compare again and target area base quality is repaiied
Just, the optimization comparison result of the optimization comparison result and normal cell of tumour cell is obtained.
4. a kind of identification method of tumor neogenetic antigen according to claim 3, it is characterised in that：In step S2, just
Short read sequence of the short read sequence alignment of normal cell sequencing result to mankind's reference gene group and tumour cell sequencing result
It compares mankind's reference gene group and synchronizes progress.
5. a kind of identification method of tumor neogenetic antigen according to claim 1, it is characterised in that：Step S3 functions are noted
The range released include sudden change region, gene, transcript, base variation, amino acid variation, clinvar, thousand human genomes,
Esp6500, dbsnp, cosmic, polyphen, sift, Membrane protein conformation, cancer related gene.
6. a kind of identification method of tumor neogenetic antigen according to claim 5, it is characterised in that：Step S3 detects institute
There is the method for believable mutation to be：
S3.1, obtain tumour cell optimization comparison result and normal cell optimization comparison result, detect tumour cell and just
The germline mutation site of normal cell；
S3.2, the believable mutation of acquisition is filtered to obtained germline mutation site and somatic mutation site；
S3.3, the somatic mutation filtered out and germline mutation are annotated.
7. a kind of identification method of tumor neogenetic antigen according to claim 1, it is characterised in that：Step S4 includes institute
There is the method for the polypeptide sequence of mutation to be：
S4.1, normal nucleotide sequence is found out from ensembl databases according to transcript number, is noted according to the function of mutation
It releases and the nucleotide of corresponding position on nucleotide sequence is made into modification, obtain a nucleotide sequence for including all mutation；Such as
That functional annotation provides is G100A, then the 100th bases G for splicing nucleotide sequence is revised as A.
S4.2, the transcript nucleotide sequence comprising all mutation is translated into polypeptide sequence；
S4.3, sequence obtain sporting in polypeptide sequence and work as premutation, centered on being currently mutated, intercept n amino forward
M amino acid acquisition polypeptide sequence of acid and backward interception, the maximum length that n can present for HLA partings, m is HLA partings institute energy
The maximum length or m of presentation are from when premutation to the length of first terminator codon；
S4.4, the length needed for the combination of HLA partings in the small peptide that polypeptide sequence successively intercepted length is N, N；To include current
N small peptide of mutation is used for step S6.
8. a kind of identification method of tumor neogenetic antigen according to claim 7, it is characterised in that：Shape in step S4.1
It is at the method for nucleotide sequence：CDS sequences are found out from database according to transcript number, and upper 3 ' are spliced after CDS sequences
The sequence of UTR region forms nucleotide sequence.
9. a kind of identification method of tumor neogenetic antigen according to claim 7, it is characterised in that：Step S4.3's cuts
The rule taken is：
A, for point mutation：Centered on the position of mutation, n amino acid is intercepted forward, intercepts m amino acid, n backward
The longest peptide fragment that=m=HLA partings can present, if when leading portion or back segment curtailment, how many cuts how many；As fruit dot is prominent
Change belongs to stop loss, then m is from when premutation to the length of first terminator codon；
B, for non-frameshift mutation：Non- frameshit insertion will intercept n amino acid forward from first amino acid of insetion sequence；From
The last one amino acid of insetion sequence intercepts m amino acid, the longest peptide fragment that n=m=HLA partings can present backward；To insert
Centered on entering sequence, m amino acid after n amino acid and insetion sequence before insetion sequence, insetion sequence and common
Form polypeptide sequence；
Non- frameshift deletion then centered on deletion segment, respectively forwardly intercepts n amino acid, intercepts m amino acid, n=m backward
The longest peptide fragment that=HLA partings can present；
C, for frameshift mutation：Centered on first amino acid for starting frameshift mutation, n amino acid, n=are intercepted forward
The longest peptide fragment that HLA partings can present；M amino acid is intercepted backward, and m is from when premutation to first terminator codon
Length is intercepted backward to first terminator codon.
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| CN110675913A (en) * | 2019-01-16 | 2020-01-10 | 倍而达药业（苏州）有限公司 | Screening method of tumor neoantigen based on HLA typing and structure |
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