CN114708905A

CN114708905A - Chromosome aneuploidy detection method, device, medium and equipment based on NGS

Info

Publication number: CN114708905A
Application number: CN202210506571.7A
Authority: CN
Inventors: 陶威; 杨滢; 陈维之; 何骥; 杜波
Original assignee: Wuxi Precision Medical Laboratory Co ltd; Zhenhe Beijing Biotechnology Co ltd
Current assignee: Wuxi Precision Medical Laboratory Co ltd; Zhenhe Beijing Biotechnology Co ltd
Priority date: 2022-05-11
Filing date: 2022-05-11
Publication date: 2022-07-05

Abstract

The invention discloses a chromosome aneuploidy detection method, a chromosome aneuploidy detection device, a chromosome aneuploidy detection medium and chromosome aneuploidy detection equipment based on NGS, and relates to the technical field of biomedicine. Comprising receiving NGS sequencing data of tumor tissue and normal tissue; preprocessing the NGS sequencing data to obtain an intermediate data file; using the intermediate data file to perform consistency evaluation on the sex and the embryo SNP; acquiring the coverage depth information and SNP genotype information on the genome of the sample to be detected by using the intermediate data file; detecting the purity, ploidy and SCNV fragments of the tumor sample; calculating SCNV of chromosome arm level of each tumor sample according to the single tumor sample and a prepared SCNV database of the pan-carcino cohort; based on SCNV at the level of the chromosome arms of the samples, the final chromosome aneuploidy score for each tumor sample was calculated. The apparatus, medium, and device are all based on the method. The invention improves the accuracy of the aneuploidy detection of the tumor chromosome.

Description

Chromosome aneuploidy detection method, device, medium and equipment based on NGS

Technical Field

The invention relates to the technical field of biomedicine, in particular to a chromosome aneuploidy detection method, a chromosome aneuploidy detection device, a chromosome aneuploidy detection medium and chromosome aneuploidy detection equipment based on NGS.

Background

Next-generation sequencing (NGS) is a high-throughput large-scale parallel sequencing technology, which is a DNA sequencing technology developed based on PCR and gene chips. The technology can acquire gene mutation and chromosome structure variation information of a plurality of target regions on a genome at one time with lower cost.

Chromosomal Aneuploidy (Aneuploidy), an imbalance in the number of chromosomes in cells, is an important feature found in approximately 90% of solid tumors. Chromosome Aneuploidy score (Aneuploidy score), defined as the number of tumor samples that develop SCNV at the level of the chromosome arm, can be used as an immune biomarker to indicate the efficacy of immunotherapy.

At present, a method for calculating chromosome Aneuploidy score (Aneuploidy score) based on second-generation sequencing SCNV data is reported in documents, and the chromosome Aneuploidy cannot be accurately detected due to the defects that an SCNV-segment detection method and a filtering standard are not uniform, tumor purity and ploidy correction are not carried out, operation steps are complicated and the like.

Disclosure of Invention

The technical problem is as follows: the invention provides a chromosome aneuploidy detection method and a chromosome aneuploidy detection device based on NGS, and aims to improve the detection accuracy of tumor chromosome aneuploidy. Also, a corresponding computer-readable storage medium and electronic device are provided.

The technical scheme is as follows: in a first aspect, the present invention provides a method for detecting chromosomal aneuploidy based on NGS, comprising:

receiving NGS sequencing data of tumor tissue and normal tissue;

preprocessing the NGS sequencing data to obtain an intermediate data file;

using the intermediate data file to perform consistency evaluation on sex and germ line SNP;

acquiring the coverage depth information and SNP genotype information on the genome of the sample to be detected by using the intermediate data file;

detecting the purity, ploidy and SCNV fragments of the tumor sample;

calculating SCNV of chromosome arm level of each tumor sample according to the single tumor sample and a prepared SCNV database of the pan-carcino cohort;

based on SCNV at the level of the chromosome arms of the samples, the final chromosome aneuploidy score for each tumor sample was calculated.

Further, NGS sequencing data for tumor and normal tissues, including whole genome sequencing and whole exon capture sequencing.

Further, using the intermediate data file, performing a consistency assessment of gender and germline SNPs includes:

estimating the sex of the sample based on the sequencing depth of the Y chromosome, and performing consistency estimation on the sex;

the identity of germline SNPs was assessed using Conpair software.

Further, the step of obtaining the coverage depth information and the SNP genotype information on the genome of the sample to be detected by using the intermediate data file comprises the following steps:

and taking the intermediate data file as input, using an SNP locus database, and acquiring the coverage depth information and the SNP genotype information on the genome of the sample to be detected by utilizing the SNP-pileup software.

Further, tumor samples were tested for purity, ploidy, and SCNV fragments using FACETS or Sequenza software.

Further, calculating SCNV at the chromosome arm level for each tumor sample from the single tumor sample and the prepared pan-carcinoma cohort SCNV database comprises:

a single tumor sample and a prepared pan-cancer queue SCNV database are used as input, and GISTIC software is used for analysis;

and setting a threshold parameter, and judging whether CNV occurs at the chromosome arm level according to the set threshold parameter.

Further, calculating a final chromosomal aneuploidy score for each tumor sample based on SCNV at the chromosome arm level of the sample comprises:

the sum of the non-0 numbers of the long and short arms of all 22 autosomes was calculated.

In a second aspect, the present invention provides an NGS-based chromosome aneuploidy detection apparatus for detecting a tumor chromosome aneuploidy by using any of the NGS-based chromosome aneuploidy detection methods, including:

a data receiving module configured to receive NGS sequencing data of tumor tissue and normal tissue;

a data preprocessing module configured to preprocess the NGS sequencing data to obtain an intermediate data file;

a consistency assessment module configured to utilize the intermediate data file for consistency assessment of gender and germline SNPs;

the information acquisition module is configured to acquire the coverage depth information and the SNP genotype information of the genome of the sample to be detected by using the intermediate data file;

an SCNV detection module configured to detect tumor sample purity, ploidy, and SCNV fragments;

an SCNV calculation module configured to calculate SCNV at the chromosome arm level for each tumor sample from the single tumor sample and a prepared pan-cancer cohort SCNV database;

a score calculation module configured to calculate a final chromosomal aneuploidy score for each tumor sample based on the SCNV for the sample chromosome arm level.

In a third aspect, the present invention provides a computer readable storage medium having stored thereon computer instructions executable by a processor to implement any of the NGS-based chromosomal aneuploidy detection methods.

In a fourth aspect, the present invention provides an electronic device comprising: the computer-readable storage medium, and a processor configured to execute computer instructions in the computer-readable storage medium.

English abbreviation is illustrated:

(1) and (3) NGS: next generation, sequencing, the second generation sequencing technology;

(2) aneuploidy: chromosome aneuploidy;

(3) SCNV: (ii) somatic copy number variants;

(4) SNP: single Nucleotide Polymorphism (SNP).

Compared with the prior art, the purity and the ploidy state of a tumor sample are considered when the SCNV is calculated, and the Aneuploidy score finally calculated has higher accuracy and robustness by combining the SCNV result of the pan-carcinoma database, so that the Aneuploidy of the tumor chromosome can be more accurately detected.

Drawings

FIG. 1 is a flow chart of a method for NGS-based chromosomal aneuploidy detection in accordance with the present invention;

FIG. 2 is a line graph of the difference between Aneuploidy score groups in a recurrent group and a non-recurrent group of lung cancer in accordance with an embodiment of the present invention;

FIG. 3 is a graph of the difference in recurrence-free survival time (RFS) for lung cancer when cutoff =18 in an example of the present invention;

FIG. 4 is a forest chart of HR values of lung cancer with cutoff =18 in accordance with an embodiment of the present invention;

fig. 5 is a block diagram of an NGS-based chromosomal aneuploidy detection apparatus according to the present invention.

Detailed Description

The invention is further described with reference to the following examples and the accompanying drawings.

Fig. 1 shows a flow chart of an NGS-based chromosomal aneuploidy detection method in an embodiment of the invention. Referring to fig. 1, the method includes steps S110 to S170, which are specifically as follows:

step S110: NGS sequencing data of tumor tissue and normal tissue (or blood cells) are received. Specifically, as in fig. 2 in the embodiment of the present invention, sequencing data may be acquired based on Whole Genome Sequencing (WGS) or whole exon capture sequencing (WES) in next generation sequencing. The sequencing data may be Paired-end sequencing or single-end sequencing, with the preferred embodiment of the invention being Paired-end sequencing, the sequencing strategy being PE150 (Paired-end). Sequencing depth: WGS sequencing is preferably >30X deep; WES sequencing is preferably >100X deep.

Step S120: and preprocessing the NGS sequencing data to obtain an intermediate data file.

Specifically, the quality control of sequencing original data of tumor tissues and normal tissues mainly comprises the steps of removing sequencing joint sequences and low-quality sequences by using Trimmomatic software, and then comparing filtered clean reads to a ginseng reference genome hg19 by using BWA software to obtain an original BAM file. The original BAM also needs to obtain the final BAM file through the following steps: 1) sequencing by Samtools software; 2) picard removes the repetitive sequence; 3) partial realignment by GATK4 software; 4) and performing base quality correction and generating an index file by using GATK4 software. Through the above operations, an intermediate data file is obtained, which is a BAM file.

Step S130: sex and germline SNPs were assessed for consistency using the intermediate data file. In the examples of the present invention, sex consistency assessment was performed by the depth of sequencing of the Y chromosome. Using the intermediate data file obtained in step S120 as an input, the sequencing depth of the Y chromosome was determined using Samtools software to perform sex consistency evaluation.

Further, the identity of germline SNPs was assessed using Conpair software. And (4) completing the consistency evaluation of the embryonic SNP through 7000 SNP sites in Conpair software. The consistency evaluation is carried out on the sex and the germ line SNP, so that the detection accuracy is improved.

Step S140: and acquiring the coverage depth information and the SNP genotype information on the genome of the sample to be detected by using the intermediate data file.

And acquiring the coverage depth information and SNP genotype information on the sample genome by using the intermediate data file and the dbSNP138 database and using the SNP-pileup software.

Step S150: tumor samples were tested for purity, ploidy and SCNV fragments. Specifically, in the examples of the present invention, based on the coverage depth information and SNP genotype information, the tumor sample purity, ploidy and SCNV fragment detection were performed using FACETS or Sequenza software. Preferably, the SCNV test is performed using facts software, with the main parameters: underpth (WGS = 10; WES = 35); np.nbhd (WGS = 500; WES = 250); cval (WGS = 600; WES = 250); nhet (WGS = 15; WES = 15). FACETS adopts a binary genome segmentation method for analyzing allele specific copy number variation and simultaneously calculating clone structure information of sample purity, ploidy, LOH and CNV, and has high analysis speed and accurate result.

Step S160: from the single tumor samples and the prepared pan-cancer cohort SCNV database, the SCNV at the chromosome arm level of each tumor sample was calculated.

Analyses were performed using the gist 2.0 software based on single tumor sample SCNV results and a prepared pan-cancer cohort SCNV database. The main parameters of the software are shown in table 1. The pan cancer cohort SCNV database is constructed based on an SCNV result file obtained from a pan cancer sample by using the method and meeting the quality control condition, and is used for horizontal CNV analysis of a cohort sample chromosome arm.

TABLE 1 GISTIC 2.0 software usage parameters

-rx	-ta	-td	-js	-qvt	-cap	-broad	-brlen	-maxseg	-conf
										1	0.1	0.1	4	0.25	1.5	1	0.8	8500	0.95

In this step, it is determined whether the threshold parameter for CNV occurrence at the chromosome arm level is 0.8, i.e., the CNV occurrence interval on each chromosome arm is greater than 80% of the length of the whole chromosome arm.

Step S170: based on SCNV at the level of the chromosome arms of the samples, the final chromosome aneuploidy score for each tumor sample was calculated. Based on the SCNV at the chromosome arm level of the sample, the sum of the number of chromosome arms with changed copy number per tumor sample is calculated as the final chromosome Aneuploidy score (Aneuploidy score) of the sample. In the calculation, the sum of the number of non-0 (0 means no occurrence of CNV) long and short arms of all 22 autosomes was calculated. The short arms of chr13, chr14, chr15, chr21 and chr22 are too short to be included in the calculation. Therefore, the value range of Aneuploidy score is 0-39. According to Aneuploidy score, the chromosome Aneuploidy state of the tumor sample can be evaluated, and the higher the Aneuploidy score is, the more unstable the chromosome of the tumor sample is.

To verify the utility of the method of the present invention, we performed we sequencing on 81 collected early lung cancer tissue and normal tissue samples (mean sequencing depth, tumor sample 257X, normal tissue sample 219X), quality control removed 2 pairs (gender or germ line consistency inconsistency), and finally calculated Aneuploidy score for 79 samples according to the above method steps. As shown in fig. 2, the relapsing group Aneuploidy score was significantly higher than the non-relapsing group (Wilcoxon rank-sum test, p = 0.012) in combination with the clinical relapsing status of the patients. Further, as shown in fig. 3 and 4, single-factor Cox regression analysis was performed using R software in conjunction with patient RFS time, and Kaplan-Meier survival curves and forest plots were plotted. The results show that the prognosis of recurrence of the Aneuploid score low group is significantly better than that of the Aneuploid score high group (p =0.0383, HR = 2.3693) when median Aneuploid score (cutoff = 18) is used as a threshold. The results further define the accuracy of the proposed method and the suggestive role of Aneuploid score for recurrence in early stage lung cancer.

In another aspect, the present invention provides an NGS-based chromosomal aneuploidy detection apparatus, which may use any of the proposed NGS-based chromosomal aneuploidy detection apparatuses, as shown in fig. 5, including: a data receiving module 210, a data preprocessing module 220, a consistency evaluation module 230, an information acquisition module 240, an SCNV detection module 250, an SCNV calculation module 260, and a score calculation module 270. Wherein the data receiving module is configured to receive NGS sequencing data of tumor tissue and normal tissue; the data preprocessing module is configured to preprocess the NGS sequencing data to obtain an intermediate data file; a consistency assessment module configured to utilize the intermediate data file for consistency assessment of gender and germline SNPs; the information acquisition module is configured to acquire the coverage depth information and the SNP genotype information of the genome of the sample to be detected by using the intermediate data file; the SCNV detection module is configured to detect tumor sample purity, ploidy and SCNV fragments; the SCNV calculation module is configured to calculate SCNV at the chromosome arm level for each tumor sample from the single tumor sample and the prepared pan-carcinoma cohort SCNV database; the score calculation module is configured to calculate a final chromosomal aneuploidy score for each tumor sample based on the SCNV at the sample chromosomal arm level.

The manner in which each module implements the corresponding function corresponds to the description of the above method, and a repeated description thereof is omitted here.

In a third aspect, the present invention provides a computer-readable storage medium having stored thereon a computer program, which, when executed, may comprise the procedures of the embodiments of the methods described above. Any reference to memory, databases, or other media used in the embodiments provided by the embodiments of the disclosure may include at least one of non-volatile and volatile memory. The nonvolatile Memory may include Read-Only Memory (ROM), magnetic tape, floppy disk, flash Memory, optical Memory, high-density embedded nonvolatile Memory, resistive Random Access Memory (ReRAM), Magnetic Random Access Memory (MRAM), Ferroelectric Random Access Memory (FRAM), Phase Change Memory (PCM), graphene Memory, and the like. Volatile Memory can include Random Access Memory (RAM), external cache Memory, and the like. By way of illustration and not limitation, RAM can take many forms, such as Static Random Access Memory (SRAM) or Dynamic Random Access Memory (DRAM), among others.

In a fourth aspect, the present invention provides an electronic device, in the disclosed embodiment, the electronic device includes any one of the computer-readable storage media and a processor, and the processor in the embodiments provided in the present disclosure may be a general processor, a central processing unit, a graphics processor, a digital signal processor, a programmable logic device, a data processing logic device based on quantum computing, and the like, but is not limited thereto. The flow steps of the above-described methods may be implemented when a computer program stored in a computer-readable storage medium is executed by a processor.

The above examples are only preferred embodiments of the present invention, it should be noted that: it will be apparent to those skilled in the art that various modifications and equivalents can be made without departing from the spirit of the invention, and it is intended that all such modifications and equivalents fall within the scope of the invention as defined in the claims.

Claims

1. A chromosome aneuploidy detection method based on NGS is characterized by comprising the following steps:

receiving NGS sequencing data of tumor tissue and normal tissue;

preprocessing the NGS sequencing data to obtain an intermediate data file;

acquiring the coverage depth information and SNP genotype information of the genome of the sample to be detected by using the intermediate data file;

detecting the purity, ploidy and SCNV fragments of the tumor sample;

calculating SCNV of chromosome arm level of each tumor sample according to single tumor sample and prepared SCNV database of pan-cancer cohort;

2. The method of claim 1, wherein NGS sequencing data of tumor and normal tissues comprises whole genome sequencing and whole exon capture sequencing.

3. The method of claim 1, wherein using the intermediate data file to perform a consistency assessment of gender and germline SNPs comprises:

the identity of germline SNPs was assessed using Conpair software.

4. The method according to any one of claims 1 to 3, wherein the obtaining of the overlay depth information and the SNP genotype information on the genome of the sample to be tested by using the intermediate data file comprises:

5. The method of claim 4, wherein the tumor sample purity, ploidy and SCNV fragments are detected using FACETS or Sequenza software.

6. The method of claim 5, wherein calculating SCNV at the chromosome arm level for each tumor sample from a single tumor sample and a prepared pan-carcinoma cohort SCNV database comprises:

7. The method of claim 6, wherein calculating a final chromosome aneuploidy score for each tumor sample based on SCNV at the chromosome arm level of the sample comprises:

8. An NGS-based chromosomal aneuploidy detection apparatus for detecting a tumor chromosomal aneuploidy by the NGS-based chromosomal aneuploidy detection method according to any one of claims 1 to 7, comprising:

a score calculation module configured to calculate a final chromosomal aneuploidy score for each tumor sample based on the SCNV at the sample chromosomal arm level.

9. A computer-readable storage medium having stored thereon computer instructions executable by a processor to implement the NGS-based chromosomal aneuploidy detection method of any of claims 1-7.

10. An electronic device, comprising: the computer-readable storage medium of claim 9, and a processor configured to execute computer instructions in the computer-readable storage medium.