CN113652484A

CN113652484A - Application of sequencing panel, kit and preparation method of sequencing library

Info

Publication number: CN113652484A
Application number: CN202110954469.9A
Authority: CN
Inventors: 胡传圣; 李华; 胡文献; 胡延平; 沈益行
Original assignee: Shanghai Yidi Biotechnology Co ltd; Suzhou Jingmai Biotechnology Co ltd
Current assignee: Shanghai Yidi Biotechnology Co ltd; Suzhou Jingmai Biotechnology Co ltd
Priority date: 2021-08-03
Filing date: 2021-08-19
Publication date: 2021-11-16
Anticipated expiration: 2041-08-19
Also published as: CN113652484B

Abstract

The application relates to application of a sequencing panel, a kit and a preparation method of a sequencing library, and belongs to the technical field of biology. The sequence design of the Y-shaped joint adopted by the sequencing library is more optimized, so that the accuracy of data analysis is improved, and the economy of sequencing is improved; the sequencing panel is obtained by a reasonable and strict screening method, can accurately distinguish gastric cancer patients from healthy people, and can be applied to cancer detection or preparation of cancer detection products; and through designing a reasonable detection model and a score formula, the detection accuracy can be improved, the high prediction accuracy is verified by clinical samples, and the method has great popularization potential.

Description

Application of sequencing panel, kit and preparation method of sequencing library

The present application claims priority from chinese patent application having application number 202110885340.7, application date 2021, 8/03, the entire contents of which are incorporated herein by reference.

Technical Field

The invention relates to application of a sequencing panel, a kit and a preparation method of a sequencing library, and belongs to the technical field of biology.

Background

Gastric cancer is one of the most common malignant tumors worldwide and is also one of the most mortality malignant tumors. According to recent statistics, gastric cancer is one of the three main causes of death of various cancers in China. At present, the treatment effect of early gastric cancer patients is remarkably improved by the remarkably improved operation technology and various newly developed treatment methods, and the life cycle is also remarkably increased; however, the survival time of the patients with the advanced gastric cancer is still very optimistic, the five-year survival rate is far lower than 50%, and the long-term high treatment cost brings heavy burden to families and society. Therefore, early detection of gastric cancer is crucial for the treatment of gastric cancer.

Gastroscopes are currently the most commonly used tools for diagnosing gastric cancer. Early gastric cancer manifestations under the gastroscope include abnormal mucosal color and superficial vessel disappearance, thickening of the mucosal lining pits or bumps, abnormal mucosal folds around the ulcer, etc.; if necessary, the stomach tissue may also be excised for biopsy. Although tissue biopsy after gastroscopy is the gold standard for diagnosis of gastric cancer, patients with non-obvious symptoms are often reluctant to receive gastroscopy due to the invasiveness of gastroscopy, often causing discomfort and fear to the patient. When the symptoms are severe, the patient is subjected to gastroscopy and is likely to be in a late stage of gastric cancer. The protein markers can be used as reference bases for gastric cancer diagnosis, and the commonly used gastric cancer protein markers comprise carcinoembryonic antigen (CEA), carbohydrate antigen 19-9(CA19-9), carbohydrate antigen 50(CA50) and pepsinogen. However, these conventional gastric cancer protein markers are not high enough in sensitivity/specificity, are not suitable for being used alone as a diagnostic standard for gastric cancer, and have a very limited effect on early gastric cancer detection.

The liquid biopsy technology is taken as a branch of in vitro diagnosis, and the liquid biopsy is used for diagnosing diseases such as cancers through blood or urine and the like, so that the method has the advantages that the damage of the biopsy can be reduced through non-invasive sampling, the life cycle of a patient can be effectively prolonged, and the cost performance is high. ctDNA detection based on blood is one of the important development branches of liquid biopsy technology, and is expected to replace tissue biopsy. The liquid biopsy products on the market can only detect one or a plurality of gene mutations of the gastric cancer basically, the mutation coverage is not high, and the sensitivity and the specificity brought by the detection of the gastric cancer (especially early gastric cancer) by the liquid biopsy products are not high. Therefore, there is a need to develop a new gastric cancer detection method and kit based on liquid biopsy for tumor detection of high-risk gastric cancer people, which facilitates early clinical intervention. .

Disclosure of Invention

The invention aims to provide an application of a sequencing panel, a kit and a preparation method of a sequencing library, which can simply and accurately distinguish gastric cancer patients from healthy people, and have great popularization potential due to high prediction accuracy verified by clinical samples.

In order to achieve the purpose, the invention provides the following technical scheme:

in a first aspect, there is provided a use of a sequencing panel for cancer detection or for the preparation of a product for cancer detection, the sequencing panel comprising the following 98 genes:

ABCC9，ACTC1，ANK2，ANK3，ANO3，APC，ARID1A，ATM，ATP10A，BRCA2，C3ORF20，CACNA1C，CACNA1E，CDH1，CDH11，CDH23，CHD6，CNTN6，CNTNAP5，COL6A3，CREBBP，CSMD1，CSMD3，CTNNB1，CUBN，DCC，DDX17，DDX51，DENND3，DIDO1，DKK2，DNAH1，DNAH11，DNAH5，DNAJC2，DPP4，ENAM，ERBB3，EVX2，EYS，FAT3，FAT4，FBN1，FBXW7，FLG，FLNC，GAREM1，GRM5，HAS3，HMCN1，HRH2，IGSF10，ITPR3，KMT2D，KRAS，LRP1，LRP1B，LRP2，LRRIQ1，LRRK2，MED12L，MFSD1，MUC16，MYH10，MYO15A，NALCN，NBEA，NIPBL，OBSCN，OR51A4，PCDH15，PCIF1，PCLO，PEG3，PHF2，PIK3CA，RHOA，RNF213，RSPH3，RYR2，SCN9A，SMAD4，SPEN，SPTA1，SYNE1，TBXT，TENM4，TNXB，TP53，TRIO，TRPS1，TTN，TUBB2B，USH2A，XIRP2，ZFHX4，ZIM3，ZNF77。

further, the sequencing region of the sequencing panel comprises the exon region of the 98 genes, 20bp upstream and downstream of the exon, 500bp upstream of the transcription start site and 100bp downstream of the transcription termination site.

Further, the cancer is gastric cancer.

Further, sequencing library preparation and sequencing data analysis were performed based on the sequencing panel.

Further, in the sequencing library preparation, the following Y-linker was used:

5’-AATGATACGGCGACCACCGAGATCTACACAATTATCGTATAGCCTCAAGTATCTGCGTTCACCGACCTGCAACGACTAGCNNNNNNNTACGGTGCGCT-3’，

5’-GCGCACCGTANNNNNNNGCTAGTCGTTGCAGACAGTCCTGATCGACAGATCACGCCAATTAGCATCGTTATCTCGTATGCCGTCTTCTGCTTG-3’；

wherein NNNNNNN is a random nucleotide sequence; ACCGGTCCGTAA is a fixed sequence of 12 bases; TATAGCCT and GCCAATTA are index sequences which can be replaced according to actual conditions.

Further, in the sequencing data analysis, SNV and InDel analysis is carried out on the sequencing panel, and the tested sample is scored according to a score formula, wherein the score formula is as follows:

Score_lung＝C₁×∑(Mu_i)+C₂×∑(Mu_j)+C₃×∑(Mu_k)；

wherein i represents the following 31 genes: ATM, TP53, FLG, PIK3CA, CSMD3, NBEA, GAREM1, PCDH15, DNAH1, TENM4, ZNF77, CACNA1C, TTN, ANK3, APC, LRRK2, ACTC1, ABCC9, DCC, DDX51, ENAM, CTNNB1, HRH2, SPEN, CHD6, MED12L, USH2A, ATP10A, ZFHX4, cntnp 5, HMCN 1;

j identifies the following 30 genes: RSPH3, LRP1B, KRAS, DNAJC2, CSMD1, DKK2, FAT4, XIRP2, TUBB2B, MYH10, DDX17, CNTN6, ERBB3, FAT3, SCN9A, OR51a4, PCIF1, ANO3, NIPBL, IGSF10, GRM5, EVX2, TRIO, CREBBP, RHOA, CACNA1E, PHF2, COL6A3, MYO15A, SPTA 1;

k identifies the following 37 genes: ANK2, ARID1A, BRCA2, C3ORF20, CDH1, CDH11, CDH23, CUBN, DENND3, DIDO1, DNAH11, DNAH5, DPP4, EYS, FBN1, FBXW7, FLNC, HAS3, ITPR3, KMT2D, LRP1, LRP2, LRRIQ1, MFSD1, MUC16, NALCN, OBSCN, PCLO, PEG3, RNF213, RYR2, SMAD4, SYNE1, TBXT, TNXB, TRPS1, ZIM 3;

mu is the total number of SNV and InDel detected in the i, j or k genes; c₁＝0.90，C₂＝0.74，C₃＝0.54。

In a second aspect, a kit is provided that includes probes, primers, and Y-linkers for the following sequencing panel, which is the following 98 genes:

further, the capture region of the probe comprises the exon region of the 98 genes, 20bp upstream and downstream of the exon, 500bp upstream of the transcription start site and 100bp downstream of the transcription termination site;

and/or the Y-shaped joint is 5 '-AATGATACGGCGACCACCGAGATCTACACAATTATCGTATAGCCTCAAGTATCTGCGTTCACCGACCTGCAACGACTAGCNNNNNNNTACGGTGCGCT-3',

wherein NNNNNNN is a random nucleotide sequence; ACCGGTCCGTAA is a fixed sequence of 12 bases; TATAGCCT and GCCAATTA are index sequences which can be replaced according to actual conditions;

and/or, the primer comprises: sequencing primers 5'-ACCGACCTGCAACGACTAGC-3' and 5'-GACTGTCTGCAACGACTAGC-3', and Index primers 5'-AGTCCTGATCGACAGATCAC-3' and 5'-TCGGTGAACGCAGATACTTG-3'.

In a third aspect, there is provided a method for preparing a sequencing library, which uses the kit, the method comprising:

s1, providing the kit and peripheral blood;

s2, extracting free DNA of the peripheral blood, breaking, purifying and screening magnetic beads to obtain DNA fragments;

s3, filling the ends of the DNA fragments, adding A to the ends of the DNA fragments, and connecting the DNA fragments with a Y-shaped joint after magnetic bead purification;

s4, amplifying the sample obtained in the step S3 by PCR, and performing magnetic bead purification and Qubit quantification;

s5, repeating the step S4, mixing the obtained different samples according to equal mass ratio, hybridizing by using the probe, and purifying the product by using magnetic beads after elution; subsequent PCR amplification and magnetic bead purification yielded the sequencing library.

Compared with the prior art, the beneficial effect of this application lies in:

1) the 98 sequencing panel genes are obtained by a reasonable and strict screening method, can accurately distinguish gastric cancer patients from healthy people, and can be applied to cancer detection or preparation of cancer detection products; and through designing a reasonable detection model and a score formula, the detection accuracy can be improved, the high prediction accuracy is verified by clinical samples, and the method has great popularization potential.

2) Compared with the conventional Y-type joint, the sequence design of the Y-type joint adopted by the sequencing library is more optimized, the sequence of a primer region is optimized, a random nucleotide sequence (A1) and a fixed sequence (A2) are introduced, and more importantly, the optimization design methods of A1 and A2 are provided, so that errors generated by subsequent data analysis can be reduced, the sequencing economy can be improved, the gastric cancer detection accuracy can be improved, and the gastric cancer detection cost can be reduced.

The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following detailed description is given with reference to the preferred embodiments of the present invention and the accompanying drawings.

Drawings

FIG. 1 is a box plot of quality control results of training set sequencing data according to the first embodiment of the present application, wherein the average coverage is logarithmically transformed to base 10000.

FIG. 2 is a graph of ROC for gastric cancer detection using a score formula in a training set according to the first embodiment of the present application.

FIG. 3 is a box plot diagram of quality control results of the validation set of sequencing data in example two of the present application, wherein the average coverage is logarithmically transformed to base 10000.

Fig. 4 is a graph showing ROC curves for gastric cancer detection obtained by using a score formula in the verification group in example two of the present application.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings, and it should be understood that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In the description of the present application, it is noted that, unless explicitly stated or limited otherwise, the terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusions. For example, a process, method, system, article, or apparatus that comprises a list of steps or elements is not limited to only those steps or elements listed, but may alternatively include other steps or elements not listed, or inherent to such process, method, article, or apparatus. The specific meaning of the above terms in the present application can be understood in a specific case by those of ordinary skill in the art. Further, in the description of the present application, "a plurality" means two or more unless otherwise specified. "and/or" describes the association relationship of the associated objects, meaning that there may be three relationships, e.g., a and/or B, which may mean: a exists alone, A and B exist simultaneously, and B exists alone. The character "/" generally indicates that the former and latter associated objects are in an "or" relationship.

The application provides an application of a sequencing panel in cancer detection or preparation of a cancer detection product, wherein the sequencing panel comprises the following 98 genes:

optionally, the sequencing region of the sequencing panel comprises exon regions of the 98 genes, 20bp upstream and downstream of the exons, 500bp upstream of the transcription start site, and 100bp downstream of the transcription termination site.

Optionally, the cancer is gastric cancer.

The application also provides a kit comprising probes, primers and Y-linkers for the above 98 sequencing panels.

Optionally, the capture region of the probe comprises exon regions of the 98 genes, 20bp upstream and downstream of the exons, 500bp upstream of the transcription initiation site, and 100bp downstream of the transcription termination site.

Optionally, the Y-shaped joint is 5 '-AATGATACGGCGACCACCGAGATCTACACAATTATCGTATAGCCTCAAGTATCTGCGTTCACCGACCTGCAACGACTAGCNNNNNNNTACGGTGCGCT-3',

Optionally, the primer comprises: sequencing primers 5'-ACCGACCTGCAACGACTAGC-3' and 5'-GACTGTCTGCAACGACTAGC-3', and Index primers 5'-AGTCCTGATCGACAGATCAC-3' and 5'-TCGGTGAACGCAGATACTTG-3'.

In the present application, sequencing library preparation and sequencing data analysis can be performed based on the sequencing panel by the following methods:

1) obtaining peripheral blood from a subject, separating the serum and extracting free DNA therefrom as a test sample;

2) taking 100 and 500ng DNA, breaking the DNA into 100 and 1000bp by ultrasonic, and purifying and screening the fragments of about 150bp by magnetic beads;

3) firstly, carrying out end filling on the screened DNA fragments and purifying the products by using magnetic beads; then, adding A at the tail end of the DNA fragment, and purifying the product by magnetic beads;

4) using a Y-shaped linker (sequence 5' -AATGATACGGCGACCACCGAGATCTACACAATTATCG)TATAGCCTCAAGTATCTGCGTTCACCGACCTGCAACGACTAGCNNNNNNNACCGGTCCGTAAT-3 'and 5' -TTACGGACCGGTN NNNNNNGCTAGTCGTTGCAGACAGTCCTGATCGACAGATCACGCCAATTAGCATCGTTATCTCGTATGCCGTCTTCTGCTTG-3') is connected with the sample obtained in the step (3); in the Y-type linker, NNNNNNN is a random nucleotide sequence of 7 bases (abbreviated as a1, and the sequence a1 can be different random sequences in two linkers to increase complexity), ACCGGTCCGTAA is a fixed sequence of 12 bases (abbreviated as a2), and TATAGCCT and GCCAATTA are index sequences for distinguishing the sequencing data of different testees, and can be replaced by 8-base index commonly used by Illumina.

5) Amplifying the sample obtained in the step 4 by PCR, and performing magnetic bead purification and Qubit quantification;

6) the different samples obtained in 3-6 steps 5 were mixed in equal mass proportions, followed by the use of a corresponding sequencing panel (containing 98 genes: ABCC, ACTC, ANK, ANK, ANO, APC, ARID1, ATM, ATP10, BRCA, C3ORF, CACNA1, CACNA1, CDH, CDH, CDH, CHD, CNTN, CNTNAP, COL6A, CREBBP, CSMD, CSMD, CTNNB, CUBN, DCC, DDX, DDX, DENND, DIDNDK, DNAH, DNAH, DNAJC, DPP, ENAM, ERBB, EYS, FAT, FAT, FBN, FBXW, FLG, FLNC, GAREM, GRM, HAS, HMCN, HRH, IGSF, ITPR, KMT2, KRAS, DNAP 1, LRP, LRRIQ, LRRK, MED12, MFSD, MECC, MYH, MUO 15, NMN, NBEA, NILCEA, PBNALRPA, PBLRPA, PHRAP, PHR 1, RNTP, RNSPF, TPRX, TRYP, TRRPS, TRRPNA, TRYP, TRPSROPA, TRYP, TRPSR, TRYP, TRPSN, TRYP, TRPSN, TRYP, TRPSN, TRYP, TRPSN, TRYP, TRPSN, TRYP, TRYPO, TRYP, TRPSN, TRYP, thereby capturing the exon regions of the 98 genes, 20bp upstream and downstream of the exons, 500bp upstream of the transcription initiation site and 100bp downstream of the transcription termination site in a targeted manner;

7) performing targeted capture by using a probe corresponding to the sequencing panel again, and purifying a product by using magnetic beads after elution; then PCR amplification is carried out, magnetic beads are purified, and the obtained product is a prepared DNA library (sequencing library);

8) for the sequencing library obtained in the step (7), after fragment length range detection is carried out by using an Agilent 2100Bioanalyzer and concentration quantification is carried out by Invitrogen Qubit, the sequencing library is sent to an Illumina NextSeq500 high-throughput sequencing platform for sequencing, a sequencing primer (5'-ACCGACCTGCAACGACTAGC-3', 5'-GACTGTCTGCAACGACTAGC-3') and an Index primer (5'-AGTCCTGATCGACAGATCAC-3', 5'-TCGGTGAACGCAGATACTTG-3') are added during sequencing, and then off-line data are obtained, the sequencing read length is 150bp, and the sequencing mode is double-ended sequencing;

9) performing data quality control and pretreatment on the off-line data obtained in the step (8) by using a quality control tool (such as FastQC, Cutaddat and Trimmomatic) to obtain effective data with low-quality sequences and sequencing joints removed, and then comparing the obtained sequences to a reference genome sequence by using sequence comparison software (such as Bowtie2) to obtain position information positioned on a reference genome;

10) and (4) removing the PCR repeated sequences of the result according to the sequence alignment position obtained in the step (9). Specifically, sequences aligned to the same position of the reference genome by the sequence alignment software (i.e., the 5 'and 3' ends of the sequences are identical at the position of the reference genome) are regarded as PCR repeats, and are combined into the same sequence;

11) filtering the sequence with removed PCR duplication obtained in the step (10) to further filter sequences with low comparison quality (MAPQ <20) to obtain sequences with high comparison quality, further counting the sequencing coverage of a target region, discarding the region with the coverage lower than 1000 times, and entering the next step for analysis;

12) performing SNV and InDel analysis on the data obtained in the step (11) by using a variation detection tool (such as Varscan2), further filtering out low-quality SNV and InDel, obtaining high-quality SNV and InDel, and calculating the total number of SNV and InDel in each target gene; at the same time, high quality SNV and InDel sites were annotated using an annotation tool (e.g., SnpEff).

13) Based on the 98 gene sequencing panel, the following stomach cancer Score formula was used (each subject received a Score)_lung)：

Score_lung＝C₁×∑(Mu_i)+C₂×∑(Mu_j)+C₃×∑(Mu_k)

Wherein i represents the following 31 genes: ATM, TP53, FLG, PIK3CA, CSMD3, NBEA, GAREM1, PCDH15, DNAH1, TENM4, ZNF77, CACNA1C, TTN, ANK3, APC, LRRK2, ACTC1, ABCC9, DCC, DDX51, ENAM, CTNNB1, HRH2, SPEN, CHD6, MED12L, USH2A, ATP10A, ZFHX4, cntnp 5, HMCN 1; j identifies the following 30 genes: RSPH3, LRP1B, KRAS, DNAJC2, CSMD1, DKK2, FAT4, XIRP2, TUBB2B, MYH10, DDX17, CNTN6, ERBB3, FAT3, SCN9A, OR51a4, PCIF1, ANO3, NIPBL, IGSF10, GRM5, EVX2, TRIO, CREBBP, RHOA, CACNA1E, PHF2, COL6A3, MYO15A, SPTA 1; k identifies the following 37 genes: ANK2, ARID1A, BRCA2, C3ORF20, CDH1, CDH11, CDH23, CUBN, DENND3, DIDO1, DNAH11, DNAH5, DPP4, EYS, FBN1, FBXW7, FLNC, HAS3, ITPR3, KMT2D, LRP1, LRP2, LRRIQ1, MFSD1, MUC16,NALCN, OBSCN, PCLO, PEG3, RNF213, RYR2, SMAD4, SYNE1, TBXT, TNXB, TRPS1, ZIM 3; mu is the total number of SNV and InDel detected in the i, j or k genes; c₁＝0.90，C₂＝0.74，C₃0.54. With Score_lungWhen the classification threshold is 2, the subject is predicted as a gastric cancer when the classification threshold is higher than 2, and the subject is predicted as a healthy person when the classification threshold is lower than or equal to 2.

The present application will be described in detail with reference to specific embodiments.

Example sequencing panel for serum free DNA determination using training set samples

The applicant collected 50 samples of peripheral venous blood from untreated stage I and II gastric cancer patients from 3 months to 1 month of 2019 in 2018, each sample containing 20ml of peripheral blood, wherein 30 cases of men and 20 cases of women have an average age of 57.8 and an age distribution of 33-79 years. Meanwhile, the applicant collected 50 healthy human peripheral venous blood samples, each containing 20ml of peripheral blood, wherein 30 males and 20 females had an average age of 58.2 and an age distribution of 33-78 years. Neither group of samples had statistically significant differences in gender and age, and therefore satisfied the principles of gender and age matching.

Design of sequencing panel:

first, the panel needs to include the following gastric cancer driver genes TP53, ARID1A, PIK3CA, CDH1, SMAD4, KRAS, APC, KMT2D, CDH11, ERBB3, RHOA, CTNNB 1;

subsequently, using TCGA gastric cancer patient (asian) whole genome/whole exome sequencing data, the mutation frequencies of different genes in the patient were calculated (the mutations included SNV and InDel only), and ranked from high to low according to the mutation frequencies, and then top-ranked genes were selected to constitute a sequencing panel containing 300 genes with the above cancer driver genes.

These 300 genes are: ABCA, ABCC, ACTC, ADCY, ADGRL, ADRB, AHNAK, AKAP, ALDH4A, ALK, ANGPT, ANK, ANK, ANO, APC, APCDD1, APOB, ARID1, ASCC, ASPG, ATM, ATP10, BCAR, BRCA, BRD, C3ORF, CACNA1, CACNA1, CAPN, CARD, CDH, CDH, CDH, CEP120, CFAP, CFHR, CGRRF, CHD, CHEK, CHI3L, CNTN, CNTNAP, COL11A, COL6A, COL9A, CREBP, CSMD, CSDD, DNAF 2, CTNB, CUBN, CYLD, CYP2F, DNA39A, DBN, DCC, DDX, DEND, PDC, DIPDDO, DIPDK, ACAK, SACK 2, SACK, HEFLXGRHP, HEFLEX, HERBAC, FAR, HERBAC, FLXGRHP, HERBAC, HOCK, HO, KMT2, KMT2, KMT2, KRAS, KRT, LANCL, LMBRD, LPCAT, LRP, LRP1, LRP, LRRIQ, LRRK, MACF, MAMLD, MAP, MAPT, MARVELD, MBOAT, MED12, MFSD, MIB, MOGS, MREG, MSS, MTR, MUC, MYD, MYH, MYNN, MYO15, MZF, N4BP, NAALADL, NALCN, NBEA, NDA, NIBAN, NIPBL, NKX-2, NME, NOP, NR1H, NRXN, NUCB, NUP, NUTM2, NXPE, OBSCN, OPLAH, OR4P, OR51A, OR5L, ORM, OS, OSBP, P3H, PCDH, PCDH, PCDH, PCGB, PCIF, PCSLC, SLC, DHSLC, SLC, PRRPSLC, SLC, PSRPSLC, SLC, PSRPRG, PSRPR 4, PSRPRG, PSRPR, PSR, PSRG SLC, PSRG SLC, PSRG SLC, PSRG SLC, PSRG SLC, PSRG SLC, PSRG SLC, PSRG SLC, PSRG SL, SPTA1, ST6GALNAC3, STAB1, SUSD1, SYNE1, SYNE2, TBXT, TENM4, TET3, TG, THSD7B, TIE1, TM9SF2, TMEM109, TMEM165, TNFAIP3, TNXB, TP53, TRDN, TRIO, TRPS1, TSPOAP1, TTN, TUBB2B, TXNDC5, UNC79, UPF2, USH2A, USP17L2, USP7, VAX1, VPS13D, XIRP2, ZFHX4, ZIM3, ZNF142, ZNF212, ZNF217, ZNF280A, ZNF667, ZNF77, ZNF790, ZPR1, ZW 10. The probe target capture region of the panel comprises exon regions of the 300 genes, 20bp upstream and downstream of the exons, 500bp upstream of a transcription initiation site and 100bp downstream of a transcription termination site; the probe is a single-stranded DNA molecule with a biotin label, and the design of the probe refers to Illumina TruSeq exosome (FC-150-1004), and a conventional method is used for chemical synthesis; genome annotation information was from the Ensembl v96 database.

The method for designing the Y-shaped joint comprises the following steps:

firstly, the length of a random nucleotide sequence (A1) in the Y-shaped joint is determined by computer simulation calculation, and the specific method comprises the following steps:

1) dividing the entry amount of free DNA (in 500 ng) by the amount of DNA of a single cell (in 6 pg) and multiplying by 2 to obtain the maximum copy number m of the DNA fragment at the same position generated by sonication;

2) assuming that A1 has a length of n, using the R language results in a length of 4²ⁿThe vector of (1) contains elements from 1 to 4²ⁿA natural number of (2);

3) performing replacement sampling on the vector by using R language, wherein the sampling frequency is m, performing duplicate removal on the m extracted elements, and calculating the proportion P of the number of the residual elements in the m elements after the duplicate removal;

4) increasing n continuously, when n is more than or equal to 7, P is more than 99.9 percent, namely the proportion of the DNA copy at the same position which is mistaken for PCR repeated removal in subsequent analysis because the DNA copy is connected with the same A1 is less than one thousandth; and since A1 should be as short as possible (reducing the likelihood that different A1 will be read as identical due to sequencing errors; reducing the proportion of A1 in the off-line data to improve economics), the length of A1 should obviously be chosen to be 7.

Secondly, the length of the fixed sequence (A2) in the Y-type linker is determined by the following method: assuming that the fixed sequence contains n nucleotides (n.gtoreq.8), when n is increased from 8 to 12 in this order, the number of base combinations of the corresponding fixed sequence is 65536, 262144, 1048576, 4194304, 16777216 in this order; all combinations were aligned to the human reference genome (hg38) using the sequence alignment software Bowtie2, all combinations were perfectly aligned (i.e. without any mismatch) to the human reference genome when n ≦ 11, and a small number (< 1%) of base combinations that could not be perfectly aligned began to appear when n ≦ 12; it is expected that when n is larger than or equal to 13, base combinations which cannot be perfectly aligned exist; to avoid erroneous sequence removal due to the fixed sequence being identical to the genomic sequence, and to make the fixed sequence as short as possible (to reduce the inability of A2 to recognize due to sequencing errors; to reduce the proportion of A2 in the off-line data to improve economy), the length of the fixed sequence A2 should obviously be chosen to be 12.

In this example, for each peripheral blood sample, the following steps (1) to (23) were used for library preparation and sequencing data analysis of serum free DNA, thereby obtaining SNV and InDel of serum free DNA:

(1) collecting peripheral blood sample with dry blood collecting tube, standing at4 deg.C for more than half an hour, centrifuging at 400g and 4 deg.C for 10min to obtain supernatant, further centrifuging at 1800g and 4 deg.C for 10min to obtain supernatant, and storing in-80 deg.C refrigerator;

(2) 100-500ng of DNA was extracted from the above serum sample using QIAamp Circulating Nucleic Acid Kit (55114, QIAGEN), diluted with ultrapure water (without DNase and RNase, the same applies below) to a total volume of 20. mu.l, then disrupted using a Bioruptor sonicator and purified using magnetic beads to a fragment length of around 150 bp;

(3) supplementing the interrupted sample to 50ul volume with RSB (15026770, Illumina), adding 100ul SPB (15052080, Illumina), mixing uniformly, incubating at room temperature for 5min, adsorbing on a magnetic frame, removing the supernatant, washing twice with 200ul 80% alcohol, drying the removed liquid, adding 62.5ul RSB, mixing uniformly, incubating at room temperature for 2min, adsorbing on the magnetic frame, and taking 60ul supernatant to a new tube;

(4) adding 40ul ERP3(15046465, Illumina), mixing, leveling at 30 deg.C for 30min, cooling to 4 deg.C, and taking out;

(5) adding 90ul of SPB, fully mixing, incubating at room temperature for 5min, adsorbing on a magnetic frame, sucking 185ul of supernatant to a new tube, adding 125ul of SPB into the new tube, fully mixing, incubating at room temperature for 5min, adsorbing on the magnetic frame, discarding the supernatant, washing twice with 200ul of 80% alcohol, discarding the liquid, drying, adding 20ul of RSB for resuspending magnetic beads, incubating at room temperature for 2min, adsorbing on the magnetic frame, and taking 17.5ul of supernatant to the new tube;

(6) adding 12.5ul ATL2(15046467, Illumina), mixing, reacting at 37 deg.C for 30min, at 70 deg.C for 5min, cooling at4 deg.C for 5min, and taking out;

(7) 2.5ul RSB, 2.5ul LIG2(15036183, Illumina), 2.5ul linker (adapter; 15 uM; linker is Y-type linker, base sequence is 5' -AATGATACGGCGACCACCGAGATCTACACAATTATCGTATAGCC TCAAGTATCTGCGTTCACCGACCTGCAACGACTAGCNNNNNNNTACGGTGCGCT-3 'and 5'-GCGCACCGTANNNNNNNGCTAGTCGTTGCAGACAGTCCTGATCGACAGATCACGCCAATTAGCATCGTTATCTCGTATGCCGTCTTCTGCTTG-3'), reacting at 30 deg.C for 10min, cooling to 4 deg.C, and taking out; wherein TATAGCCT and GCCAATTA are index sequences used for distinguishing sequencing data of different testees, and different index sequences are used for different samples needing to be mixed subsequently (the alternative sequences refer to 8-base index sequences commonly used by Illumina).

(8) Adding 5ul STL (15012546, Illumina), mixing, adding 39ul SPB, mixing, incubating at room temperature for 5min, adsorbing on a magnetic frame, removing supernatant, washing twice with 200ul 80% alcohol, removing liquid, drying, adding 52.5ul RSB, incubating at room temperature for 2min, adsorbing on the magnetic frame, adsorbing 50ul of supernatant to a new tube, adding 45ul of SPB into the new tube, mixing, incubating at room temperature for 5min, adsorbing on the magnetic frame, removing supernatant, washing twice with 200ul 80% alcohol, drying, adding 27.5ul of RSB, incubating at room temperature for 2min, adsorbing on the magnetic frame, and adsorbing 25ul of supernatant to the new tube;

(9) 5ul of PPC (15031748, Illumina) and 20ul of EPM (15041027, Illumina) were added and mixed for PCR: pre-denaturation at 95 ℃ for 3min, denaturation at 98 ℃ for 20s, annealing at 60 ℃ for 15s, extension at 72 ℃ for 30s, executing 11 cycles, extension at 72 ℃ for 5min, cooling to 4 ℃, and taking out;

(10) adding 35ul of SPB, fully mixing, incubating at room temperature for 5min, adsorbing on a magnetic frame, taking 82ul of supernatant to a new tube, adding 82ul of SPB, mixing, incubating at room temperature for 5min, adsorbing on the magnetic frame, discarding the supernatant, washing twice with 200ul of 80% alcohol, discarding the liquid, drying, adding 17.5ul of RSB heavy suspension magnetic beads, incubating at room temperature for 2min, adsorbing on the magnetic frame, taking 15ul of supernatant to the new tube, and measuring the concentration by an Invitrogen Qubit;

(11) mixing 5 samples according to equal mass proportion, ensuring that the total sample amount is between 900ng and 1500ng, and supplementing the volume to 40ul by RSB;

(12) adding 50ul CT3(15048799, Illumina) and 10ul DNA probe solution (0.5 uM; the probe is a single-stranded DNA molecule with a biotin label, and the design of the probe refers to Illumina TruSeq Exome, and the chemical synthesis uses a conventional method), mixing uniformly, and then carrying out hybridization reaction: at 95 ℃ for 10 min; 94 ℃,1min, 92 ℃,1min, 90 ℃,1min, …, 60 ℃,1min (1 min reaction at every 2 ℃); taking out after 90min at 58 ℃;

(13) taking an EP tube, immediately adding 100ul of the sample obtained in the previous step, adding 250ul of SMB (15015927, Illumina), incubating at room temperature for 25min, adsorbing on a magnetic frame, removing supernatant, adding 200ul of SWS (15052987, Illumina), uniformly mixing, incubating at 50 ℃ for 30min, immediately placing on the magnetic frame, adsorbing, removing supernatant, and repeating once;

(14) preparing a working solution: blending 28.5ul EE1(15037034, Illumina) and 1.5ul HP3(11324596, Illumina); taking 23ul of the resuspended beads, incubating at room temperature for 2min, adsorbing on a magnetic frame, taking 21ul of the supernatant to a new tube, adding 4ul of ET2(15013008, Illumina), and mixing uniformly;

(15) adding 15ul RSB, 50ul CT3 and 10ul DNA probe solution (same as above), mixing uniformly, and carrying out hybridization reaction: at 95 ℃ for 10 min; 94 ℃,1min, 92 ℃,1min, 90 ℃,1min, …, 60 ℃,1min (1 min reaction at every 2 ℃); the material can be taken out after 14.5h at 58 ℃;

(16) taking an EP tube, immediately adding 100ul of the sample obtained in the previous step, adding 250ul of SMB, incubating at room temperature for 25min, adsorbing on a magnetic frame, discarding the supernatant, adding 200ul of SWS, mixing, incubating at 50 ℃ for 30min, immediately placing on the magnetic frame, adsorbing, discarding the supernatant, and repeating once;

(17) preparing a working solution: mixing 28.5ul EE1 and 1.5ul HP 3; taking 23ul of the obtained heavy suspension magnetic beads, incubating at room temperature for 2min, adsorbing on a magnetic frame, taking 21ul of supernatant to a new tube, adding 4ul of ET2, and mixing uniformly; adding 45ul SPB, mixing, incubating at room temperature for 5min, adsorbing on a magnetic frame, removing supernatant, washing with 200ul 80% ethanol twice, removing liquid, drying, adding 27.5ul RSB, incubating at room temperature for 2min, adsorbing on the magnetic frame, and collecting supernatant 25ul in a new tube;

(18) PCR was carried out by adding 5ul of PPC and 20ul of NEM (15037047, Illumina) and mixing: pre-denaturation at 98 ℃ for 30s, denaturation at 98 ℃ for 10s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 30s, performing 11 cycles, extension at 72 ℃ for 5min, cooling to 4 ℃, taking out, adding 45ul SPB, incubating at room temperature for 5min, adsorbing on a magnetic frame, discarding the supernatant, washing twice with 200ul 80% alcohol, adding 22ul RSB after discarding the liquid and drying, incubating at room temperature for 2min, adsorbing on the magnetic frame, taking 20ul of the supernatant to a new tube, and obtaining the prepared exon library.

(19) After detecting the fragment length range (the fragment length is basically distributed between 200 and 400 bp) by using an Agilent 2100Bioanalyzer and quantifying the concentration of Invitrogen Qubit (more than 1 ng/. mu.l), sending the fragments to an Illumina NextSeq500 sequencing platform for sequencing, wherein the sequencing read length is 150bp, the sequencing mode is double-ended sequencing, adding a sequencing primer (5'-ACCGACCTGCAACGACTAGC-3', 5'-GACTGTCTGCAACGACTAGC-3') and an Index primer (5'-AGTCCTGATCGACAGATCAC-3', 5'-TCGGTGAACGCAGATACTTG-3'), and obtaining off-line data.

(20) Performing data quality control and pretreatment (using default parameters) by using FastQC, Cutaddat and Trimmomatic to obtain effective data from which low-quality sequences and sequencing adapters are removed, then removing a random nucleotide sequence A1 and a fixed base sequence A2 from the 5 'and 3' ends of the sequence of the effective data by identifying a fixed base sequence A2, and then re-aligning the obtained sequence onto a human reference genome sequence by using sequence alignment software Bowtie2 (hg 38; using default parameters) to obtain position information located on the reference genome;

(21) and removing the PCR repetitive sequence of the result according to the sequence alignment position. Specifically, sequences aligned to the same position in the reference genome by Bowtie2 (i.e., the 5 'and 3' ends of the sequences are identical in position in the reference genome) are considered PCR repeats if they carry the same random nucleotide sequence a1, and are combined into the same sequence;

(22) further filtering sequences with low comparison quality (only sequences with MAPQ being more than or equal to 20 are reserved) from the sequences with removed PCR repetition, further counting the sequencing coverage of a target region, discarding the region with the coverage being less than 1000 times, and entering the next step for analysis;

(23) SNV and InDel analysis are carried out on the data obtained in the last step by using a mutation detection tool Varscan2, then common mutations in a dbSNP (v151) database are further filtered out to obtain high-quality SNV and InDel, and then high-quality SNV and InDel sites are annotated by using an annotation tool SnpEff (default parameters).

Sequencing data quality control analysis shows that the off-line data Q30 of 100 samples is greater than 85%, the comparable sequence exceeds 95%, the average coverage of the target capture area reaches more than 4300 times (a boxplot of data distribution is shown in figure 1), and the data quality is qualified.

For any one of gene X and subject N, define T_XNThe total number of SNV and InDel detected in the gene of the subject in step (23). At the same time, a variable S is defined for each subject_NWhen the subject is a healthy person, a stage I patient and a stage II patient, S_N0, 1 and 2, respectively. Subsequently, for gene X, T was calculated_XNAnd S_NPearson's correlation coefficient R_XRetention of the correlation coefficient R_XGenes greater than 0.7 are useful for the prediction of gastric cancer status. In this example, the total number of remaining genes is 98, which constitutes the final 98 gene sequencing panel, including ABCC, ACTC, ANK, ANK, ANO, APC, ARID1, ATM, ATP10, BRCA, C3ORF, CACNA1, CACNA1, CDH, CDH, CDH, CHD, CNTN, COL6A, CREBP, CSMD, CSMD, CTNNB, CUBN, DCC, DDX, DDX, DDXD, DENND, DIDO, DKK, DNAH, DNAH, JC, DNAH, DPP, ENAM, ERBB EYS, FAT, FBN, FBXW, FLG, FLNC, GAREM, GRM, HMCN, HRH, IGSF, ITPR, KMT2, KRAS, HAS, FAT1, LRP, RIQ, SPENFR, SPENSD, MUSD 12, LRZI, MFRB, HMCN, SACK, PSRHO, SARB, PSRHO, PSR, PSRXO, PSRXN, PSRXO, PSRXN, PSRXO, PSRXN, PSRXO, PSN, PSRXN, PSRXO, PSN, PSRXN, PSN, PSRXO, PSN, PSRXN, PSN, PSK, PSR, PSRXO, PSN, PSR, PSN, PSK, PSR 2, PSR, PSRXO, PSR, PSRXO, PSR, PSRX, PSR, PSRX, PSR, PSRXO, PSR, PS; the probe target capture region comprises the exon region of the 98 genes, 20bp upstream and downstream of the exon, 500bp upstream of the transcription initiation site and 100bp downstream of the transcription termination site.

Based on the 98 gene sequencing panel, the applicant designed a formula for the gastric cancer development of the sample, and each subject obtained a Score (Score)_lung) The specific score formula is as follows:

Score_lung＝C₁×∑(Mu_i)+C₂×∑(Mu_j)+C₃×∑(Mu_k)

where i is R_XGenes greater than 0.9 (31 in total, including ATM, TP53, FLG, PIK3CA, CSMD3, NBEA, GAREM1, PCDH15, DNAH1, TENM4, ZNF77, CACNA1C, TTN, ANK3, APC, LRRK2, ACTC1ABCC9, DCC, DDX51, ENAM, CTNNB1, HRH2, SPEN, CHD6, MED12L, USH2A, ATP10A, ZFHX4, CNTNAP5, HMCN1), j is R_XMore than 0.8 and less than OR equal to 0.9 (30 genes in total, including RSPH3, LRP1B, KRAS, DNAJC2, CSMD1, DKK2, FAT4, XIRP2, TUBB2B, MYH10, DDX17, CNTN6, ERBB3, FAT3, SCN9A, OR51A4, PCIF1, ANO3, NIPBL, IGSF10, GRM5, EVX2, TRIO, CREBP, RHOA, CACNA1E, PHF2, COL6A3, MYO15A, SPTA1), k is R H_XGenes greater than 0.7 and equal to or less than 0.8 (including the remaining 37 genes); mu is the total number of SNV and InDel detected in the i, j or k genes; c₁＝∑(R_i ²)/28＝0.90，C₂＝∑(R_j ²)/33＝0.74，C₃＝∑(R_k ²)/41＝0.54。

With Score_lungWhen the classification threshold is 2, the subject is judged to be gastric cancer when the classification threshold is higher than 2, and the subject is judged to be healthy when the classification threshold is lower than or equal to 2. Based on the score formula and classification threshold, the detection sensitivity and specificity of gastric cancer were 0.92 and 0.92, respectively. Subsequently, an ROC Curve (all chinese is called a receiver operating characteristic Curve) is plotted using the R language ROCR package, and the corresponding AUC (all english is called Area Under Curve) value is 0.934, as shown in fig. 2, which shows that the score formula can accurately distinguish stage I and II gastric cancer patients from healthy people in the training set.

Example two determination of reliability of sequencing panel Using validation set samples

The applicant collected 50 samples of peripheral venous blood from untreated stage I and II gastric cancer patients from month 2 to month 2020 in 2019, each containing 20ml of peripheral blood, of which 29 in men and 21 in women, the mean age was 62.2 years and the age distribution was 40-81 years, all of which were chinese. Meanwhile, the applicant collected 50 healthy human peripheral venous blood samples, each containing 20ml of peripheral blood, of which 29 males and 21 females had an average age of 61.9 years and an age distribution of 41-80 years, all of which were Chinese. Neither group of samples had statistically significant differences in gender and age, and therefore satisfied the principles of gender and age matching.

Similarly, the validation set samples were subjected to library preparation and sequencing data analysis (except for targeted captureThe region was changed from the corresponding region of the original 300 genes to the final 98 genes of the sequencing panel, and the remaining steps were the same as in steps (1) to (23) of example one). The quality control result shows that the off-line data Q30 of 100 samples is greater than 85%, the comparable sequence exceeds 95%, the average coverage of the target capture area reaches 4600 times or more (as shown in figure 3), and the data quality is qualified. Subsequently, the Score obtained in example one was used_lungCalculation formula, calculating the Score of each tested person_lung. Also as Score_lungClassifying the gastric cancer patients and healthy people by taking 2 as a classification threshold, wherein the corresponding gastric cancer detection sensitivity is 0.92, and the specificity is 0.92; the ROC curve based on the validation set samples was plotted using the R language ROCR package and had an AUC value of 0.922, as shown in fig. 4. Again, the results of this example demonstrate that 98 genes were sequenced panel and Score_lungThe calculation formula can accurately distinguish the stage I and II gastric cancer patients from healthy people.

In summary, the following steps: 1) the 98 sequencing panel genes are obtained by a reasonable and strict screening method, can accurately distinguish gastric cancer patients from healthy people, and can be applied to cancer detection or preparation of cancer detection products; and through designing a reasonable detection model and a score formula, the detection accuracy can be improved, the high prediction accuracy is verified by clinical samples, and the method has great popularization potential.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. Use of a sequencing panel for cancer detection or for the preparation of a product for cancer detection, wherein the sequencing panel comprises the following 98 genes:

2. the use of claim 1, wherein the sequencing region of the sequencing panel comprises the exon region of the 98 genes, 20bp upstream and downstream of the exon, 500bp upstream of the transcription start site, and 100bp downstream of the transcription termination site.

3. The use of claim 1, wherein the cancer is gastric cancer.

4. The use of any of claims 1 to 3, wherein sequencing library preparation and sequencing data analysis is performed based on the sequencing panel.

5. The use of claim 4, wherein in the sequencing library preparation, the following Y-linkers are used:

5’-

AATGATACGGCGACCACCGAGATCTACACAATTATCGTATAGCCTCAAGTATCTGCGTTCACCGACCTGCAACGACTAGCNNNNNNNTACGGTGCGCT-3’，

5’-

GCGCACCGTANNNNNNNGCTAGTCGTTGCAGACAGTCCTGATCGACAGATCACGCCAATTAGCATCGTTATCTCGTATGCCGTCTTCTGCTTG-3’；

6. The use of claim 4, wherein in the sequencing data analysis, the sequencing panel is subjected to SNV and InDel analysis and is configured to score the sample being tested according to a score formula:

Score_lung＝C₁×∑(Mu_i)+C₂×∑(Mu_j)+C₃×∑(Mu_k)；

7. A kit comprising probes, primers and Y-linkers for sequencing a panel of 98 genes:

8. the kit of claim 7, wherein the capture region of the probe comprises the exon region of the 98 genes, 20bp upstream and downstream of the exon, 500bp upstream of the transcription start site, and 100bp downstream of the transcription termination site;

and/or the Y-type linker is 5-

5’-

9. A method for preparing a sequencing library, wherein the kit of claim 7 or 8 is used, the method comprising:

s1, providing the kit and peripheral blood;

10. The method of claim 9, wherein the capture region of the probe comprises the exon region of the 98 genes, 20bp upstream and downstream of the exon, 500bp upstream of the transcription start site, and 100bp downstream of the transcription termination site;

and/or the Y-type linker is 5-

5’-