CN114317731A - Method for detecting hepatocellular carcinoma by using whole exon sequencing - Google Patents
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Abstract
The invention discloses a method for detecting hepatocellular carcinoma by sequencing with a whole exon, which comprises the following steps: s1, extracting liver cancer cell genome DNA: extracting genome DNA by using a kit and a standard DNA extraction method; s2, gene capture: carrying out ultrasonic treatment breaking, end repairing, joint connection, hybridization capturing and PCR (polymerase chain reaction) amplification on genome DNA; s3, sequencing: performing high-throughput sequencing by adopting an Illumina Solexa platform to perform whole exon sequencing; s4, data analysis: carrying out standard information analysis on the data, and simultaneously carrying out quality control detection on the data; s5, screening: comparing the obtained data with a database to obtain high-availability and high-quality SNP and Indel; s6, analyzing result biological information: determining the mutation position and mutation type of the corresponding gene segment, and performing biological analysis. The method can facilitate diagnosis of hepatocellular carcinoma and is beneficial to follow-up research of hepatocellular carcinoma signal mechanism.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a method for detecting hepatocellular carcinoma by sequencing of a whole exon.
Background
Hepatocellular carcinoma (HCC) is a primary liver cancer with a high mortality rate. It is one of the most common malignancies worldwide, especially in asia, africa and southern europe. Hepatocellular carcinoma is the major histological subtype of liver cancer, accounting for 90% of primary liver cancer, and is the third most common cause of cancer-related mortality worldwide. Inheritance, epigenetic change, chronic hepatitis B, hepatitis C virus infection, aflatoxin exposure, smoking, obesity, diabetes and the like are main risk factors of liver cancer. The poor prognosis of liver cancer is due to high recurrence rate and high metastasis rate. The mechanism of production and transfer of hepatocellular carcinoma is very complex, and is still one of the problems which are eagerly and seriously overcome in medicine. Modern medicine finds that genetic changes affect the development of all diseases. The relationship between the gene mutation and the disease-related pathway in which the mutation affects the dysregulation of the corresponding protein or the corresponding molecular channel is important for the research.
Most of the gene functions are carried by exons (expressed region), which are part of eukaryotic genes, which are preserved after Splicing (Splicing) and can be expressed as proteins during protein biosynthesis. Exons are the gene sequences that are eventually found in mature RNA, also known as expression sequences. A nucleotide sequence present in both the original transcript and the mature RNA molecule. The term exon also refers to a region in DNA that encodes an exon of the corresponding RNA. All exons together constitute the genetic information that will be represented on the protein. There are approximately 180000 exons in the human gene, accounting for 1% of the human genome, about 30MB, and the protein-coding region of the human genome contains approximately 85% of the causative mutations.
Exon Sequencing (WES) refers to the process of capturing and enriching DNA in genomic exon regions by using a nucleic acid sequence capture technology, and then constructing a library for NGS sequencing analysis. The method has unique advantages in SNP change and insertion deletion of genes. The exon sequencing has the advantages of deeper coverage, high accuracy, simplicity, convenience and the like when the protein codes of a plurality of individuals are sequenced, and the exon sequencing only aims at 1% of the human genome sequence. However, the expression of hepatocellular carcinoma and related genes is not clear, and if exon sequencing can be applied to probing mutant genes and corresponding mutant sites of hepatocellular carcinoma tissues, the personalized treatment of hepatocellular carcinoma provides molecular means which has great guiding significance for clinical diagnosis and treatment of hepatocellular carcinoma.
Disclosure of Invention
The present invention is directed to overcoming at least one of the drawbacks of the prior art described above, and to provide a method for detecting hepatocellular carcinoma by whole exon sequencing, which can screen out a target gene close to hepatocellular carcinoma and SNP sites on the gene, thereby facilitating diagnosis of hepatocellular carcinoma, providing a molecular means for personalized treatment of hepatocellular carcinoma, facilitating subsequent research for clarifying a signal mechanism of hepatocellular carcinoma, and also providing a molecular means for personalized treatment of hepatocellular carcinoma.
The technical scheme adopted by the invention is as follows:
a method for detecting hepatocellular carcinoma by whole exon sequencing comprises the following steps:
s1, extracting liver cancer cell genome DNA: extracting genome DNA by using a kit and a standard DNA extraction method;
s2, gene capture: carrying out ultrasonic treatment breaking, end repairing, joint connection, hybridization capturing and PCR (polymerase chain reaction) amplification on genome DNA;
s3, sequencing: performing high-throughput sequencing by adopting an Illumina Solexa platform to perform whole exon sequencing;
s4, data analysis: carrying out standard information analysis on the data, and simultaneously carrying out quality control detection on the data;
s5, screening: comparing the obtained data with a database to obtain high-availability, high-quality single nucleotide variation, small fragment sequence insertion and deletion;
s6, analyzing result biological information: determining the mutation position and mutation type of the corresponding gene segment, and performing biological analysis.
Preferably, the step S1 specifically includes: extracting sample genomic DNA by using a QIAamp genomic DNA extraction kit:
s1.1, unfreezing a blood sample, sucking 200ul of blood sample, adding 20ul of protein K solution, and uniformly mixing; adding 200ul buffer solution GB, shaking for 15s, standing at 70 deg.C for 10min, and centrifuging to remove water droplets on the inner wall of the tube cover; adding 200 μ L of anhydrous ethanol, shaking thoroughly and mixing for 15 s; after simple centrifugation, the solution was transferred to a 2mL spin column;
s1.2, centrifuging the centrifugal column at 12000rpm for 30S, pouring waste liquid, and transferring the centrifugal column into a new centrifugal tube;
s1.3, adding 500ul of rinsing liquid GD into a new centrifugal tube, centrifuging at 12000rpm for 30S, pouring out waste liquid, and replacing the centrifugal tube again;
s1.4, adding 600ul of rinsing liquid PW again, centrifuging at 12000rpm for 30S, pouring out waste liquid, replacing a centrifuge tube, and repeating the step once;
s1.5, centrifuging the replaced centrifuge tube at 12000rpm for 2min, pouring off waste liquid, placing at room temperature for 3 min, and airing residual rinsing liquid in the material; dropping 200ul of elution buffer TE into the centrifugal column, standing at room temperature for 5min, and centrifuging at 12000rpm for 2 min; the DNA concentration was measured with a spectrophotometer, normalized to 50 ng/. mu.l and stored at-30 ℃ until use.
Preferably, the genomic DNA obtained by the disruption of the ultrasonication in step S2 is a DNA fragment of 180-250 bp.
Preferably, the end repair in step S2 is to connect the sequencing adaptor A base at the 3' end of the fragment DNA.
Preferably, the capture Kit used for hybrid capture in step S2 is the Sure Select Human All Exon 50Mb Kit Exon capture Kit.
Preferably, the PCR amplification conditions in step S2 are: pre-denaturation at 95 ℃ for 2 min; denaturation at 98 ℃ for 20s, 5mim at 70 ℃ and 25-30 cycles; the resulting amplification product was stored at 0 ℃.
Preferably, the raw sequence data is obtained after high throughput sequencing of step S3.
Preferably, the database used in the data analysis of step S4 is a single nucleotide polymorphism database or a thousand-person gene database.
Preferably, the quality control detection in step S4 includes sequencing depth, coverage uniformity, and contrast ratio analysis.
Compared with the prior art, the invention has the beneficial effects that: the invention provides a method for detecting hepatocellular carcinoma by using whole exon sequencing, which can screen out a target gene close to the hepatocellular carcinoma and an SNP site on the gene, is convenient for diagnosing the hepatocellular carcinoma, is convenient for researching a subsequent clear hepatocellular carcinoma signal mechanism, has important significance for clinical diagnosis and treatment of diseases, and can also provide a molecular means for personalized treatment of the hepatocellular carcinoma.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail with reference to specific embodiments. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.
The experimental raw materials, reagent materials and the like used in the following examples are all common commercial products unless otherwise specified.
Examples
The sources of the study subjects were as follows: 3 patients for surgery treatment of hepatocellular carcinoma at certain affiliated hospital of Zhongshan university in Guangzhou City.
Collecting samples: 2mL of peripheral blood of a hepatocellular carcinoma patient is collected, and EDTA is adopted for anticoagulation and low-temperature refrigeration storage.
A method for detecting hepatocellular carcinoma by whole exon sequencing comprises the following steps:
s1, extracting liver cancer cell genome DNA: extracting the genomic DNA by using a QIAamp genomic DNA extraction kit, wherein the specific process comprises the following steps:
s1.1, unfreezing a blood sample, sucking 200ul of blood sample, adding 20ul of protein K solution, and uniformly mixing; adding 200ul buffer solution GB, shaking for 15s, standing at 70 deg.C for 10min, and centrifuging to remove water droplets on the inner wall of the tube cover; adding 200 μ L of anhydrous ethanol, shaking thoroughly and mixing for 15 s; after simple centrifugation, the solution was transferred to a 2mL spin column;
s1.2, centrifuging the centrifugal column at 12000rpm for 30S, pouring waste liquid, and transferring the centrifugal column into a new centrifugal tube;
s1.3, adding 500ul of rinsing liquid GD into a new centrifugal tube, centrifuging at 12000rpm for 30S, pouring out waste liquid, and replacing the centrifugal tube again;
s1.4, adding 600ul of rinsing liquid PW again, centrifuging at 12000rpm for 30S, pouring out waste liquid, replacing a centrifuge tube, and repeating the step once;
s1.5, centrifuging the replaced centrifuge tube at 12000rpm for 2min, pouring off waste liquid, placing at room temperature for 3 min, and airing residual rinsing liquid in the material; dropping 200ul of elution buffer TE into the centrifugal column, standing at room temperature for 5min, and centrifuging at 12000rpm for 2 min; the DNA concentration was measured with a spectrophotometer, normalized to 50 ng/. mu.l and stored at-30 ℃ until use.
S2, gene capture: the genome DNA is subjected to ultrasonic treatment breaking, end repairing, joint connection, hybridization capture and PCR amplification.
More specifically, the obtained 180-and 250-bp DNA fragment is obtained by breaking the genome DNA through ultrasonic treatment in the gene capturing process of the step S2; the end repair is that the 3' end of the fragment DNA is connected with a sequencing adaptor A base; the capture Kit adopted by the hybrid capture is a Sure Select Human All Exon 50Mb Kit Exon capture Kit; the PCR amplification conditions were: pre-denaturation at 95 ℃ for 2 min; denaturation at 98 ℃ for 20s, 5mim at 70 ℃ and 25-30 cycles; the resulting amplification product was stored at 0 ℃.
S3, sequencing: and (3) carrying out high-throughput sequencing by adopting an Illumina Solexa platform to carry out whole exon sequencing, and obtaining original sequence data after sequencing.
S4, data analysis: performing standard information analysis on the single nucleotide polymorphism database and the thousand-person gene database, and performing quality control detection on the data, including sequencing depth, coverage uniformity and contrast ratio analysis;
s5, screening: comparing the obtained data with a database to obtain high-availability, high-quality single nucleotide variation, small fragment sequence insertion and deletion;
s6, analyzing result biological information: and (3) determining the mutation position and the mutation type of the corresponding gene fragment, and screening the gene with SNP site change which has influence on the protein through bioinformatics analysis on the gene with SNP site change in the exon sequencing result.
Through Sanger sequencing verification, sequencing results are respectively compared with PTEN and FHIT reference sequences, and through comparison, PTEN genes of two patients have gene mutation in the three tested patients, and the two patients have missense mutation in exon 5 and exon 8 of the PTEN gene; one patient had a genetic mutation in the FHIP gene and was heterozygous for the deletion.
It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the technical solutions of the present invention, and are not intended to limit the specific embodiments of the present invention. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention claims should be included in the protection scope of the present invention claims.
Claims (9)
1. A method for detecting hepatocellular carcinoma by using whole exon sequencing is characterized by comprising the following steps:
s1, extracting liver cancer cell genome DNA: extracting genome DNA by using a kit and a standard DNA extraction method;
s2, gene capture: carrying out ultrasonic treatment breaking, end repairing, joint connection, hybridization capturing and PCR (polymerase chain reaction) amplification on genome DNA;
s3, sequencing: performing high-throughput sequencing by adopting an Illumina Solexa platform to perform whole exon sequencing;
s4, data analysis: carrying out standard information analysis on the data, and simultaneously carrying out quality control detection on the data;
s5, screening: comparing the obtained data with a database to obtain high-availability, high-quality single nucleotide variation, small fragment sequence insertion and deletion;
s6, analyzing result biological information: determining the mutation position and mutation type of the corresponding gene segment, and performing biological analysis.
2. The method of claim 1, wherein the step S1 comprises the following steps: extracting sample genomic DNA by using a QIAamp genomic DNA extraction kit:
s1.1, unfreezing a blood sample, sucking 200ul of blood sample, adding 20ul of protein K solution, and uniformly mixing; adding 200ul buffer solution GB, shaking for 15s, standing at 70 deg.C for 10min, and centrifuging to remove water droplets on the inner wall of the tube cover; adding 200 μ L of anhydrous ethanol, shaking thoroughly and mixing for 15 s; after simple centrifugation, the solution was transferred to a 2mL spin column;
s1.2, centrifuging the centrifugal column at 12000rpm for 30S, pouring waste liquid, and transferring the centrifugal column into a new centrifugal tube;
s1.3, adding 500ul of rinsing liquid GD into a new centrifugal tube, centrifuging at 12000rpm for 30S, pouring out waste liquid, and replacing the centrifugal tube again;
s1.4, adding 600ul of rinsing liquid PW again, centrifuging at 12000rpm for 30S, pouring out waste liquid, replacing a centrifuge tube, and repeating the step once;
s1.5, centrifuging the replaced centrifuge tube at 12000rpm for 2min, pouring off waste liquid, placing at room temperature for 3 min, and airing residual rinsing liquid in the material; dropping 200ul of elution buffer TE into the centrifugal column, standing at room temperature for 5min, and centrifuging at 12000rpm for 2 min; the DNA concentration was measured with a spectrophotometer, normalized to 50 ng/. mu.l and stored at-30 ℃ until use.
3. The method for detecting hepatocellular carcinoma by using whole exon sequencing as claimed in claim 1, wherein the obtained DNA fragment of 180-250bp obtained by disrupting genomic DNA by sonication in step S2.
4. The method for detecting hepatocellular carcinoma in accordance with claim 1, wherein the end repair in step S2 is performed by connecting sequencing adaptor A base to the 3' end of the fragment DNA.
5. The method for detecting hepatocellular carcinoma using whole Exon sequencing as claimed in claim 1, wherein the capture Kit used for hybrid capture in step S2 is Sure Select Human All Exon 50Mb Kit Exon capture Kit.
6. The method for detecting hepatocellular carcinoma in accordance with claim 1, wherein the PCR amplification conditions in step S2 are as follows: pre-denaturation at 95 ℃ for 2 min; denaturation at 98 ℃ for 20s, 5mim at 70 ℃ and 25-30 cycles; the resulting amplification product was stored at 0 ℃.
7. The method for detecting hepatocellular carcinoma in accordance with claim 1, wherein the original sequence data is obtained after high throughput sequencing in step S3.
8. The method for detecting hepatocellular carcinoma in accordance with claim 1, wherein the database used in the data analysis of step S4 is SNP database or thousand-human gene database.
9. The method of claim 1, wherein the quality control test of step S4 includes analysis of sequencing depth, coverage uniformity and contrast ratio.
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