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CN112226496A - Method and system for applying DNA and RNA double-built library to NGS (Next Generation System) to calibrating lung cancer targeted drug and chemotherapy drug genomes - Google Patents

Method and system for applying DNA and RNA double-built library to NGS (Next Generation System) to calibrating lung cancer targeted drug and chemotherapy drug genomes Download PDF

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CN112226496A
CN112226496A CN202011086713.6A CN202011086713A CN112226496A CN 112226496 A CN112226496 A CN 112226496A CN 202011086713 A CN202011086713 A CN 202011086713A CN 112226496 A CN112226496 A CN 112226496A
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李志强
李晓华
陈浩
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Abstract

The invention discloses a method and a system for using a DNA and RNA double-built library for NGS to calibrate lung cancer targeted drug and chemotherapy drug genomes, wherein the method comprises the following steps: directly amplifying genes of known mutation sites, then establishing a library, carrying out sequencing detection on NGS, and determining and verifying the existence of the mutation genes by combining bioinformatics analysis; the method comprises the following steps: s1: extracting nucleic acid and determining the concentration of DNA/RNA; s2: constructing a DNA or RNA library; s3: and (3) machine sequencing: the high-throughput sequencing library is loaded on a computer, and equipment is operated to carry out NGS sequencing; s4: data bioinformatics processing analysis: uploading the Fastq format file obtained by sequencing to a living letter analysis software system matched with the real solid organism for automatic analysis; s5: and (4) quality control. The invention solves the problem that the existing lung cancer drug gene detection system only can aim at targeted drugs or chemotherapeutic drugs, realizes lung cancer chemotherapy and targeted drug gene detection in one project, and has low detection cost and high detection efficiency.

Description

Method and system for applying DNA and RNA double-built library to NGS (Next Generation System) to calibrating lung cancer targeted drug and chemotherapy drug genomes
Technical Field
The invention belongs to the technical field of gene sequencing, and particularly relates to a method and a system for using a DNA and RNA double-built library for NGS to calibrate lung cancer targeted drug and chemotherapy drug genomes.
Background
Tumors are a complex disease with associated gene dysfunction caused by changes in multiple genes, which are the most fundamental causes of tumorigenesis. The tumor patients have obvious individual difference to various medicines and are also related to the individual tumor-related gene differential expression and polymorphism of the patients. The mutation state of tumor related genes plays an important role in the personalized treatment process of diagnosis, treatment, prognosis and the like of tumors, as more and more markers are identified, the treatment mode of the tumors becomes more and more personalized according to patient-specific biomarkers, and on the way of realizing the personalized treatment of the tumors, the Next Generation Sequencing (NGS) technology plays an irreplaceable role, and the method helps researchers and pathologists to realize the perfect conversion of single-gene detection to multi-gene detection.
Drug safety is the field in which patients first benefit from individualized medication, malignant tumors are the most demanding disease to be treated by individualized drugs at present, however, in tumor chemotherapy, there are great individual differences in both curative effect and toxic and side effects. Through individual gene polymorphism detection and analysis, powerful evidence and information can be provided for doctors, and when a treatment scheme is implemented, a most effective drug treatment scheme for patients can be selected, so that the curative effect is improved, and the toxic and side effects of chemotherapy can be reduced. Currently, even if not all drugs can carry out gene-oriented personalized therapy, safe drug personalized therapy under the guidance of gene detection has significant clinical application significance.
Lung cancer is one of the most rapidly growing malignancies that threaten human health and life. In many countries, the incidence and mortality of lung cancer have been reported to be significantly higher in recent 50 years, with lung cancer incidence and mortality in men accounting for the first of all malignancies, in women accounting for the second, and mortality accounting for the second. The etiology of lung cancer is not completely clear up to now, and a large amount of data show that a large amount of smoking for a long time has a very close relationship with the occurrence of lung cancer. Currently, the current practice is. Chemotherapy is the most main treatment means of lung cancer, more than 90% of lung cancer patients need to receive chemotherapy treatment, the curative effect of chemotherapy on lung cancer is more certain in early stage or late stage, even about 1% of early stage small cell lung cancer is cured by chemotherapy, and the tumor remission rate on non-small cell lung cancer is 40% -50%. The existing lung cancer drug gene detection system focuses on detection of a single targeted drug or a chemotherapy drug gene, and individual detection combinations aiming at gene mutation also appear, but the existing lung cancer drug gene detection system is mainly designed aiming at pan-cancer species, is limited to conventional mutations such as detection point mutation and the like, cannot detect gene variation related to diseases caused by gene fusion mutation and splicing variation, and has low detection efficiency and high detection cost.
The detection system of the invention is to use PCR to amplify DNA to construct DNA library, and simultaneously use reverse transcription of mRNA to complementary DNA (cDNA), and then PCR amplification fusion and different shearing recombination of DNA (RNA library construction). And performing high-throughput sequencing and analysis on the target sequence by the two kinds of library nucleic acids by adopting an Illumina platform so as to obtain mutation information of the target gene. The Illumina new generation sequencing technology can carry out deep sequencing on nucleic acid fragments in high throughput and in parallel, the technical principle of sequencing is that reversible terminal sequencing reaction is adopted while synthesis is carried out, firstly, universal joints with known sequences and beacon sequences are added at two ends of a DNA fragment to construct a library, the library is loaded on a sequencing chip Flowcell, the known sequences at the two ends of the library are complementary with an Oligo sequence on a Flowcell substrate, each library fragment is amplified through bridge PCR to form a cluster, sequencing reaction while synthesis is carried out, namely, in the base extension process, only one correct complementary base can be extended in each cycle reaction, the base types are confirmed according to different fluorescent signals, the quality of the final nucleic acid sequence is ensured, and the nucleic acid sequence is completely read after multiple cycles.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a method and a system for applying a DNA and RNA double-built library to NGS (natural gas chromatography) to demarcate drug genomes for targeting and chemotherapy of lung cancer.
The technical scheme of the invention is summarized as follows:
a method for using a DNA and RNA double-built library for NGS to calibrate lung cancer targeted drug and chemotherapy drug genomes is provided: directly amplifying genes of known mutation sites, then establishing a library, carrying out sequencing detection on NGS, and determining and verifying the existence of the mutation genes by combining bioinformatics analysis; the method specifically comprises the following steps:
s1: nucleic acid extraction: extracting DNA according to FFPE tissue genome DNA extraction standard operation process, extracting RNA according to tissue RNA extraction standard operation process, and determining the concentration of DNA/RNA;
s2: library construction:
construction of a DNA library: carrying out specific PCR amplification reaction and specific primer digestion on the genome DNA, purifying the product, carrying out secondary PCR amplification reaction on the product, and purifying and controlling the library;
the specific PCR reaction system comprises a high-fidelity PCR mixed solution, a CHS298 primer and DNA according to the ratio of (10-14): (3-5): (6.5-9.5) by volume ratio;
the secondary PCR reaction system comprises an annealing PCR Master Mix, nuclease-free water, a Pi700 sequence tag, a Pi500 sequence tag and a purified product according to the ratio of (20-30): (12-16): (3-5): (3-5): (2.5-3.5) by volume ratio;
construction of RNA library: reverse transcription is carried out by taking RNA as a template to synthesize cDNA, and then a specific PCR amplification reaction and a specific primer digestion are carried out, after a product is purified, a secondary PCR amplification reaction is carried out on the product, and a library is purified and quality controlled;
wherein the specific PCR reaction system comprises a high-fidelity PCR mixed solution, 5 xSF primers and cDNA according to the ratio of (20-30): (8-12): (14-16) by volume;
the secondary PCR reaction system comprises an annealing PCR Master Mix, nuclease-free water, a Pi700 sequence tag, a Pi500 sequence tag and a purified product according to the ratio of (20-30): (4-6): (3-5): (3-5): (11-13) by volume;
s3: and (3) machine sequencing: loading the high-throughput sequencing library on a computer, operating equipment, and sequencing on a Miniseq platform of Illumina;
s4: data bioinformatics processing analysis: uploading the Fastq format file obtained by sequencing to a living letter analysis software system matched with the real solid organism for automatic analysis;
s5: quality control: positive quality control: taking nucleic acid of a known mutation sample in the panel detection range as a template, and carrying out parallel experiments with the sample, wherein 1 nucleic acid is obtained in each batch; negative quality control: and (3) taking the mutation sample nucleic acid within the non-detection range or outside the detection range as a template, and carrying out parallel experiments with the sample, wherein 1 in each batch.
Preferably, the operation method for determining the concentration of DNA/RNA comprises: use of
Figure BDA0002720601900000021
dsDNA/
Figure BDA0002720601900000022
RNA high-sensitivity kit and
Figure BDA0002720601900000023
3.0Fluorometer to determine the DNA/RNA concentration, the specific procedure is as follows:
a) adding 1 mu L of the Qubit Reagent into 199 mu L of the Qubit Buffer, fully oscillating, and carrying out microcentrifugation to obtain a Qubit fluorescent mixture;
b) to 2 Qubit concentration determination dedicated tubes were added 190. mu.L of the Qubit fluorescent mixture, labeled S1, S2, to S1 labeled dedicated tube 10. mu.L of Standard 1, to S2 labeled dedicated tube 10. mu.L of Standard 2, respectively;
c) adding 199 mu L of the Qubit fluorescent mixture into the other 2 tubes special for the Qubit concentration determination, and adding 1 mu L of the DNA/RNA sample;
d) is connected to
Figure BDA0002720601900000024
3.0Fluorometer Power supply, select Main worldThe sample type on the face, such as DNA or RNA, was selected for dsDNA/RNA High Sensitivity option, and the S1, S2 assay tubes and sample tubes were added in the order suggested and concentration data recorded.
Preferably, the method for purifying and controlling the library in the DNA or RNA library construction comprises the following steps:
a. mixing 50.0 μ L of Agencour AMPure XP beads with the product at room temperature, and incubating at room temperature for 5 min;
b. placing on magnetic frame for 5min, and discarding supernatant;
c. adding 150.0 μ L70% ethanol, standing for 30s, and removing supernatant;
d. adding 150.0 μ L70% ethanol, standing for 30s, and removing supernatant;
e. standing at room temperature for 2-5min until the magnetic beads are dried;
f. adding 32.0 μ L ddH2O, resuspending magnetic beads, standing at room temperature for 5min, centrifuging, and standing on magnetic frame for 2min until the supernatant is clear;
g. transfer 30.0. mu.L of the supernatant to a new 0.2mL PCR tube;
h. the final library was concentration-measured with qubit 3.0;
i. library fragment sizes were measured using an Agilent 2100 bioanalyzer.
Preferably, the specific primer digestion method in the DNA library construction comprises the following steps: and (3) dropwise adding Exonase and 10x Exonase I buffer into the mixed solution obtained after 25 mu L of specific PCR amplification, performing oscillation reaction at 37 ℃ for 20min, heating to 80 ℃, performing oscillation reaction for 10min, and cooling to 8 ℃ for later use.
Preferably, the method for purifying the product in the DNA library construction comprises the following steps:
a. adding 20 μ L of nuclease-free water to the specific primer digest;
b. balancing Agencour AMPure XP beads to room temperature in advance, adding 60 mu L of the Agencour AMPure XP beads into a product, uniformly mixing, and incubating for 5min at room temperature;
c. placing on magnetic frame for 5min, and discarding supernatant;
d. adding 150.0 μ L of freshly prepared 70% ethanol, standing for 30s, and removing the supernatant;
e. adding 150.0 μ L of freshly prepared 70% ethanol, standing for 30s, and removing the supernatant;
f. standing at room temperature for 2-5min, and adding 32 μ L ddH after the magnetic beads are dried2And O, resuspending the magnetic beads, standing for 5min at room temperature, centrifuging, and placing on a magnetic frame for 5min until the supernatant is clear.
Preferably, the specific primer digestion method in the RNA library construction comprises the following steps: and (3) dropwise adding Exonase and 10x Exonase I buffer into the mixed solution after 50 mu L of specific PCR amplification, performing oscillation reaction at 37 ℃ for 20min, heating to 80 ℃, performing oscillation reaction for 10min, and cooling to 8 ℃ for later use.
Preferably, the DNA library is constructed, wherein the specific PCR reaction procedure is: hot start at 95 ℃ for 15 minutes; denaturation at 98 ℃ for 1 min; annealing at 58 ℃ for 2 minutes; annealing at 60 ℃ for 4 minutes; annealing at 64 ℃ for 1 minute; extension at 72 ℃ for 1 min; amplifying for 5 cycles; denaturation at 95 ℃ for 30 seconds; annealing and extending at 66 ℃ for 3 minutes; amplifying for 18 cycles;
preserving at 8 ℃ and circulating infinitely;
the secondary PCR reaction procedure was: hot start at 95 ℃ for 2 minutes; denaturation at 95 ℃ for 30 seconds; annealing at 66 ℃ for 30 seconds; extension at 72 ℃ for 60 seconds; amplifying for 6 cycles; final extension at 72 ℃ for 5 min; preserving at 8 ℃ and circulating infinitely;
constructing the RNA library, wherein a specific PCR reaction program comprises the following steps: hot start at 95 ℃ for 15 minutes; denaturation at 95 ℃ for 1 min; annealing at 58 ℃ for 1 minute; annealing at 60 ℃ for 2 minutes; annealing at 64 ℃ for 30 seconds; extension at 72 ℃ for 1 min; amplifying for 5 cycles; denaturation at 95 ℃ for 30 seconds; annealing and extending for 3 minutes at 66 ℃; amplifying for 24 cycles; preserving at 8 ℃ and circulating infinitely;
the secondary PCR reaction procedure was: hot start at 95 ℃ for 2 minutes; denaturation at 95 ℃ for 30 seconds; annealing at 66 ℃ for 30 seconds; extension at 72 ℃ for 60 seconds; amplifying for 5 cycles; final extension at 72 ℃ for 5 min; storing at 8 deg.C, and circulating indefinitely.
Preferably, the method for purifying the product in the construction of the RNA library comprises the following steps:
a. balancing the Agencour AMPure XP beads to room temperature in advance, adding 66 mu L of the Agencour AMPure XP beads into a product, uniformly mixing, and incubating for 5min at room temperature;
b. placing on magnetic frame for 5min, and discarding supernatant;
c. adding 150.0 μ L of freshly prepared 70% ethanol, standing for 30s, and removing the supernatant;
d. adding 150.0 μ L of freshly prepared 70% ethanol, standing for 30s, and removing the supernatant;
e. standing at room temperature for 2-5min, and adding 32 μ L ddH after the magnetic beads are dried2And O, resuspending the magnetic beads, standing for 5min at room temperature, centrifuging, and placing on a magnetic frame for 5min until the supernatant is clear.
Preferably, the high throughput sequencing library is prepared by the following method:
a. the library was first diluted to 1.0nM with RSB buffer;
b. mixing 5 μ L of 1.0nM library with 5 μ L of 0.2N NaOH, standing at room temperature for 5min for denaturation;
c. adding 5 μ L of 200mM Tris-HCl, adding 985 μ L of Hybridization Buffer into the denatured library, shaking and mixing, placing on ice to prevent renaturation, and waiting for further dilution;
d. 120 μ L of the diluted library was mixed with 380 μ L Hybridization Buffer to obtain 0.5mL high throughput sequencing library.
Also provides a system for using the DNA and RNA double-built library for NGS to calibrate lung cancer targeted drug and chemotherapy drug genomes, which comprises the following steps:
the nucleic acid extraction module is used for extracting DNA according to FFPE tissue genome DNA extraction standard operation flow, extracting RNA according to tissue RNA extraction standard operation flow and determining the concentration of the DNA/RNA;
a library construction module for:
construction of a DNA library: carrying out specific PCR amplification reaction and specific primer digestion on the genome DNA, purifying the product, carrying out secondary PCR amplification reaction on the product, and purifying and controlling the library;
the specific PCR reaction system comprises a high-fidelity PCR mixed solution, a CHS298 primer and DNA according to the ratio of (10-14): (3-5): (6.5-9.5) by volume ratio;
the secondary PCR reaction system comprises an annealing PCR Master Mix, nuclease-free water, a Pi700 sequence tag, a Pi500 sequence tag and a purified product according to the ratio of (20-30): (12-16): (3-5): (3-5): (2.5-3.5) by volume ratio;
construction of RNA library: reverse transcription is carried out by taking RNA as a template to synthesize cDNA, and then a specific PCR amplification reaction and a specific primer digestion are carried out, after a product is purified, a secondary PCR amplification reaction is carried out on the product, and a library is purified and quality controlled;
wherein the specific PCR reaction system comprises a high-fidelity PCR mixed solution, 5 xSF primers and cDNA according to the ratio of (20-30): (8-12): (14-16) by volume;
the secondary PCR reaction system comprises an annealing PCR Master Mix, nuclease-free water, a Pi700 sequence tag, a Pi500 sequence tag and a purified product according to the ratio of (20-30): (4-6): (3-5): (3-5): (11-13) by volume;
a loading sequencing module, which is used for loading the high-throughput sequencing library, running the equipment and sequencing on a Miniseq platform of Illumina;
the processing and analyzing module is used for uploading the Fastq format file obtained by sequencing to a living letter analysis software system matched with the real and solid organisms for automatic analysis; and
a quality control module for:
positive quality control: taking nucleic acid of a known mutation sample in the panel detection range as a template, and carrying out parallel experiments with the sample, wherein 1 nucleic acid is obtained in each batch; negative quality control: and (3) taking the mutation sample nucleic acid within the non-detection range or outside the detection range as a template, and carrying out parallel experiments with the sample, wherein 1 in each batch.
The invention has the beneficial effects that:
the invention solves the problem that the existing lung cancer drug gene detection system can only detect and use the non-structural gene mutation (point mutation, deletion and insertion mutation, frameshift mutation and the like) for targeted drugs or chemotherapeutic drugs, can discover and quantitatively detect the gene fusion mutation reflected by a new transcript and abnormal mutation formed by different shearing, combines the structural gene mutation (gene fusion mutation, gene shearing mutation and the like) and the change of the expression quantity thereof with the non-structural gene mutation for lung cancer chemotherapy and targeted drug gene detection, and detects multiple mutation types of lung cancer chemotherapy and targeted drug genes in one project; secondly, the invention solves the problems that the detection range of the small set of lung cancer is insufficient and the cost is increased due to excessive detection content of the large set of lung cancer; according to the invention, the nucleic acid in the detection area is subjected to gene amplification firstly, and then the nucleic acid is subjected to on-machine sequencing analysis, so that the detection quality is ensured, meanwhile, the output of irrelevant data volume is greatly reduced, the detection can be completed on a Miniseq desktop instrument of a relatively low-flux Illumina platform, the detection cost and the equipment requirement are greatly reduced, and the detection and utilization efficiency of the equipment is improved; the invention combines the DNA library construction technology and the RNA library construction technology to select the targeted drug and the chemotherapeutic drug for treating the lung cancer, and is suitable for various medical institutions to apply the NGS technology to guide clinical accurate medication.
Drawings
Fig. 1 is a flowchart of a method for using a DNA and RNA duplex library for NGS targeting drug and chemotherapy drug genomes for NGS calibration.
FIG. 2 is a system of a DNA and RNA dual library for NGS targeting drug and chemotherapy drug genome according to an embodiment of the present invention
Detailed Description
The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.
Example 1
As shown in figure 1, a method for using a DNA and RNA double-built library for NGS to calibrate drug genomes for targeting and chemotherapy of lung cancer is provided: directly amplifying genes of known mutation sites, then establishing a library, carrying out sequencing detection on NGS, and determining and verifying the existence of the mutation genes by combining bioinformatics analysis; the method specifically comprises the following steps:
s1: nucleic acid extraction: extracting DNA according to FFPE tissue genome DNA extraction standard operation process, extracting RNA according to tissue RNA extraction standard operation process, and determining the concentration of DNA/RNA;
the operation method for determining the concentration of the DNA/RNA comprises the following steps: use of
Figure BDA0002720601900000051
dsDNA/
Figure BDA0002720601900000052
RNA HS Assay Kit(
Figure BDA0002720601900000053
dsDNA/
Figure BDA0002720601900000054
RNA high sensitivity kit) and
Figure BDA0002720601900000055
3.0Fluorometer to determine the DNA/RNA concentration, the specific procedure is as follows:
a) adding 1 mu L of the Qubit Reagent into 199 mu L of the Qubit Buffer, fully oscillating, and carrying out microcentrifugation to obtain a Qubit fluorescent mixture;
b) to 2 Qubit concentration determination dedicated tubes were added 190. mu.L of the Qubit fluorescent mixture, labeled S1, S2, to S1 labeled dedicated tube 10. mu.L of Standard 1, to S2 labeled dedicated tube 10. mu.L of Standard 2, respectively;
c) adding 199 mu L of the Qubit fluorescent mixture into the other 2 tubes special for the Qubit concentration determination, and adding 1 mu L of the DNA/RNA sample;
d) is connected to
Figure BDA0002720601900000056
3.0Fluorometer power, selecting sample type on the main interface, such as DNA or RNA, selecting dsDNA/RNA High Sensitivity option, adding S1 and S2 measuring tube and sample tube according to the prompt sequence, and recording concentration data;
s2: library construction:
construction of a DNA library: carrying out specific PCR amplification reaction and specific primer digestion on the genome DNA, purifying the product, carrying out secondary PCR amplification reaction on the product, and purifying and controlling the library;
wherein, the specific PCR reaction system is as follows: 12.5 μ L GS PCR MMX (high fidelity PCR cocktail) +4.5 μ L CHS298 oligo pool (CHS298 primer) +8 μ L DNA;
the specific PCR reaction program is as follows:
[95℃,15min]；
[98℃,1min；58℃,2min；60℃,4min；64℃,1min；72℃,1min]5cycles；
[95℃,30s；66℃,3min]18cycles；
[8℃,∞]；
the specific primer digestion method comprises the following steps: dripping 3 mu L of Exonaclease and 2 mu L of 10x Exonaclease I buffer into 25 mu L of the mixed solution after the specific PCR amplification, performing oscillation reaction at 37 ℃ for 20min, heating to 80 ℃, performing oscillation reaction for 10min, and cooling to 8 ℃ for later use;
a. adding 20 μ L of nuclease-free water to the specific primer digest;
b. balancing Agencour AMPure XP beads to room temperature in advance, adding 60 mu L of the Agencour AMPure XP beads into a product, uniformly mixing, and incubating for 5min at room temperature;
c. placing on magnetic frame for 5min, and discarding supernatant;
d. adding 150.0 μ L of freshly prepared 70% ethanol, standing for 30s, and removing the supernatant;
e. adding 150.0 μ L of freshly prepared 70% ethanol, standing for 30s, and removing the supernatant;
f. standing at room temperature for 2-5min, and adding 32 μ L ddH after the magnetic beads are dried2O, resuspending the magnetic beads, standing for 5min at room temperature, centrifuging, and standing for 5min on a magnetic frame until the supernatant is clear;
the secondary PCR reaction system is as follows: 25 μ L of Indexing PCR Master Mix +14 μ L of nucleic-free water) +4 μ L of Pi700 Pillar Index (Pi700 sequence tag) +4 μ L of Pi500 Pillar Index (Pi500 sequence tag) +3 μ L of purified product;
the secondary PCR reaction procedure was:
[95℃,2min]；
[95℃,30s；66℃,30s；72℃,60s]6cycles；
[72℃,5min]；
[8℃,∞]；
construction of RNA library: reverse transcription is carried out by taking RNA as a template to synthesize cDNA, and then a specific PCR amplification reaction and a specific primer digestion are carried out, after a product is purified, a secondary PCR amplification reaction is carried out on the product, and a library is purified and quality controlled;
wherein, the specific PCR reaction system is as follows: 25 μ L GS PCR MMX (high fidelity PCR cocktail) +10 μ L5 × SF oligo pool (SF primer) +15 μ LcDNA;
the specific PCR reaction program is as follows:
[95℃,15min]；
[95℃,1min；58℃,1min；60℃,2min；64℃,30s；72℃,1min]5cycles；
[95℃,30s；66℃,3min]24cycles；
[8℃,∞]；
the specific primer digestion method in the construction of the RNA library comprises the following steps: dripping 3 mu L of Exonaclease and 2 mu L of 10x Exonaclease I buffer into 50 mu L of the mixed solution after the specific PCR amplification, performing oscillation reaction at 37 ℃ for 20min, heating to 80 ℃, performing oscillation reaction for 10min, and cooling to 8 ℃ for later use;
the method for purifying the product in the construction of the RNA library comprises the following steps:
a. balancing the Agencour AMPure XP beads to room temperature in advance, adding 66 mu L of the Agencour AMPure XP beads into a product, uniformly mixing, and incubating for 5min at room temperature;
b. placing on magnetic frame for 5min, and discarding supernatant;
c. adding 150.0 μ L of freshly prepared 70% ethanol, standing for 30s, and removing the supernatant;
d. adding 150.0 μ L of freshly prepared 70% ethanol, standing for 30s, and removing the supernatant;
e. standing at room temperature for 2-5min, and adding 32 μ L ddH after the magnetic beads are dried2O, resuspending the magnetic beads, standing for 5min at room temperature, centrifuging, and standing for 5min on a magnetic frame until the supernatant is clear;
the secondary PCR reaction system is as follows: 25 μ L of Indexing PCR Master Mix +5 μ L of Nuclear-free water +4 μ L of Pi700 Pillar Index +4 μ L of Pi500 Pillar Index +12 μ L of purified product;
the secondary PCR reaction procedure was:
[95℃,2min]；
[95℃,30s；66℃,30s；72℃,60s]5cycles；
[72℃,5min]；
[8℃,∞]；
the method for purifying and controlling the library in the construction of the DNA or RNA library comprises the following steps:
a. mixing 50.0 μ L of Agencour AMPure XP beads with the product at room temperature, and incubating at room temperature for 5 min;
b. placing on magnetic frame for 5min, and discarding supernatant;
c. adding 150.0 μ L70% ethanol, standing for 30s, and removing supernatant;
d. adding 150.0 μ L70% ethanol, standing for 30s, and removing supernatant;
e. standing at room temperature for 2-5min until the magnetic beads are dried;
f. adding 32.0 μ L ddH2O, resuspending magnetic beads, standing at room temperature for 5min, centrifuging, and standing on magnetic frame for 2min until the supernatant is clear;
g. transfer 30.0. mu.L of the supernatant to a new 0.2mL PCR tube;
h. the final library was concentration-measured with qubit 3.0;
i. detecting the size of the library fragment by adopting an Agilent 2100 bioanalyzer;
s3: and (3) machine sequencing: loading the high-throughput sequencing library on a computer, operating equipment, and performing Illumina platform sequencing;
the preparation method of the high-throughput sequencing library comprises the following steps:
a. the library was first diluted to 1.0nM with RSB buffer;
b. mixing 5 μ L of 1.0nM library with 5 μ L of 0.2N NaOH, standing at room temperature for 5min for denaturation;
c. adding 5 μ L of 200mM Tris-HCl, adding 985 μ L of Hybridization Buffer into the denatured library, shaking and mixing, placing on ice to prevent renaturation, and waiting for further dilution;
d. taking 120 mu L of the diluted library and 380 mu L of Hybridization Buffer to be fully and uniformly mixed, and obtaining 0.5mL of high-throughput sequencing library;
s4: data bioinformatics processing analysis: uploading the Fastq format file obtained by sequencing to a living letter analysis software system matched with the real solid organism for automatic analysis;
s5: quality control:
positive quality control: taking nucleic acid of a known mutation sample in the panel detection range as a template, and carrying out parallel experiments with the sample, wherein 1 nucleic acid is obtained in each batch;
negative quality control: and (3) taking the mutation sample nucleic acid within the non-detection range or outside the detection range as a template, and carrying out parallel experiments with the sample, wherein 1 in each batch. The list of reagents is shown in table 1:
TABLE 1 list of reagents for testing
Figure BDA0002720601900000081
Table 2 lists the detected gene names and mutation types:
TABLE 2 detection of Gene name and mutation type
Figure BDA0002720601900000082
Figure BDA0002720601900000091
Referring to fig. 2, another embodiment of the present invention further provides a system for dual DNA and RNA library for NGS targeting drug and chemotherapy drug genomes for lung cancer, comprising:
the nucleic acid extraction module is used for extracting DNA according to FFPE tissue genome DNA extraction standard operation flow, extracting RNA according to tissue RNA extraction standard operation flow and determining the concentration of the DNA/RNA;
a library construction module for:
construction of a DNA library: carrying out specific PCR amplification reaction and specific primer digestion on the genome DNA, purifying the product, carrying out secondary PCR amplification reaction on the product, and purifying and controlling the library;
the specific PCR reaction system comprises a high-fidelity PCR mixed solution, a CHS298 primer and DNA according to the ratio of (10-14): (3-5): (6.5-9.5) by volume ratio;
the secondary PCR reaction system comprises an annealing PCR Master Mix, nuclease-free water, a Pi700 sequence tag, a Pi500 sequence tag and a purified product according to the ratio of (20-30): (12-16): (3-5): (3-5): (2.5-3.5) by volume ratio;
construction of RNA library: reverse transcription is carried out by taking RNA as a template to synthesize cDNA, and then a specific PCR amplification reaction and a specific primer digestion are carried out, after a product is purified, a secondary PCR amplification reaction is carried out on the product, and a library is purified and quality controlled;
wherein the specific PCR reaction system comprises a high-fidelity PCR mixed solution, 5 xSF primers and cDNA according to the ratio of (20-30): (8-12): (14-16) by volume;
the secondary PCR reaction system comprises an annealing PCR Master Mix, nuclease-free water, a Pi700 sequence tag, a Pi500 sequence tag and a purified product according to the ratio of (20-30): (4-6): (3-5): (3-5): (11-13) by volume;
a loading sequencing module, which is used for loading the high-throughput sequencing library, running the equipment and sequencing on a Miniseq platform of Illumina;
the processing and analyzing module is used for uploading the Fastq format file obtained by sequencing to a living letter analysis software system matched with the real and solid organisms for automatic analysis; and
a quality control module for:
positive quality control: taking nucleic acid of a known mutation sample in the panel detection range as a template, and carrying out parallel experiments with the sample, wherein 1 nucleic acid is obtained in each batch; negative quality control: and (3) taking the mutation sample nucleic acid within the non-detection range or outside the detection range as a template, and carrying out parallel experiments with the sample, wherein 1 in each batch.
The implementation process and the working principle of the system for applying the DNA and RNA double-built library to the NGS-calibrated lung cancer targeted drug and chemotherapy drug genome according to the embodiment of the invention can refer to any one of the above descriptions of the method for applying the DNA and RNA double-built library to the NGS-calibrated lung cancer targeted drug and chemotherapy drug genome, and are not described herein again.
While embodiments of the invention have been disclosed above, it is not limited to the applications listed in the description and the embodiments, which are fully applicable in all kinds of fields of application of the invention, and further modifications may readily be effected by those skilled in the art, so that the invention is not limited to the specific details without departing from the general concept defined by the claims and the scope of equivalents.

Claims (10)

1.一种DNA和RNA双建库用于NGS标定肺癌靶向用药和化疗用药基因组的方法，其特征在于：通过直接对已知突变位点的基因进行扩增后建库，并进行NGS的测序检测，再结合生物信息学分析确定并验证突变基因的存在；具体包括以下步骤：1. a kind of DNA and RNA double-building library is used for NGS to demarcate the method for lung cancer targeted medicine and chemotherapeutic medicine genome, it is characterized in that: build library after the gene of known mutation site is directly amplified, and carry out NGS Sequencing detection, combined with bioinformatics analysis to determine and verify the existence of the mutated gene; specifically include the following steps: S1：核酸提取：按照《FFPE组织基因组DNA提取标准操作流程》提取DNA，按照《组织RNA提取标准操作流程》提取RNA，并测定DNA/RNA的浓度；S1: Nucleic acid extraction: DNA is extracted according to the "Standard Operating Procedures for FFPE Tissue Genomic DNA Extraction", RNA is extracted according to the "Standard Operating Procedures for Tissue RNA Extraction", and the concentration of DNA/RNA is determined; S2：文库构建：S2: Library Construction: DNA文库构建：对基因组DNA进行特异性PCR扩增反应、特异性引物消化，纯化产物后，再对其进行二次PCR扩增反应，纯化并质控文库；DNA library construction: perform specific PCR amplification on genomic DNA, digest with specific primers, purify the product, and then perform a secondary PCR amplification reaction to purify and quality control the library; 其中，特异性PCR反应体系由高保真PCR混合液、CHS298引物、DNA按照(10～14)：(3～5)：(6.5～9.5)的体积比混合而成；Wherein, the specific PCR reaction system is formed by mixing high-fidelity PCR mixture, CHS298 primers and DNA according to the volume ratio of (10-14):(3-5):(6.5-9.5); 二次PCR反应体系由Indexing PCR Master Mix、无核酸酶水、Pi700序列标签、Pi500序列标签、纯化后产物按照(20～30)：(12～16)：(3～5)：(3～5)：(2.5～3.5)的体积比混合而成；The secondary PCR reaction system consists of Indexing PCR Master Mix, nuclease-free water, Pi700 sequence tag, Pi500 sequence tag, and purified products according to (20-30): (12-16): (3-5): (3-5 ): (2.5～3.5) volume ratio is mixed; RNA文库构建：以RNA为模板反转录合成cDNA，并进行特异性PCR扩增反应、特异性引物消化，纯化产物后，再对其进行二次PCR扩增反应，纯化并质控文库；RNA library construction: cDNA is synthesized by reverse transcription using RNA as a template, and specific PCR amplification reaction, specific primer digestion is performed, and the product is purified, and then a secondary PCR amplification reaction is performed to purify and control the library; 其中，特异性PCR反应体系由高保真PCR混合液、5x SF引物、cDNA按照(20～30)：(8～12)：(14～16)的体积比混合而成；The specific PCR reaction system is composed of high-fidelity PCR mixture, 5x SF primers, and cDNA in a volume ratio of (20-30): (8-12): (14-16); 二次PCR反应体系由Indexing PCR Master Mix、无核酸酶水、Pi700序列标签、Pi500序列标签、纯化后产物按照(20～30)：(4～6)：(3～5)：(3～5)：(11～13)的体积比混合而成；The secondary PCR reaction system is composed of Indexing PCR Master Mix, nuclease-free water, Pi700 sequence tag, Pi500 sequence tag, and purified products according to (20-30): (4-6): (3-5): (3-5 ): (11～13) volume ratio is mixed; S3：上机测序：将高通量测序文库上机，并运行设备，在Illumina的Miniseq平台测序；S3: On-board sequencing: put the high-throughput sequencing library onboard, run the equipment, and sequence on the Illumina Miniseq platform; S4：数据生物信息学处理分析：将测序得到的Fastq格式文件上传至真固生物配套的生信分析软件系统进行自动分析即可；S4: Data bioinformatics processing and analysis: Upload the Fastq format file obtained by sequencing to the bioinformatics analysis software system of True Solid Biotechnology for automatic analysis; S5：质控：阳性质控：以panel检测范围内已知突变标本核酸为模板，与样本平行实验，每批次1个；阴性质控：以无检测范围内或检测范围外突变标本核酸为模板，与样本平行实验，每批次1个。S5: Quality control: Positive quality control: Take the nucleic acid of the known mutation sample within the detection range of the panel as the template, and parallel experiments with the sample, 1 per batch; Template, parallel experiments with samples, 1 per batch. 2.根据权利要求1所述一种DNA和RNA双建库用于NGS标定肺癌靶向用药和化疗用药基因组的方法，其特征在于，所述测定DNA/RNA的浓度操作方法为：使用
Figure FDA0002720601890000011
高灵敏试剂盒及
Figure FDA0002720601890000012
Fluorometer测定DNA/RNA浓度，具体操作如下：2. a kind of DNA and RNA double-building library according to claim 1 is used for NGS demarcation lung cancer targeted medicine and the method for chemotherapy medicine genome, it is characterized in that, the concentration operation method of described measuring DNA/RNA is: use
Figure FDA0002720601890000011
Highly sensitive kits and
Figure FDA0002720601890000012
Fluorometer measures DNA/RNA concentration as follows: a)将1μL Qubit Reagent加入199μL Qubit Buffer，充分震荡，微离心，得到Qubit荧光混合物；a) Add 1 μL of Qubit Reagent to 199 μL of Qubit Buffer, shake well, and microcentrifuge to obtain Qubit fluorescence mixture; b)向2个Qubit浓度测定专用管中分别加入190μL Qubit荧光混合物，标记为S1、S2，向S1标记专用管中加入10μL Standard 1，向S2标记专用管中加入10μL Standard 2；b) Add 190 μL of Qubit fluorescence mixture to 2 special tubes for measuring Qubit concentration, marked as S1 and S2, add 10 μL of Standard 1 to the special tube marked with S1, and add 10 μL of Standard 2 to the special tube marked with S2; c)向另外2个Qubit浓度测定专用管中分别加入199μL Qubit荧光混合物，加入1μLDNA/RNA样本；c) Add 199 μL of Qubit fluorescence mixture and 1 μL of DNA/RNA samples to the other 2 tubes dedicated to the determination of Qubit concentration; d)接通
Figure FDA0002720601890000013
Fluorometer电源，选择主界面上的样本类型，如DNA或RNA，再选择dsDNA/RNA High Sensitivity选项，按提示顺序加入S1、S2测定管及样品管，记录浓度数据。d) switch on
Figure FDA0002720601890000013
Fluorometer power supply, select the sample type on the main interface, such as DNA or RNA, and then select the dsDNA/RNA High Sensitivity option, add S1, S2 measuring tubes and sample tubes in the order indicated, and record the concentration data. 3.根据权利要求1所述一种DNA和RNA双建库用于NGS标定肺癌靶向用药和化疗用药基因组的方法，其特征在于，所述DNA或RNA文库构建中纯化并质控文库方法为：3. a kind of DNA and RNA double building library according to claim 1 is used for NGS demarcation lung cancer targeted medicine and the method for chemotherapy medicine genome, it is characterized in that, in described DNA or RNA library construction, purify and quality control library method is : a.室温下，取50.0μL Agencourt AMPure XP beads与产物混匀，室温孵育5min；a. At room temperature, mix 50.0 μL Agencourt AMPure XP beads with the product, and incubate at room temperature for 5 minutes; b.置于磁力架上5min，弃上清；b. Place on a magnetic stand for 5 minutes, discard the supernatant; c.加入150.0μL 70％乙醇，静置30s，弃上清；c. Add 150.0 μL of 70% ethanol, let stand for 30s, and discard the supernatant; d.加入150.0μL 70％乙醇，静置30s，弃上清；d. Add 150.0 μL of 70% ethanol, let stand for 30s, and discard the supernatant; e.室温放置2～5min，待磁珠干燥；e. Place at room temperature for 2-5 minutes, and wait for the magnetic beads to dry; f.加入32.0μL ddH2O，重悬磁珠，室温静置5min，离心后磁力架上放置2min至上清澄清；f. Add 32.0 μL ddH 2 O, resuspend the magnetic beads, let stand for 5 min at room temperature, and place on the magnetic stand for 2 min after centrifugation until the supernatant is clear; g.转移30.0μL上清至新0.2mL PCR管中；g. Transfer 30.0 μL of supernatant to a new 0.2 mL PCR tube; h.用Qubit3.0将终文库进行浓度测定；h. Use Qubit3.0 to determine the concentration of the final library; i.采用Agilent 2100生物分析仪检测文库片段大小。i. Use an Agilent 2100 Bioanalyzer to detect the size of the library fragments. 4.根据权利要求1所述一种DNA和RNA双建库用于NGS标定肺癌靶向用药和化疗用药基因组的方法，其特征在于，所述DNA文库构建中特异性引物消化方法为：向25μL特异性PCR扩增后混合液中滴加3μL Exonuclease、2μL10x Exonuclease I buffer，于37℃震荡反应20min，升温至80℃，震荡反应10min，再降温至8℃，备用。4. a kind of DNA and RNA double-building library according to claim 1 is used for NGS to demarcate the method for lung cancer targeted medicine and chemotherapy medicine genome, it is characterized in that, in described DNA library construction, specific primer digestion method is: to 25 μL After specific PCR amplification, 3 μL of Exonuclease and 2 μL of 10x Exonuclease I buffer were added dropwise to the mixture, shaken at 37°C for 20 minutes, heated to 80°C, shaken for 10 minutes, and then cooled to 8°C for later use. 5.根据权利要求1所述一种DNA和RNA双建库用于NGS标定肺癌靶向用药和化疗用药基因组的方法，其特征在于，所述DNA文库构建中纯化产物方法为：5. a kind of DNA and RNA double-building library according to claim 1 is used for NGS to demarcate the method for lung cancer targeted medicine and chemotherapeutic medicine genome, it is characterized in that, in described DNA library construction, purifying product method is: a.加入20μL无核酸酶水至特异性引物消化产物中；a. Add 20 μL of nuclease-free water to the specific primer digestion product; b.将Agencourt AMPure XP beads提前平衡至室温，加入60μL至产物中，混匀，室温孵育5min；b. Equilibrate Agencourt AMPure XP beads to room temperature in advance, add 60 μL to the product, mix well, and incubate at room temperature for 5 minutes; c.置于磁力架上5min，弃上清；c. Place on a magnetic stand for 5 minutes, discard the supernatant; d.加入150.0μL新鲜配置的70％乙醇，静置30s，弃上清；d. Add 150.0 μL of freshly prepared 70% ethanol, let stand for 30s, and discard the supernatant; e.加入150.0μL新鲜配置的70％乙醇，静置30s，弃上清；e. Add 150.0 μL of freshly prepared 70% ethanol, let stand for 30s, and discard the supernatant; f.室温放置2-5min，待磁珠干燥，再加入32μL ddH2O，重悬磁珠，于室温静置5min，离心后磁力架上放置5min至上清澄清。f. Leave at room temperature for 2-5min. After the magnetic beads are dry, add 32 μL of ddH 2 O to resuspend the magnetic beads and let stand for 5 minutes at room temperature. After centrifugation, place on a magnetic stand for 5 minutes until the supernatant is clear. 6.根据权利要求1所述一种DNA和RNA双建库用于NGS标定肺癌靶向用药和化疗用药基因组的方法，其特征在于，所述RNA文库构建中特异性引物消化方法为：向50μL特异性PCR扩增后混合液中滴加3μL Exonuclease、2μL10x Exonuclease I buffer，于37℃震荡反应20min，升温至80℃，震荡反应10min，再降温至8℃，备用。6. a kind of DNA and RNA double building library according to claim 1 is used for NGS demarcation lung cancer targeted medicine and the method for chemotherapy medicine genome, it is characterized in that, in described RNA library construction, specific primer digestion method is: to 50 μL After specific PCR amplification, 3 μL of Exonuclease and 2 μL of 10x Exonuclease I buffer were added dropwise to the mixture, shaken at 37°C for 20 minutes, heated to 80°C, shaken for 10 minutes, and then cooled to 8°C for later use. 7.根据权利要求1所述一种DNA和RNA双建库用于NGS标定肺癌靶向用药和化疗用药基因组的方法，其特征在于，所述RNA文库构建中纯化产物方法为：7. a kind of DNA and RNA double-building library according to claim 1 is used for NGS to demarcate the method for lung cancer targeted medicine and chemotherapy medicine genome, it is characterized in that, in described RNA library construction, purifying product method is: a.将Agencourt AMPure XP beads提前平衡至室温，加入66μL至产物中，混匀，室温孵育5min；a. Equilibrate Agencourt AMPure XP beads to room temperature in advance, add 66 μL to the product, mix well, and incubate at room temperature for 5 minutes; b.置于磁力架上5min，弃上清；b. Place on a magnetic stand for 5 minutes, discard the supernatant; c.加入150.0μL新鲜配置的70％乙醇，静置30s，弃上清；c. Add 150.0 μL of freshly prepared 70% ethanol, let stand for 30s, and discard the supernatant; d.加入150.0μL新鲜配置的70％乙醇，静置30s，弃上清；d. Add 150.0 μL of freshly prepared 70% ethanol, let stand for 30s, and discard the supernatant; e.室温放置2～5min，待磁珠干燥，再加入32μL ddH2O，重悬磁珠，于室温静置5min，离心后磁力架上放置5min至上清澄清。e. Leave at room temperature for 2 to 5 minutes. After the magnetic beads are dry, add 32 μL of ddH 2 O to resuspend the magnetic beads and let stand for 5 minutes at room temperature. After centrifugation, place on a magnetic stand for 5 minutes until the supernatant becomes clear. 8.根据权利要求1所述一种DNA和RNA双建库用于NGS标定肺癌靶向用药和化疗用药基因组的方法，其特征在于，所述DNA文库构建，其中，特异性PCR反应程序为：95℃热启动，15分钟；98℃变性，1分钟；58℃退火，2分钟；60℃退火，4分钟；64℃退火，1分钟；72℃延伸，1分钟；扩增5个循环；95℃变性，30秒；66℃退火延伸，3分钟；扩增18个循环；8℃保存，无限循环；8. a kind of DNA and RNA double building library according to claim 1 is used for NGS demarcation lung cancer targeted medicine and the method for chemotherapy medicine genome, it is characterized in that, described DNA library is constructed, wherein, specific PCR reaction program is: 95°C hot start, 15 minutes; 98°C denaturation, 1 minute; 58°C annealing, 2 minutes; 60°C annealing, 4 minutes; 64°C annealing, 1 minute; 72°C extension, 1 minute; 5 cycles of amplification; 95 Denaturation at ℃, 30 seconds; annealing and extension at 66℃, 3 minutes; amplification for 18 cycles; storage at 8℃, infinite cycle; 二次PCR反应程序为：95℃热启动，2分钟；95℃变性，30秒；66℃退火，30秒；72℃延伸，60秒；扩增6个循环；72℃终延伸，5分钟；8℃保存，无限循环；The secondary PCR reaction program was: 95°C hot start, 2 minutes; 95°C denaturation, 30 seconds; 66°C annealing, 30 seconds; 72°C extension, 60 seconds; 6 cycles of amplification; 72°C final extension, 5 minutes; Store at 8℃, infinite cycle; 所述RNA文库构建，其中，特异性PCR反应程序为：95℃热启动，15分钟；95℃变性，1分钟；58℃退火，1分钟；60℃退火，2分钟；64℃退火，30秒；72℃延伸，1分钟；扩增5个循环；95℃变性，30秒；66℃退火延伸3分钟；扩增24个循环；8℃保存，无限循环；The RNA library construction, wherein the specific PCR reaction program is: 95°C hot start, 15 minutes; 95°C denaturation, 1 minute; 58°C annealing, 1 minute; 60°C annealing, 2 minutes; 64°C annealing, 30 seconds ; 72°C extension, 1 minute; 5 cycles of amplification; 95°C denaturation, 30 seconds; 66°C annealing and extension for 3 minutes; 24 cycles of amplification; 8°C storage, infinite cycle; 二次PCR反应程序为：95℃热启动，2分钟；95℃变性，30秒；66℃退火，30秒；72℃延伸，60秒；扩增5个循环；72℃终延伸，5分钟；8℃保存，无限循环。The secondary PCR reaction program was: 95°C hot start, 2 minutes; 95°C denaturation, 30 seconds; 66°C annealing, 30 seconds; 72°C extension, 60 seconds; 5 cycles of amplification; 72°C final extension, 5 minutes; Store at 8°C and cycle indefinitely. 9.根据权利要求1所述一种DNA和RNA双建库用于NGS标定肺癌靶向用药和化疗用药基因组的方法，其特征在于，所述高通量测序文库的制备方法为：9. a kind of DNA and RNA double-building library according to claim 1 is used for NGS demarcation lung cancer targeted medicine and the method for chemotherapy medicine genome, it is characterized in that, the preparation method of described high-throughput sequencing library is: a.用RSB buffer先将文库稀释成1.0nM；a. Dilute the library to 1.0nM with RSB buffer; b.将5μL 1.0nM文库与5μL 0.2N NaOH充分混匀，室温静置5min变性；b. Thoroughly mix 5μL of 1.0nM library with 5μL of 0.2N NaOH, and let stand at room temperature for 5min to denature; c.加入5μL 200mM Tris-HCl，将985μL Hybridization Buffer加入到变性完毕的文库中，振荡混匀，放置冰上防止复性，待进一步稀释；c. Add 5μL of 200mM Tris-HCl, add 985μL of Hybridization Buffer to the denatured library, shake and mix well, place on ice to prevent renaturation, and wait for further dilution; d.取120μL上述稀释的文库与380μL Hybridization Buffer充分混匀，即得0.5mL高通量测序文库。d. Take 120 μL of the above-diluted library and mix well with 380 μL of Hybridization Buffer to obtain 0.5 mL of high-throughput sequencing library. 10.一种DNA和RNA双建库用于NGS标定肺癌靶向用药和化疗用药基因组的系统，其特征在于，包括：10. A system of DNA and RNA double building library for NGS demarcation of lung cancer targeted drug and chemotherapy drug genome, is characterized in that, comprising: 核酸提取模块，用于按照《FFPE组织基因组DNA提取标准操作流程》提取DNA，按照《组织RNA提取标准操作流程》提取RNA，并测定DNA/RNA的浓度；The nucleic acid extraction module is used to extract DNA according to the "Standard Operating Procedure for Extracting FFPE Tissue Genomic DNA", extract RNA according to the "Standard Operating Procedure for Tissue RNA Extraction", and determine the concentration of DNA/RNA; 文库构建模块，用于：Library building blocks for: DNA文库构建：对基因组DNA进行特异性PCR扩增反应、特异性引物消化，纯化产物后，再对其进行二次PCR扩增反应，纯化并质控文库；DNA library construction: perform specific PCR amplification on genomic DNA, digest with specific primers, purify the product, and then perform a secondary PCR amplification reaction to purify and quality control the library; 其中，特异性PCR反应体系由高保真PCR混合液、CHS298引物、DNA按照(10～14)：(3～5)：(6.5～9.5)的体积比混合而成；Wherein, the specific PCR reaction system is formed by mixing high-fidelity PCR mixture, CHS298 primers and DNA according to the volume ratio of (10-14):(3-5):(6.5-9.5); 二次PCR反应体系由Indexing PCR Master Mix、无核酸酶水、Pi700序列标签、Pi500序列标签、纯化后产物按照(20～30)：(12～16)：(3～5)：(3～5)：(2.5～3.5)的体积比混合而成；The secondary PCR reaction system consists of Indexing PCR Master Mix, nuclease-free water, Pi700 sequence tag, Pi500 sequence tag, and purified products according to (20-30): (12-16): (3-5): (3-5 ): (2.5～3.5) volume ratio is mixed; RNA文库构建：以RNA为模板反转录合成cDNA，并进行特异性PCR扩增反应、特异性引物消化，纯化产物后，再对其进行二次PCR扩增反应，纯化并质控文库；RNA library construction: cDNA is synthesized by reverse transcription using RNA as a template, and specific PCR amplification reaction, specific primer digestion is performed, and the product is purified, and then a secondary PCR amplification reaction is performed to purify and control the library; 其中，特异性PCR反应体系由高保真PCR混合液、5x SF引物、cDNA按照(20～30)：(8～12)：(14～16)的体积比混合而成；The specific PCR reaction system is composed of high-fidelity PCR mixture, 5x SF primers, and cDNA in a volume ratio of (20-30): (8-12): (14-16); 二次PCR反应体系由Indexing PCR Master Mix、无核酸酶水、Pi700序列标签、Pi500序列标签、纯化后产物按照(20～30)：(4～6)：(3～5)：(3～5)：(11～13)的体积比混合而成；The secondary PCR reaction system is composed of Indexing PCR Master Mix, nuclease-free water, Pi700 sequence tag, Pi500 sequence tag, and purified products according to (20-30): (4-6): (3-5): (3-5 ): (11～13) volume ratio is mixed; 上机测序模块，用于将高通量测序文库上机，并运行设备，在Illumina的Miniseq平台测序；On-board sequencing module, which is used to put high-throughput sequencing libraries on the machine, run the equipment, and sequence on the Illumina Miniseq platform; 处理分析模块，用于将测序得到的Fastq格式文件上传至真固生物配套的生信分析软件系统进行自动分析即可；以及The processing and analysis module is used to upload the Fastq format file obtained by sequencing to the bioinformatics analysis software system of True Solid Biotechnology for automatic analysis; and 质控模块，用于：Quality Control Module for: 阳性质控：以panel检测范围内已知突变标本核酸为模板，与样本平行实验，每批次1个；阴性质控：以无检测范围内或检测范围外突变标本核酸为模板，与样本平行实验，每批次1个。Positive quality control: Take the nucleic acid of the sample with known mutation within the detection range of the panel as the template, and experiment with the sample in parallel, one per batch; Experiment, 1 per batch.
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