Targeted sequencing panel, kit and method for obtaining targeted sequencing panel for lung cancer detection
A detection reagent and targeted capture technology, which is applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., can solve the problems of unsatisfactory sensitivity and specificity of lung cancer detection and low mutation coverage , to achieve the effects of large promotion potential, sequence design optimization, and error reduction
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Embodiment 1
[0092] Embodiment 1: Obtain peripheral blood of training group
[0093] The applicant collected 50 peripheral venous blood samples from untreated stage I and II non-small cell lung cancer patients from March 2018 to January 2019, and each sample contained 20ml of peripheral blood, including 30 males , 20 females, average age 57.8, age distribution 33-79 years old, all Chinese. During the same period, the applicant collected a total of 50 samples of peripheral venous blood from healthy people, each of which contained 20ml of peripheral blood, including 30 males and 20 females, with an average age of 58.2, and an age distribution of 33-78 years old. Chinese people. These two groups of samples are used as training group samples, and there is no statistically significant difference in gender and age of these two groups of samples, so the principle of gender and age matching is satisfied.
[0094] For each piece of peripheral blood, free DNA was extracted, fragments were interrup...
Embodiment 2
[0095] Example 2: Preparation of samples to be captured
[0096] For each piece of peripheral blood, the following procedures were performed: extracting free DNA, filling in fragments and adding A, connecting Y-shaped adapters, amplification, purification and quantification, and the obtained DNA was used as the sample to be captured:
[0097] (1) For each piece of peripheral blood, after collection with a dry blood collection tube, let stand at 4°C for more than half an hour, then centrifuge at 400g for 10 minutes at 4°C to take the supernatant, and then centrifuge at 1800g for 10 minutes at 4°C to take the supernatant, Serum samples were obtained and stored in a -80°C refrigerator;
[0098] (2) Use the QIAamp Circulating Nucleic Acid Kit (55114, QIAGEN) to extract 100–500 ng of DNA from the above serum sample, dilute it to a total volume of 20 μl with ultrapure water (no DNase and RNase, the same below), and then use The Covaris ultrasonic breaker interrupts it to a fragment...
Embodiment 3
[0107] Example 3: Select 500 genes with high mutation frequency
[0108] First, the following common cancer driver genes were selected: AKT1, BRAF, CDKN2A, CTNNB1, EGFR, ERBB2, HRAS, KEAP1, KRAS, MEK1, MET, NFE2L2, NOTCH1, NRAS, PIK3CA, PTEN, STK11, TP53.
[0109]Then, using the whole genome / whole exome sequencing data of TCGA lung cancer patients (Asians), calculate the mutation frequency of different genes in the patient, the mutation is only SNV and InDel, and sort according to the mutation frequency from high to low , select the top 500 genes, these 500 genes contain the above-mentioned 18 common cancer driver genes, these 500 genes are: ABCA13, ABCA9, ABCB1, ABCB5, ACAN, ACTN2, ADAMTS12, ADAMTS16, ADAMTS18, ADAMTS20, ADAMTSL3, ADCY2, ADCY8, ADGRB3, ADGRG4, ADGRL3, ADGRL4, ADGRV1, AFF2, AHNAK, AHNAK2, AIDA, AKAP6, AKT1, ALMS1, ALPK2, AMER3, ANK1, ANK2, ANK3, ANKRD30A, ANKRD30B, APOB ARID1A, ASAH2, ASPM, ASTN1, ASTN2, ASXL3, ATM, ATRNL1, BCHE, BCLAF1, BEST1, BIRC6, BOD1L1,...
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