Coding PCR next-generation sequencing library construction method, kit and detection method

Abstract

The invention discloses a coding PCR second-generation sequencing library construction method. The steps include: 1) extracting DNA; 2) performing a forward single-strand linear amplification reaction with a single-end specific primer with a random molecular coding sequence; 3) using a pair The side single-end specific primer and the common adapter primer constitute a primer pair for single-end specific exponential amplification reaction; 4) use the sequencing double-end common adapter primer pair for exponential amplification reaction; 5) quantitative quality inspection to obtain a DNA library. The invention also discloses a method for sequencing detection by using the above-mentioned DNA library, and a coded PCR next-generation sequencing library construction kit containing the DNA library. In the present invention, the original DNA template sequence of the sample is coded in the initial complementary synthesis, so that the sequencing results can detect the real sequence of the original DNA template and the number of real detected molecules according to the coding traceability, thereby significantly reducing mutation false positives and false negatives.

Description

technical field [0001] The invention relates to the technical field of DNA deep sequencing detection, in particular to the high-fidelity deep sequencing detection of DNA fragments with a low proportion of 0.03% to 1% target sequence variation sites in tissue, plasma or serum. Background technique [0002] Gene science is an important field of life science. The genetic code sequence and its variation of living organisms will affect various biological functions of living organisms, resulting in functional variation. By detecting the genetic variation of living organisms, the functional variation of life can be understood and estimated. By quantitatively detecting the genetic variation of living organisms, it is also possible to accurately understand the disease progression and prognosis of diseased living organisms, such as tumor progression, recurrence or early, middle and late stages, and treatment effects. At present, the detection of gene variation has become an important ...

AI Technical Summary

Problems solved by technology

With such 10,000 original copies, about 2,000 DNA molecules derived from the original template can be obtained. Although PCR amplification can achieve enough sequences for 5,000 times and 10,000 times of sequencing, it is due to the fact that the number of sequences of the original template has not increased. Although the depth Very deep, but not representative of the depth of 10,000 original templates

More than 30 cycles of PCR will also produce non-negligible mismatch errors, which seriously affect the fidelity and sensitivity of sequencing

The obvious limitations of this method are: 1) digital PCR, one reaction can only detect 1-2 known mutation genes at certain sites, and it cannot reliably detect multiple unknown mutations at the same sample at the same time

Because tumor mutations are complex, multi-gene and multi-locus mutations are an important feature of tumors, so the detection of multi-gene and multi-locus mutations is very important, so the current digital PCR cannot well meet the needs of plasma multi-locus mutation detection

2) The amount of DNA required for digital PCR is relatively large. Generally, 10ML of blood is only enough for one reaction at a time, and 1 to 2 sites are measured. If more sites are to be tested, the sample volume is not enough

3) Only 1 to 2 sites are detected. For tumors, the detection coverage is too low and the application area is too narrow

In the detection of tumor tissue and peripheral blood of tumor patients, similar multi-site hot spot mutations are the most common, while the proportion of a single site mutation is low, so the applicability of digital PCR is narrow, and it has not been widely used so far.

4) The cost of digital PCR is too high. To detect one site, the material cost alone needs 200 to 400 yuan

Therefore, digital PCR has only been applied in the field of scientific research so far, and cannot be used for actual medical detection.

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