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Method for constructing sequencing library and application of sequencing library
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A sequencing library and sequencing technology, applied in the field of biomedicine, can solve the problems of low-frequency mutations that need to be improved, and achieve the effects of reducing false positives, improving quality, and improving efficiency
Active Publication Date: 2015-01-21
天津华大基因科技有限公司 +1
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[0002] High-throughput sequencing is attracting increasing attention, but the current high-throughput sequencing for the detection of low-frequency mutations still needs to be improved
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preparation example Construction
[0076] 2. Preparation of sample library:
[0077] The cfDNA extracted from the plasma was followed by a 3-step enzymatic reaction according to the KAPA LTP Library Preparation Kit library construction instructions.
[0078] 1) End repair
[0079]
[0080] Afterwards, add 120 μL of Agencourt AMPure XP reagent for magnetic bead purification, and finally redissolve 42 μL of ddH 2 O, carry out the next reaction with magnetic beads.
[0081] 2) add A
[0082]
[0083] Then add 90 μL of PEG / NaCl SPRI solution, mix well, perform magnetic bead purification, and finally redissolve (35-linker) μL ddH 2 O, carry out the next reaction with magnetic beads.
[0084] 3) Joint connection
[0085]
[0086] Afterwards, 50 μL of PEG / NaCl SPRI solution was added twice for two times of magnetic bead purification, and finally 25 μL of ddH was dissolved back. 2 O.
[0119] 1) Design of tumor individualized genechip:
[0120] Based on TCGA, ICGC, COSMIC and other databases and related literature references, an iterative algorithm was used to design the gene chip CANPer-YY for individualized drug guidance for 12 common cancers. The CANPer-YY chip includes: oncogenes, tumor suppressor genes, 12 common cancer high-frequency genes, important genes in 12 cancer signaling pathways, target drug and chemotherapydrug genes, etc., a total of 524 genes, 750KB.
[0121] The main chip design process is divided into 4 steps:
[0122] 1. Count the number of mutation samples in each exon region of the 12 cancer-related driver genes (driver genes) in the cosmic database, the number of mutation samples, the number of samples where the hottest mutation is located, and the PI value (to evaluate the frequency of patient response in each Exon levels, PI = cumulative number ...
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Abstract
The invention discloses a method for constructing a sequencing library and an application of the sequencing library. The method comprises the following steps: (a) connecting linkers with the two ends of double-stranded DNA fragments respectively so as to obtain linking products; (b) pyrolyzing the linking products into single-stranded DNA fragments; (c) screening the single-stranded DNA fragments by utilizing a probe; (d) carrying out chain extension reaction on the single-stranded DNA fragments by utilizing a first primer so as to obtain chain extension products; and (e) amplifying the chain extension products so as to obtain amplification products, wherein the amplification products form the sequencing library. The invention also discloses a sequencing method, a method for determining a nucleotide sequence, a device for constructing the sequencing library, sequencing equipment and a system for determining the nucleotide sequence.
Description
technical field [0001] The present invention relates to the field of biomedicine. Specifically, the present invention relates to a method for constructing a sequencing library, a sequencing method, a method for determining a nucleic acid sequence, a device for constructing a sequencing library, a sequencing device, and a system for determining a nucleic acid sequence. Background technique [0002] High-throughput sequencing has attracted increasing attention, but the current high-throughput sequencing for the detection of low-frequency mutations still needs to be improved. Contents of the invention [0003] The present invention aims to solve at least one of the technical problems existing in the prior art. Therefore, according to the embodiments of the present invention, the present invention proposes a method for constructing a sequencing library and a means for detecting low-frequency mutations. [0004] In the first aspect of the present invention, the present invent...
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