Blood plasma DNA library building method and kit

A plasma and library technology, applied in the field of plasma DNA library construction, can solve the problem of low efficiency of plasma library construction, and achieve the effect of increasing the amount of effective data and improving the efficiency of library construction

Active Publication Date: 2019-07-05
NANODIGMBIO (NANJING) BIOTECHNOLOGY CO LTD
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AI Technical Summary

Problems solved by technology

[0006] The main purpose of the present invention is to provide a method for building a library of plasma DNA an...

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  • Blood plasma DNA library building method and kit
  • Blood plasma DNA library building method and kit
  • Blood plasma DNA library building method and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0121] Example 1 Efficient plasma library construction kit

[0122] The following is an experimental comparison example of the inventor optimizing the composition of the reaction enzyme and the compatibility of the reaction buffer according to the characteristics of the plasma fragment DNA. The comparative library construction kit is currently the best library construction kit KAPA Hyper library construction reagent on the market box (Cat. No. kk8505), and the comparison experiment KAPA Hyper library construction kit was operated strictly according to the instructions, and the input amount of plasma fragment DNA was exactly the same as that of the linker.

[0123] 1. Plasma DNA Extraction

[0124] Take 2-5mL of plasma, and use the QIAamp Circulating Nucleic Acid Kit (Product No. 55114) to extract plasma DNA. Finally, 100 μl of elution buffer was used to elute the DNA, and 2 μl of Qubit was used to measure the concentration.

[0125] 2. Plasma DNA filling plus A and phosphory...

Embodiment 2

[0168] The optimal concentration test of embodiment 2 T4 DNA polymerase

[0169] The steps of this example are the same as those in Example 1, the only difference is that when constructing a 5ng plasma DNA library, the amount of T4 DNA polymerase in the two libraries is 0.5 μl (1.5U), and the amount of T4 DNA polymerase in the two libraries is It is 1 μl (3U), and the amount of T4 DNA polymerase in the other two libraries is 2 μl (6U). Finally, the output of the library was compared, and the comparison results are shown in Table 2-1.

[0170] It can be seen from Table 2-1 that the yield of T4 DNA polymerase at 1.5U and 3U is significantly higher than that at 6U, and the library yield is significantly reduced when the amount of T4 DNA polymerase is greater than 3U. Moreover, the inventors found in experiments that the library conversion efficiency is relatively highest when the amount of T4 DNA polymerase is 1U-1.5U.

[0171] Table 2-1. Library yields under two concentration ...

Embodiment 3

[0174] Example 3 Effective Detection of Low-Frequency Mutations

[0175] The steps of this example are the same as in Example 1, the only difference is that the initial amount of plasma is 10ng and 25ng respectively, and the two plasma DNAs are mixed with plasma A and plasma B, and the types and ratios of the plasma mixture are shown in Table 3-1 10ng of plasma is mixed according to the ratio of A plasma and B plasma 99:1, and 25ng of plasma is mixed according to the ratio of A plasma and B plasma 998:2. Plasma A is homozygous wild type, half of the sites in plasma B are heterozygous, and the other half are homozygous mutants, so there are two combinations of 1% and 0.5% of the mutation sites in 10ng of plasma samples, and 25ng of plasma samples There are two combinations of 0.1% and 0.2%

[0176] Table 2-1 Plasma mixing ratio

[0177]

[0178] 10ng and 25ng were mixed together for hybridization and capture after building the library. The capture chip was designed for the...

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Abstract

The invention provides a blood plasma DNA library building method and kit. The library building method comprises the following steps: repairing the tail end of blood plasma DNA and adding A to obtainrepaired DNA; and sequentially carrying out joint connection and PCR amplification on the repaired DNA by adopting a joint with a unique tag sequence to obtain a sequencing library of the blood plasmaDNA. The sequencing library of the blood plasma DNA is constructed by joint connection of the blood plasma DNA with unique tag sequence after repairing and A addition. By the formed library, repeatsgenerated by library-building amplification or sequencing amplification according to the unique tag sequence are conveniently distinguished from real repeat sequences in the blood plasma DNA when sequencing data are analyzed at a later stage, then amplification repeats are removed, repeats exist in blood plasma fragments DNA retains, thus, the effective data size of blood plasma-derived DNA is increased, and the blood plasma DNA library building efficiency is improved to a certain extent.

Description

technical field [0001] The invention relates to the field of plasma DNA library construction, in particular to a plasma DNA library construction method and a library construction kit. Background technique [0002] With the advancement of technology and the deepening of people's understanding of tumors and genetic diseases, traditional technologies can no longer meet the needs of accurate diagnosis. Accurate diagnosis is increasingly dependent on next-generation sequencing technology to solve problems, and the second-generation technology is also constantly improving, with more and more sequencing throughput and lower and lower sequencing costs. At present, there are three mainstream second-generation sequencers in the market, namely Illumina sequencers of various models, MGI sequencers and life sequencers. Both Illumina's sequencer and MGI's sequencer rely on T-A cloning to build a library. The difference is that Illumina uses a Y-shaped adapter, and MGI's intermediate bubb...

Claims

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Application Information

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IPC IPC(8): C12Q1/6806C12N15/10C40B50/06
CPCC12N15/1093C12Q1/6806C40B50/06C12Q2525/191C12Q2531/113C12Q2521/501
Inventor 唐守运曲艳汪彪胡玉刚郑文莉吴强
Owner NANODIGMBIO (NANJING) BIOTECHNOLOGY CO LTD
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