Constructing method of amplification sublibrary for precise sequencing of ctDNA

An amplicon library and construction method technology, applied in DNA preparation, recombinant DNA technology, chemical library and other directions, can solve problems such as cost increase, and achieve the effects of cost saving, improving sensitivity and efficiency, and easy operation

Inactive Publication Date: 2017-12-29
厦门燕旭安生物科技有限公司
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The above-mentioned methods all use exogenously added tags to mark and identify cfDNA,

Method used

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  • Constructing method of amplification sublibrary for precise sequencing of ctDNA
  • Constructing method of amplification sublibrary for precise sequencing of ctDNA
  • Constructing method of amplification sublibrary for precise sequencing of ctDNA

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Experimental program
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Effect test

specific Embodiment 1

[0109] S01: Random ligation of cfDNA followed by random fragmentation and addition of adapters to build a library;

[0110] After extracting and purifying cfDNA, use Agilent 2100 or similar instruments to check the quality of cfDNA. The main peak of qualified samples should be distributed around 166bp. Take 10ng of cfDNA that has passed the quality inspection, and use T4 ligase to ligate for 5 hours, then use magnetic beads to purify the cfDNA, and dissolve it in 8 μL ddH2O; the ligation effect is judged by the distribution of fragment sizes.

[0111] Take 8ng of the above randomly linked cfDNA, add Tn5 transposome fragmentation system (5*lm lysis buffer 6μL, tn5 transposon 11μL, 10ng linked cfDNA, ddH2O supplemented to 30ul), react at 60°C for 10min, then The fragmented product was purified using magnetic beads and the purified product was dissolved in 35 μL ddHO.

[0112] Before random fragmentation of the cfDNA and addition of adapters, preparation of transposomes was also...

specific Embodiment 2

[0113] S01: Random ligation of cfDNA followed by random fragmentation and addition of adapters to build a library;

[0114] After extracting and purifying cfDNA, use Agilent 2100 or similar instruments to check the quality of cfDNA. The main peak of qualified samples should be distributed around 166bp. Take 30ng of cfDNA that has passed the quality inspection, and use T4 ligase to ligate for 3 hours, then use magnetic beads to purify the cfDNA, and dissolve it in 12 μL ddH2O; the ligation effect is judged by the distribution of fragment sizes.

[0115] Take 12ng of the above randomly linked cfDNA, add Tn5 transposome fragmentation system (5*lm lysis buffer 6μL, tn5 transposon 11μL, 10ng linked cfDNA, ddH2O supplemented to 30ul), react at 50°C for 5min, then The fragmented product was purified using magnetic beads and the purified product was dissolved in 45 μL ddHO.

[0116] Before the random fragmentation of the cfDNA and the addition of adapters, the preparation of transpos...

specific Embodiment 3

[0117] S01: Random ligation of cfDNA followed by random fragmentation and addition of adapters to build a library;

[0118] After extracting and purifying cfDNA, use Agilent 2100 or similar instruments to check the quality of cfDNA. The main peak of qualified samples should be distributed around 166bp. Take 20ng of cfDNA that has passed the quality inspection, and use T4 ligase to ligate for 4 hours, then use magnetic beads to purify the cfDNA, and dissolve it in 10 μL ddH2O; the ligation effect is judged by the distribution of fragment sizes.

[0119] Take 10ng of the above randomly linked cfDNA, add it to the Tn5 transposome fragmentation system (5*lm lysis buffer 6μL, tn5 transposon 11μL, 10ng linked cfDNA, add ddH2O to 30ul), react at 55°C for 8min, and then The fragmented product was purified using magnetic beads and the purified product was dissolved in 40 μL ddHO.

[0120] Before the random fragmentation of the cfDNA and the addition of adapters, the preparation of tra...

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Abstract

The invention discloses a constructing method of an amplification sublibrary for precise sequencing of ctDNA. The constructing method comprises the following steps: S01, randomized ligation and randomized fragmentation of cfDNA, adaptor adding and library building; and S02, non-targeted/targeted library amplification of the cfDNA. According to the constructing method disclosed by the invention, firstly, the problem that a large amount of tag sequences need to be synthesized by adopting an exogenous tag method, so that the cost is relatively-high is solved; secondly, the problems that low-abundance cfDNA fragments are small, and characteristic primers for designing a target gene often cannot amplify out a target sequence under the condition that broken sites are randomly distributed are solved.

Description

technical field [0001] The invention belongs to the field of high-throughput sequencing of circulating tumor DNA (circulating tumor DNA, ctDNA), and in particular relates to a method for constructing an amplicon library for precise sequencing of ctDNA. Background technique [0002] cfDNA is the DNA in the plasma of peripheral blood, which contains DNA fragments shed from various human tissues, and its peak length is generally about 166 bases, corresponding to the length of the coiled histone DNA. cfDNA contains a small portion of DNA fragments from tumors or circulating tumor cells, that is, circulating tumor DNA (ctDNA), which carries information consistent with the genetic information of tumor cells, including mutations, methylation, etc. Information available for molecular diagnosis of tumors. ctDNA as a tumor marker has the following applications: 1) identification of tumor-specific mutations; 2) detection of tumor burden and response after tumor treatment; 3) detection...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C40B50/06
CPCC12N15/10C12N15/1093C12Q1/6806C40B50/06C12Q2525/191C12Q2563/143C12Q2563/149
Inventor 不公告发明人
Owner 厦门燕旭安生物科技有限公司
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