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Trace DNA-based next-generation sequencing library construction method

A next-generation sequencing library and next-generation sequencing technology, which is applied in the field of next-generation sequencing library construction based on trace DNA, can solve problems such as research limitations and large demand for initial DNA quantity

Inactive Publication Date: 2014-07-23
BEIJING INST OF GENOMICS CHINESE ACAD OF SCI CHINA NAT CENT FOR BIOINFORMATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the standard library construction method is suitable for most studies that require sequencing, it requires more DNA input, usually 1-10ug, so when the input DNA is not enough for standard library construction, related research will be restricted
For example, in the study of tumor genomes, although the next-generation sequencing technology has provided us with a lot of new understanding of tumors, and screened out many tumors related to breast cancer, rectal cancer, leukemia, lung cancer, pancreatic cancer, brain tumor, liver cancer, Renal cancer and other related genes and important signaling pathways; but at the same time, it is also recognized that the occurrence of tumors is highly heterogeneous, even if there is high heterogeneity in different tumors of the same person or even in different parts of the same tumor

Method used

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  • Trace DNA-based next-generation sequencing library construction method
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  • Trace DNA-based next-generation sequencing library construction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1. Tumor cell isolation

[0056] (1) Collect tumor samples from clinical operations and store them on ice immediately after obtaining the samples. Samples were kept in a -80°C freezer until tumor cell isolation. The tumor was frozen and embedded in OTC embedding medium, and the tumor was manually cut into thin slices with a thickness of 1 mm using a 121E Xiaoman knife frozen meat slicer (Yigang Machinery, Guangzhou). The cut samples could be stored at -80°C for a long time.

[0057] (2) Use a Micro-punch sampler with an inner diameter of 0.5mm to remove the tumor cells from the frozen state and place them in a 1.5ml centrifuge tube. The number of tumor cells in each tube is about 10,000-20,000.

Embodiment 2

[0058] Example 2. Extraction of Total DNA in Trace Cells

[0059] (1) Add 180 ul of GA buffer solution from TIANamp Micro DNA Kit (Tiangen, Beijing, China) to the cryopreserved cell sample (from Example 1), place at room temperature, and allow the temperature of the centrifuge tube to balance to room temperature.

[0060] (2), add 20ul proteinase K (20mg / ml) solution, vortex mix for 10s.

[0061] (3) Place the centrifuge tube in a 56°C water bath and incubate until the sample is fully degraded and digested. During this period, vortex and mix well every 15 minutes, centrifuge briefly, and collect all the liquid in the collection tube.

[0062] (4) Add 200ul buffer solution GB (TIANamp Micro DNA Kit), mix thoroughly by inversion, place at 70°C for 10min, and vortex mix for 10s every 3min during this period, the solution becomes clear, centrifuge briefly, and collect all the liquid in the tube.

[0063] (5) Add 200ul of absolute ethanol pre-cooled at -20°C, mix the s...

Embodiment 3

[0071] Example 3. Inserting DNA1 and Inserting DNA2 anneal with the EZ-Tn5 transposon sequence to form a double strand

[0072] 1. In a 200ul PCR tube, add

[0073] (1), 10ul 5×DNA Oligos annealing buffer;

[0074] (2), 5ul of Inserting DNA1 or Inserting DNA2 with a concentration of 20uM / ul;

[0075] (3), 5 ul of the transposon inverted repeat sequence (Complementary_Transposon) CTGTCTCTTATACACATCT with a concentration of 20uM / ul and a length of 19 bp;

[0076] (4) 30ul double distilled water without nuclease contamination;

[0077] 2. Mix the solution evenly, place it on the PCR instrument, and run the following program:

[0078] (1) 95°C for 5 minutes;

[0079] (2) Lower the temperature to 4°C at a cooling rate of 0.1°C / min.

[0080] In step 1, Inserting DNA1 consists of sequencing platform adapter1, PCR amplification primer 1, sequencing primer 1, and a 19bp transposon recognition sequence, the sequence of which is:

[0081] 5'- AATGATACGGCGACC...

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Abstract

The invention relates to a next-generation sequencing library construction method aiming at a trace DNA sample. The trace DNA-based next-generation sequencing library construction method comprises the following steps: standardizing each original molecule by utilizing random base, and thus obtaining a corresponding DNA library through the existing next-generation sequencing platform. According to the method, the whole-genome next-generation sequencing library beginning with 10-40ngDNA can be successfully constructed through the steps of trace cell separation, total DNA extraction, construction of insertion fragments, PCR amplification of a genome library, agarose gel electrophoresis target fragrant recovery and the like.

Description

technical field [0001] The present invention relates to a kind of using EZ-Tn5 TM Transposase is a method of constructing a small amount of next-generation DNA sequencing library. Transposons are used to insert and break DNA, and at the same time, the adapter and sequencing primers of the sequencing platform are added to the DNA fragments, and each original molecule is calibrated with random bases. . Background technique [0002] DNA sequencing has become an indispensable and important technology in biological research, fundamentally changing the way people study the blueprint of life. With the optimization of sequencing platform hardware and corresponding software, it is not the sequencing technology itself that hinders the progress of research, but the library construction and data analysis and interpretation related to it. [0003] 454Life Sciences (Roche) first introduced the revolutionary pyro-sequencing-based ultra-high-throughput genome sequencing system, pioneering...

Claims

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Application Information

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IPC IPC(8): C40B50/06C12Q1/68
Inventor 杨祖玉王开乐吴大飞吕雪梅吴仲义
Owner BEIJING INST OF GENOMICS CHINESE ACAD OF SCI CHINA NAT CENT FOR BIOINFORMATION