Method for establishing high-throughput sequencing library and application thereof
A sequencing library, high-throughput technology, applied in the biological field, can solve problems that need to be improved
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Embodiment 1
[0053] In this embodiment, 2 μg of human peripheral blood mononuclear cell genomic DNA was used as a sample, and the implementation was carried out according to the following steps.
[0054] 1. Genomic DNA fragmentation:
[0055] Use the covaris-S2 fragmentation instrument to fragment the genomic DNA of the sample according to the parameters set in the table below, so as to obtain DNA fragments.
[0056] processing 1
Load ratio (%)
10
intensity
5
cycle / pulse
200
time(s)
50
processing 2
time(s)
0
processing 3
time(s)
0
Process 4
time(s)
0
cycle
3
[0057] The obtained DNA fragments are detected by electrophoresis, and the main band of the DNA fragments is required to be concentrated between 150-300bp, without protein and RNA contamination. Using QIAquick PCR Purification Kit (Qiagen) or magnetic bead purification, the DNA fragments th...
Embodiment 2
[0156] Using the Hiseq2000 sequencer, the high-throughput sequencing library of the specific genome region of the sample constructed in Example 1 was sequenced according to the read length of 90 bases at both ends, so as to obtain the sequencing results.
[0157] After the above-mentioned sequencing, the raw data is directly obtained, and the above-mentioned sequencing results can be obtained by performing basic analysis on the raw data, wherein the basic analysis process includes the following main steps: First, distinguish different samples through the sequence tags on the adapters or PCR primers library data; then, decontamination, joint removal, and low-quality filtering are performed on the raw data obtained by sequencing; finally, base conversion is performed on the previously processed data, specifically, all Cs of the positive strand are converted into Ts, All the Gs of the complementary strands were converted into A, thus, the sequencing results of the high-throughput ...
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