DNA library construction method for high-throughput sequencing
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A technology for sequencing library and construction method, which is applied in the field of high-throughput sequencing DNA library construction, and can solve the problems of wasting time, heavy workload, cumbersome steps, etc.
Active Publication Date: 2017-01-25
承启医学(深圳)科技有限公司
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[0007] The current amplicon sequencing is based on conventional library construction methods. After PCR amplification and enrichment to the target region, the sample is mixed, then the end is repaired and A is added, and the adapter is added, the sample is purified, and the target band is recovered. , then PCR amplification, and then run the gel to recover the main band. This conventional library construction method is cumbersome, time-consuming, and heavy workload, which is not conducive to the sequencing of a large number of samples.
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Embodiment 1
[0087] Example 1: Forensic kinship identification
[0088] The purpose of this example is to detect the number of repeats of the repeat sequence in the STR, wherein the NGS sequencing protocol is used.
[0089] 1. Collection and processing of oral epithelial cells
[0090] (1) Collect human oral epithelial cells with a buccal swab collection tube.
[0091] (2) Oral epithelial cell DNA was extracted with buccal swab genome extraction kit (DP322).
[0092] (3) OD260nm and OD280nm were measured using a Nanodrop 2000 spectrophotometer to confirm that the purity was high, and the concentration was measured.
[0093] 2. Design of primers for multiplex PCR reaction
[0094] (1) The primers are designed as fusion primers, including the specific primer sequence of the target fragment and the sequencing linker sequence, which contains the index sequence.
[0095] (2) The fusion primer structure is:
[0096] Upstream primer: 5'-CCTCTCTATGGGGCAGTCGGTGAT-3'+ target fragment specific f...
Embodiment 2
[0127] Example 2: Detection of which cell line an unknown cell line belongs to
[0128] In this example, two unknown human cell lines were used, and the number of repetitions of the repeat sequence in the STR was detected to confirm which cell line the unknown cell line belonged to. Which uses the NGS sequencing protocol.
[0129] 1. DNA extraction from human cell lines
[0130] (1) Human cell line DNA was extracted using a peripheral blood DNA kit (Qiagen, Cat. No. 937236).
[0131] (2) OD260nm and OD280nm were measured using a Nanodrop 2000 spectrophotometer to confirm that the purity was high, and the concentration was measured. The final concentration was 51 ng / μl.
[0132] 2. Multiplex PCR reaction library construction and polyacrylamide PAGE gel nucleic acid electrophoresis
[0133] (1) The primers are designed as fusion primers, including the specific primer sequence of the target fragment and the sequencing linker sequence, which contains the index sequence.
[013...
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Abstract
The invention discloses a DNA library construction method for high-throughput sequencing. The DNA library construction method includes the steps of carrying out multiple PCR (polymerase chain reaction) to a DNA template obtained from a to-be-tested sample through multiple pairs of fusion primers, then recycling PCR products to obtain a sequencing library. The multiple pairs of fusion primers respectively aim at different target fragments of the DNA template, and each fusion primer comprises specificity primer sequence aiming at the target fragment and linker sequence for sequencing. The multiple PCR is carried out on reaction conditions: reacting at 95 DEG C for 2 minutes; reacting within 38 cycles, wherein each cycle includes processes of preserving at the temperature of 95 DEG C for 30 seconds, cooling to the temperature from 76 DEG C to 55-58 DEG C slowly at the speed of 0.1 DEG C each second, holding the temperature for 20 seconds once cooling to the temperature of 58 DEG C, and then preserving at 72 DEG C for 30 seconds; after that, preserving at 72 DEG C for 2 minutes, and finally preserving at 4 DEG C. The invention further discloses application of the library construction method in testing STR locus and paternity identification on the basis of high-throughput sequencing.
Description
technical field [0001] The invention relates to the field of biological detection, in particular to a DNA library construction method for high-throughput sequencing. Background technique [0002] The research and analysis objects of forensic DNA testing technology are mainly DNA polymorphisms in living organisms. In general, the location on the chromosome of a DNA marker of a gene or noncoding region is called a locus, while genes with different sequences at the same locus are called alleles. DNA polymorphism refers to the existence of multiple alleles at a locus as a genetic marker, and analyzing the differences in alleles at a locus is the biological basis for achieving the same identification [1]. [0003] Short tandem repeats (STR) [2] in microsatellite DNA is a DNA polymorphism that exists widely on 23 pairs of chromosomes in the human genome, usually consisting of 2-6 bp repeat units (or cores). Sequence, coresequence) composition, the differences between different i...
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