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Construction method suitable for 16S rDNA high-throughput sequencing library

A sequencing library and construction method technology, applied in the field of 16SrDNA high-throughput sequencing library construction, can solve problems such as insufficient coverage of amplified sequences, low library recovery efficiency, and cumbersome steps, so as to shorten the time for library construction and save libraries Effect of purification time and simplified operation steps

Inactive Publication Date: 2017-01-04
厦门基源医疗科技有限公司
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  • Abstract
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Problems solved by technology

[0007] The purpose of the present invention is to overcome the shortcomings and deficiencies of several existing library construction methods: cumbersome steps, long time-consuming, high cost, or insufficient coverage of amplified sequences, low efficiency of library recovery, etc.

Method used

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  • Construction method suitable for 16S rDNA high-throughput sequencing library
  • Construction method suitable for 16S rDNA high-throughput sequencing library
  • Construction method suitable for 16S rDNA high-throughput sequencing library

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] (1) Collect 10 cases of human alveolar lavage fluid samples, about 2ml per sample, centrifuge at room temperature, 14000rpm, 30min, discard the supernatant after centrifugation, and save the precipitate. Using the QIAamp® UCP Pathogen Mini kit from Qiagen, the genomic DNA was extracted according to the protocol.

[0026] (2) Use nanodrop 2000 to detect the DNA concentration and quality, and select DNA with a concentration greater than 10 ng / ul and OD260 / 280=1.8-2.0 for subsequent library construction operations, with a total of 10 DNA samples.

[0027] (3) The primers used in the PCR amplification of 10 samples were randomly combined with the aforementioned 8 upstream primers and 12 downstream primers, as shown in the following table:

[0028]

[0029] (4) Using Takara Premix Ex Taq TM Hot Start Version for PCR amplification.

[0030] The PCR reaction system is as follows:

[0031] Premix Ex Taq TM Hot Start Version 25μl

[0032] Forward primer (10μM) 1μl

[...

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Abstract

The invention provides a construction method suitable for a 16S rDNA high-throughput sequencing library. An upstream primer of primers is adopted as any piece in SEQ ID NO.1-8, and a downstream primer of the primers is adopted as any piece in SEQ ID NO.9-20. The provided primers include a joint sequence, an index sequence, a sequencing primer sequence and a target fragment amplification sequence at the same time, the whole library construction process can be completed only through one-step PCR, and compared with a two-step amplification method, the library construction time is obviously shortened. After the library is constructed, a beads purification method is adopted, the beads use level and the elution mode are optimized, and compared with a traditional glue recovery and purification method, time is saved, and library purification efficiency is improved.

Description

technical field [0001] The invention relates to the field of high-throughput sequencing, in particular to a method for constructing a 16S rDNA high-throughput sequencing library. Background technique [0002] 16S rDNA is the DNA sequence encoding 16S rRNA, which exists in all bacterial genomes and generally consists of 9 conserved regions and 10 variable regions. The conserved regions are not significantly different among bacteria and can be used to construct a unified evolutionary tree of all life. There are certain differences in the variable region in different bacteria. Sequencing the variable region of 16S rDNA can identify the bacterial flora down to the taxonomic genus or even species level, which is very important for the study of marine, soil, intestinal feces and other environments. The composition of microorganisms has important guiding significance. [0003] At present, the Illumina Miseq / Hiseq sequencer is mainly used to sequence 16S rDNA, but the structure of...

Claims

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Application Information

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IPC IPC(8): C12N15/11C40B50/06C12N15/10
CPCC12Q1/6869C12N15/11C12N2310/10C12Q1/6806C40B50/06C12Q2535/122C12Q2531/113
Inventor 刘青青谢文龙李珊王松林陈荣山肖辛野李奇渊姚迅
Owner 厦门基源医疗科技有限公司
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