Primer applicable to amplicon sequencing library construction, construction method, amplicon library and kit comprising amplicon library

A technology of amplicon library and amplification primer, which is applied in libraries, chemical libraries, nucleotide libraries, etc., can solve the problems of overestimating microbial diversity and low accuracy of sequencing data, and increase the number of mixed samples , Improve throughput and improve accuracy

Inactive Publication Date: 2015-01-21
天津诺禾致源生物信息科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This chimeric molecule, which was not present in the original sample, can lead to an overestimation of microbial diversity in the environment
[0007] For the library constructed from the above-mentioned templates such as 16S rDNA, there is no good solution to distinguish the above-mentioned chimeras, which makes the accuracy of the obtained sequencing data low

Method used

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  • Primer applicable to amplicon sequencing library construction, construction method, amplicon library and kit comprising amplicon library
  • Primer applicable to amplicon sequencing library construction, construction method, amplicon library and kit comprising amplicon library
  • Primer applicable to amplicon sequencing library construction, construction method, amplicon library and kit comprising amplicon library

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] A method for constructing a 16S rDNA v4 region amplicon sequencing library applied to Illumina MiSeq sequencing, comprising the following experimental steps:

[0048] (1) According to the sample 16S rDNA DNA concentration, use sterile water to dilute the sample to 1ng / μl. If there is a large amount of RNA contamination in the sample, it needs to be digested with RNase before dilution.

[0049] (2) PCR amplification. Use the synthesized primers shown in Table 1 to amplify according to the combinations shown in Table 2.

[0050] Table 1:

[0051] Numbering

Primer sequence

SEQ ID NO.1

AATGATACGGCGACCACCGAGATTCACTCTTTCCCTACACGACGCTCTTCCGATCTATCACGGTGCCAGCMGCCGCGGTAA

SEQ ID NO.2

AATGATACGGCGACCACCGAGATTCACTCTTTCCCTACACGACGCTCTTCCGATCTCGATGTGTGCCAGCMGCCGCGGTAA

SEQ ID NO.3

AATGATACGGCGACCACCGAGATTCACTACTCTTTCCCTACACGACGCTCTTCCGATCTTTAGGCGTGCCAGCMGCCGCGGTAA

SEQ ID NO.4

AATGATACGGCGACCACCGAGATTCACTCTTTCCCTACACGACGCTCTTCC...

Embodiment 2 and

Embodiment 2

[0069] Example 2 uses the same steps as Example 1, only the primers are different. The specific primer sequences are shown in Table 3, and the amplification results are shown in image 3 . image 3 The order of the samples from left to right is the order listed in Table 4.

[0070] table 3

[0071] Numbering

[0072] Table 4

[0073] Sample serial number

[0074] image 3 Among them, the size of the target fragment of the amplified 16S rDNA is about 300bp, and the total length of the upstream primer and downstream primer is about 380bp, excluding 19bp of the upstream primer and 20bp of the downstream primer of the target fragment. The icon size is also consistent with the expected size. From image 3 It can be seen from the figure that in the two-step amplification library construction method, the target fragment can be amplified well by having internal tag sequences at both ends of the target fragment amplification primer sequence. The linker sequ...

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Abstract

The invention discloses a primer applicable to amplicon sequencing library construction, a construction method, an amplicon library and a kit comprising the amplicon library. The primer comprises a target fragment amplification primer sequence, a sequencing primer sequence and an adaptor sequence, wherein at least one end of a target fragment amplification primer sequence, obtained from the target fragment amplification primer sequence, the sequencing primer sequence and the adaptor sequence by virtue of an amplification step, in the amplicon library is provided with an internal label sequence; the internal label sequence is formed by using 6-10 basic groups in different permutation and combination patterns. The primer enables the constructed amplicon library to have the internal label, and therefore, the number of the mixed samples of the library can be increased and the flux of sequencing can be increased, and besides, contaminated data generated in the library construction process can be differentiated and removed and the accuracy of late data analysis can be improved; as a result, the abundance ratio of different floras in an original sample can be reflected more really.

Description

technical field [0001] The invention relates to the field of high-throughput sequencing, in particular, to a primer suitable for constructing an amplicon sequencing library, a construction method, an amplicon library and a kit containing the same. Background technique [0002] Amplicon sequencing is the sequencing of PCR products or captured fragments of a specific length, mainly including 16S rDNA sequencing, 18S rDNA sequencing, ITS sequencing and functional gene detection. The sequence of a certain hypervariable region of 16S / 18S / ITS determined by the Illumina MiSeq second-generation high-throughput sequencing platform is used to reflect the differences between species in environmental samples in terms of bacterial, fungal, and archaeal classification, which is useful for the study of oceans and soils. The composition of microorganisms in environments such as , intestinal feces, etc. plays an important guiding role; similarly, more biological information can also be mined...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C40B50/06C40B40/06C12Q1/68
Inventor 蒋智曹志生朱海浩王大伟李明洲刘运超
Owner 天津诺禾致源生物信息科技有限公司
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