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Indexes and Primers for Multiplex Nucleic Acid Sequencing

A technology of nucleic acid sequence and nucleotide sequence, which is applied in the direction of recombinant DNA technology, biochemical equipment and methods, measurement/inspection of microorganisms, etc., and can solve the problem of adding Index kits to multiple sequencing library samples without fungal ITS sequences, etc.

Inactive Publication Date: 2019-11-15
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

[0006] On the other hand, in the prior art, there are only kits for adding Index to bacterial 16SrRNA sequencing library samples, Nextera XT Index Kit and Nextera XT v2Index Kit from Illunima; Kits for adding Index

Method used

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  • Indexes and Primers for Multiplex Nucleic Acid Sequencing
  • Indexes and Primers for Multiplex Nucleic Acid Sequencing
  • Indexes and Primers for Multiplex Nucleic Acid Sequencing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0094] Example 1 A group of PCR primers for constructing ITS sequence multiple sequencing library

[0095] Consists of 20 primers,

[0096] The forward primer set consists of the following 10 primers:

[0097] According to the present invention:

[0098] Primer F1, the nucleotide sequence of which is shown in SEQ ID NO: 1;

[0099] Primer F2, the nucleotide sequence of which is shown in SEQ ID NO: 2;

[0100] Primer F3, the nucleotide sequence of which is shown in SEQ ID NO: 3;

[0101] Primer F7, the nucleotide sequence of which is shown in SEQ ID NO: 7;

[0102] Primer F8, the nucleotide sequence of which is shown in SEQ ID NO: 8;

[0103] Primer F9, the nucleotide sequence of which is shown in SEQ ID NO: 9;

[0104] Primer F12, the nucleotide sequence of which is shown in SEQ ID NO: 12;

[0105] Primer F13, the nucleotide sequence of which is shown in SEQ ID NO: 13;

[0106] Primer F14, the nucleotide sequence of which is shown in SEQ ID NO: 14;

[0107] Primer F23...

experiment example 1

[0120] Experimental Example 1 Application of Fungal Diversity Detection

[0121] Construct ITS sequence multiplex sequencing library for 100 different samples

[0122] Experimental samples: 100 soil samples from different sources

[0123] Experimental steps:

[0124] (1), sample genomic DNA extraction:

[0125] use Soil DNA Kit extracts genomic DNA from soil samples and obtains 100 different genomic DNA samples. For the extraction steps, please refer to its product manual;

[0126] (2), PCR amplifies the ITS1-ITS4 sequence, while using PCR primers to add 2 Indexes to each library sample:

[0127] Using the 100 different genomic DNA samples obtained in step (1) as templates, pair the 20 PCR primers provided in Example 1 of the present invention in pairs to form 100 different primer pairs for amplifying the ITS1- ITS4 sequence;

[0128] The PCR reaction system is shown in the table below:

[0129] Element content Genomic DNA (10ng / μL) 2μL Primer (...

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Abstract

The invention relates to the technical field of molecular biology, and particularly relates to a label for multiple nucleic acid sequencing and a PCR primer used for constructing a fungus ITS sequence multi-sequencing library. The Index provided by the invention is subjected to massive actual sequencing tests and then is contained into a sequencing primer or a library-constructing primer, and then sequencing reaction is performed, so that sequencing data is relatively good in balance performance, which further can truly and reliably reflect the fungus composition situation of a sample. Compared with existing conventional ITS primers, the PCR primer used for constructing the fungus ITS sequence multi-sequencing library is relatively high in specificity, not only can be used for obtaining relatively high specific amplified products for low-complexity samples such as animal intestinal tracts, water and koji, but also has relatively high specificity on high-complexity samples such as soil and sludge, and thus is capable of ensuring the accuracy of follow-up sequencing results, and further obtaining relatively accurate analysis results and relatively true fungus composition situations.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to tags for multiple nucleic acid sequencing and PCR primers for constructing multiple sequencing libraries of fungal ITS sequences. Background technique [0002] Microbial diversity, especially fungal diversity, is closely related to many fields such as agriculture, medicine, food and industrial production. With the continuous development of molecular biology techniques, research methods for microbial diversity have gradually improved and matured. In the process of microbial evolution, ribosomal RNA has a certain degree of evolutionary conservation. The sequence of the conserved region is shared by all microorganisms of the same kind. There are variable regions of sequence differences between species due to evolution between the conserved sequences, so it is considered It is one of the most suitable genes for systematic classification. Through the determination and comp...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6869C12Q1/04C12N15/11C40B50/06
CPCC12Q1/6869C12Q1/6895C40B50/06C12Q2537/143C12Q2535/122C12Q2531/113
Inventor 王绪敏刘贵明赵倍伦宋利璞于军
Owner BEIJING INST OF GENOMICS CHINESE ACAD OF SCI CHINA NAT CENT FOR BIOINFORMATION