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Method for obtaining bar code area of COI (C oxidase I) gene of insect in batched and high-accuracy ways by using PacBio monomolecule sequencing

A barcode and insect technology, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of not being able to obtain the full length of the barcode area, affecting data integrity, and failing to meet the requirements of species identification, and achieve major applications The effect of promoting value, reducing time and labor costs, and improving efficiency

Inactive Publication Date: 2018-01-30
BGI SHENZHEN CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, even with the longest sequencing capability of 300 PE, the 658 bp COI barcode still has a gap of 100 bp, so the full length of the barcode region cannot be obtained by one sequencing
The existence of Gap greatly affects the integrity of the data and cannot meet the requirements of species identification

Method used

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  • Method for obtaining bar code area of COI (C oxidase I) gene of insect in batched and high-accuracy ways by using PacBio monomolecule sequencing
  • Method for obtaining bar code area of COI (C oxidase I) gene of insect in batched and high-accuracy ways by using PacBio monomolecule sequencing
  • Method for obtaining bar code area of COI (C oxidase I) gene of insect in batched and high-accuracy ways by using PacBio monomolecule sequencing

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1, the preparation of primer

[0051] The nucleotide sequences of primer LCO1490 and primer HCO2198 are as follows:

[0052] LCO1490 (sequence 1 of the sequence listing): 5'-GGTCAACAAATCATAAAGATATTGG-3';

[0053] HCO2198 (sequence 2 of the sequence listing): 5'-TAAACTTCAGGGTGACCAAAAAAATCA-3';

[0054] The target sequence is 658bp.

[0055] A forward tag was added to the 5' end of primer LCO1490, and a reverse tag was added to the 5' end of primer HCO2198. The nucleotide sequences of the forward tag and the reverse tag are shown in Table 1. For example, add forward label For001 to the 5' end of primer LCO1490 to obtain primer For001 (forward primer); add reverse label Rev001 to the 5' end of primer HCO2198 to obtain primer Rev001 (reverse primer); primers For001 and Primer Rev001 constitutes primer pair 001. By analogy, 96 primer pairs were obtained, sequentially from primer pair 001 to primer pair 096.

[0056] Table 1

[0057]

[0058]

[0059] ...

Embodiment 2

[0060] Example 2, the insect COI sequence obtained by applying the primers of Example 1 and the third generation sequencing

[0061] 1. Extraction of insect DNA

[0062] Insect Lysis Buffer (i.e. "Insect Lysis Buffer+Na 2 SO 3 "), binding mix (ie "BindingBuffer(BB)"), PWB washing liquid and WB washing liquid formulations see:

[0063] http: / / ccdb.ca / site / wp-content / uploads / 2016 / 09 / CCDB_DNA_Extraction-Plants.pdf.

[0064] 1. Clean the test bench with alcohol, and cover it with aluminum foil or plastic wrap to prevent contamination. In order to prevent the static electricity from making the insect legs jump around, about 30 μl of pure alcohol should be added to each well of the 96-well plate first. According to the sample information sheet, add 95 insect samples to 95 wells of a 96-well plate with tweezers (sampling principle: for very tiny insects, the whole body should be taken, multiple legs can be taken for tiny insects, and one leg can be taken for small insects For la...

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Abstract

The invention discloses a method for obtaining a bar code area of a COI (C oxidase I) gene of insect in batched and high-accuracy ways by using PacBio monomolecule sequencing. The method comprises thefollowing steps of (1) respectively performing PCR (polymerase chain reaction) amplification on DNA (deoxyribonucleic acid) samples of a plurality of insects, wherein the forward primers sequentiallycomprise a forward label and a forward segment corresponding to the COI gene of the insect, and the reverse primers sequentially comprise a reverse label and a reverse segment corresponding to the COI gene of the insect; the forward segments in the forward primers are the same, and the reverse segments in the reverse primers are the same; the difference of at least two nucleotides exists betweenthe forward labels of every two forward primers; the difference of at least two nucleotides exists between the reverse labels of every two reverse primers; (2) respectively obtaining the amplified products after PCR amplification, and mixing, so as to obtain a mixed sample; (3) performing third-generation sequencing on the mixed sample. The method has the advantage that the important application and popularization value on obtaining the bar code area of the COI gene of the insect at high flux is realized.

Description

technical field [0001] The invention relates to a method for obtaining insect COI gene barcode regions in batches with high precision by using PacBio single-molecule sequencing. Background technique [0002] DNA barcodes (DNA barcodes) refer to standard, sufficiently variable, easily amplified and relatively short DNA fragments that can represent the species in an organism. Canadian zoologist Paul Hebert et al. compared and analyzed the mitochondrial cytochrome C oxidase subunit gene sequences of 11 phyla and 13,320 species in the animal kingdom, including vertebrates and invertebrates, and found that, except for the coelenterate Cnidaria, 98% of the species The difference in genetic distance within a species is 0% to 2%, and the average between species can reach 11.3%. Based on this, it is proposed that a single small fragment gene can be used to represent a species, and as the barcode of the species, it is a DNA barcode. [0003] Over the past decade or so, scientists hav...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6869
Inventor 刘山林杨琛涛周程冉周欣
Owner BGI SHENZHEN CO LTD
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