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Transcriptome sequencing method for microviridae and geminivirus viral genome splice

A technology of transcriptome sequencing and viral genome, applied in the field of genome sequencing and bioinformatics, can solve the problems of time-consuming and laborious, large homology differences, and inability to obtain the whole genome sequence of new viruses well, reaching the scope of application wide effect

Pending Publication Date: 2017-12-15
INST OF TROPICAL BIOSCI & BIOTECH CHINESE ACADEMY OF TROPICAL AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the large homology differences between different species of Dwarfviridae and among different genera of Geminiviridae, the whole genome sequence of the new virus cannot be obtained well by designing degenerate primers; and Traditional virus identification methods, such as virus purification, electron microscope observation and virus genome sequencing are time-consuming and laborious

Method used

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  • Transcriptome sequencing method for microviridae and geminivirus viral genome splice
  • Transcriptome sequencing method for microviridae and geminivirus viral genome splice
  • Transcriptome sequencing method for microviridae and geminivirus viral genome splice

Examples

Experimental program
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Effect test

Embodiment 1

[0043] Both Dwarfviridae and Geminiviridae DNA viruses produce RNA from the non-coding regions:

[0044] Dwarf Viridae:

[0045] Banana bunchy top virus (BBTV) is the causative agent of banana bunchy top virus (BBTD), and its genome consists of at least six circular ssDNA components ranging in size from 1.0 to 1.1 kb. In 2015, the International Committee on Taxonomy of Viruses assigned it to the genus Babuvirus of the family Nanaviridae and became a representative member of the genus. In this example, taking banana bunchy top virus as an example, it is confirmed that the non-coding regions of Dwarfviridae members can produce RNA, and the steps are as follows:

[0046] (1) Using the extracted total RNA of the B2 sample as a template, after being treated with DNase I, the first-strand cDNA was synthesized using the ReverseTranscriptase M-MLV reverse transcription kit;

[0047] (2) Then design the following primer sequences according to the DNA-R non-coding region (SEQ ID NO.1)...

Embodiment 2

[0059] A method for sequencing virus genomes suitable for Dwarfviridae and Geminiviridae, comprising the following steps (this embodiment takes the banana sample infected by BBTV as an example to illustrate the method):

[0060] (1) Using a commercial TRIzol Reagent kit, extract total RNA from infected BBTV banana leaves (B2);

[0061] (2) Adopt commercialization Ultra TM RNA Library Prep Kit for (NEB, USA) kit: Firstly, mRNA is enriched by magnetic beads with Oligo(dT), and the mRNA is broken into short fragments; then the short fragment mRNA is used as a template to reverse transcribe into the first-strand cDNA; The kit further synthesizes double-stranded cDNA, that is, a cDNA library, which can be used for high-throughput sequencing on a computer (Illumina HiSeqTM 2000 high-throughput sequencing technology platform).

[0062] (3) Run Bowtie software (default parameters), compare the transcriptome sequencing fragments in step (2) with the nucleotide sequences of all kn...

Embodiment 3

[0080] Supplement to the gap area:

[0081] In this embodiment, PCR supplementation is performed on the two blank regions of the DNA-N component, and the steps are as follows:

[0082] (1) Using the extracted total RNA of the B2 sample as a template, use the Reverse Transcriptase M-MLV reverse transcription kit to synthesize the first-strand cDNA, and then design the following primer sequences for PCR amplification:

[0083] Forward primer DNA-N 23F: 5'-CCAGCGCTCGGGACGGG-3';

[0084]Reverse primer DNA-N 878R: 5'-GCCCAATCAACCCCCGATTCTTC-3'.

[0085] (2) PCR amplification was performed using DNA-N 23F / DNA-N 878R, and the results of electrophoresis analysis were as follows: Figure 4 shown. It can be seen from the figure that the size of the amplified fragment is about 850bp, which is consistent with the actual size;

[0086] (3) After the PCR amplification product is recovered, it is connected to the pMD18T vector;

[0087] (4) The ligation product was transformed into Esch...

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Abstract

The invention provides a transcriptome sequencing method for microviridae and geminivirus viral genome splice. The method comprises the three steps of performing transcriptome sequencing on an infected DNA virus sample, comparing and splicing viral sequences and filling a blank area of the viral genome sequence. According to the method provided by the invention, the whole genome sequence of the banana bunchy top virus can be acquired and the other microviridae or geminivirus viral genomes can be spliced so as to acquire the whole genome sequence of the virus. The invention firstly reports the method for sequencing the spliced DNA viral genome in high throughput by utilizing transcriptome at home and abroad and fills the blank of the field at home and abroad. The method provided by the invention can be used for identifying and detecting DNA virus.

Description

technical field [0001] The application belongs to the technical field of genome sequencing and bioinformatics, and specifically relates to a transcriptome sequencing method suitable for genome splicing of Dwarfviridae and Geminiviridae viruses. Background technique [0002] DNA virus refers to a class of biological viruses containing deoxyribonucleic acid genetic material, which widely exists in humans, vertebrates, insects, plants and microorganisms. According to the latest ICTV international virus classification standard, DNA viruses can be divided into vertebrate DNA viruses, plant DNA viruses, invertebrate DNA viruses, prokaryotic microbial (bacteria and archaea) DNA viruses and eukaryotic microbial DNA (algae, fungi) according to the host type. and protozoa) viruses. Genes are structurally divided into coding regions and non-coding regions. The coding region refers to the part that can transcribe messenger RNA (mRNA), which can guide the synthesis of proteins with cer...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68G06F19/22C12R1/94
CPCC12Q1/6869G16B30/00C12Q2535/122
Inventor 余乃通刘志昕熊忠国张雨良周朋
Owner INST OF TROPICAL BIOSCI & BIOTECH CHINESE ACADEMY OF TROPICAL AGRI SCI
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