Method and system for determining nucleic acid sequence

A nucleic acid sequence and sequence technology, used in sequence analysis, biochemical equipment and methods, and microbial determination/inspection, etc., can solve the problems of low total cellular RNA content, easy degradation, and difficult RNA.

Inactive Publication Date: 2016-09-07
GUANGZHOU JINGKE DX CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Enzyme-labeled detection technology has great limitations. In most cases, it can only detect the corresponding antigen and antibody of known microorganisms; while the superiority of fluorescent quantitative PCR technology itself is understandable, but for cerebrospinal fluid (CFS) samples Said that it is difficult to detect RNA in CSF by fluorescent quantitative PCR because of its low total cellular RNA content and easy degradation

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  • Method and system for determining nucleic acid sequence
  • Method and system for determining nucleic acid sequence
  • Method and system for determining nucleic acid sequence

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Example 1 Construction of a sequencing library and acquisition of sequencing data.

[0082] (1) Sample preparation

[0083] 1. Take 5-10mL of peripheral blood from the host, store it in EDTA anticoagulant tubes, and separate the peripheral blood within 4-6 hours;

[0084] 2. According to the instructions of the QIAamp Circulating Nucleic Acid Kit extraction reagent, plasma free DNA was extracted, and Qubit (Invitrogen, the Quant-iT TM dsDNA HS Assay Kit) was used to quantify the extracted DNA, with a total amount of about 5-50 ng. Plasma cell-free DNA (cfDNA) was obtained.

[0085] 3. Use an ultrasonic breaker to break the synthesized second-strand nucleic acid sample into fragments of about 200-300 bp in size. The breaking procedure is 15 s of breaking, 15 s of resting, and a total of 10 minutes of breaking. The product synthesized by the reaction was purified with a QIAquick PCR purification kit, and finally dissolved in 78 μL of ultrapure water.

[0086] (2) Libra...

Embodiment 2

[0101] Example 2 Determining the nucleic acid sequence, the specific process is detailed in figure 1 .

[0102]1. Obtain sequencing data according to the method of Example 1.

[0103] 2. Filter the sequencing data. Remove reads with an uncertain base ratio greater than 1% and / or reads with a base quality value not greater than 5 and a ratio of not less than 50% of the reads to obtain the filtered sequencing results.

[0104] 3. The first comparison. Use the BWA comparison software to compare the filtered sequencing results with the host gene database as the first database, such as the human genome (hg19). After comparison, remove the matching sequencing sequences, that is, exclude the host gene sequence, and obtain non-matching sequence.

[0105] 4. The second comparison. Using the BWA comparison software, the obtained unmatched sequencing sequences are compared with the second database to obtain a second comparison result. The second database is a bacteria database or a...

Embodiment 3

[0108] Example 3 Construction of Nucleic Acid Sequence Expression Abundance Map

[0109] 1. Obtain the abundance of bacterial or viral species. According to the method of embodiment two, bacteria or virus comparison results are obtained, and the abundance of bacteria or virus species is calculated according to formula 1, and said formula 1 is:

[0110] 1

[0111] i is the species in the second database; N is the length of the entire sequence compared to the second database; Ni is the length of the sequence compared to the species; Li is the genome length of species i; bi is the abundance.

[0112] Formula 1 is the abundance of double normalization: for the abundance of a certain bacterial or viral species in a sample, it is the amount of data (bp) per thousand (bp) length from a certain species in the amount of data per million, In this way, the influence of species genome length and sample data volume is eliminated.

[0113] 2. Obtain the relative abundance of bacterial ...

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Abstract

The present invention provides a method for determining a nucleic acid sequences. The method includes the steps of: acquiring nucleic acids in a sample to be measured, sequencing the nucleic acids to obtain a sequencing result composed of a plurality of sequencing sequences; filtering the sequencing result to exclude read segments with uncertain base ratio greater than 1% and / or read segments with base weight value of no higher than 5 and base ratio of no less than 50%, so as to obtain a filtered the sequencing result; conducting a first comparison on the filtered sequencing result with a first database to obtain a the first comparison result; conducting a second comparison the first comparison result and a second database to obtain a second comparison result; and analyzing the second comparison result, and determining the nucleic acid sequence of the sample to be measured. The invention also provides a system for determining the nucleic acid sequence. The method is based on the biological information analysis and powerful database platform to identify the microbial species in the sample, and has the advantages of high sensitivity and strong specificity.

Description

technical field [0001] The present invention relates to the field of biotechnology, specifically, a method and system for determining nucleic acid sequences, and a method and system for calculating the abundance and relative abundance of nucleic acid sequences. Background technique [0002] There are a large number of microorganisms such as bacteria or viruses parasitic in the cerebrospinal fluid of humans or animals. The study of microorganisms in cerebrospinal fluid can promote a better understanding of the parasitic conditions of microorganisms in cerebrospinal fluid. [0003] At present, the methods mainly used to study microorganisms in cerebrospinal fluid include antigen-antibody combined detection (enzyme labeling method) and fluorescent quantitative PCR technology. These technologies have played a huge role in the research field, but there are still some shortcomings. Enzyme-labeled detection technology has great limitations. In most cases, it can only detect the cor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G06F19/22
CPCC12Q1/6869G16B30/00
Inventor 张印新韩颖鑫王佳伟高晓峘张春生李胜
Owner GUANGZHOU JINGKE DX CO LTD
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