Analysis method for detecting microorganisms by using metagenome or metatranscriptome

A macrotranscriptome and metagenome technology, which is applied in the field of analysis of microorganisms using metagenome or macrotranscriptome, can solve the problems of speeding up the analysis speed, poor specificity, and many false positives in the test results, so as to reduce the search speed and avoid redundant The effect of searching and ensuring integrity

Active Publication Date: 2019-10-18
湖南赛哲医学检验所有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The sequences of some microorganisms have multiple repetitive sequences, and the sequence analysis after a single assembly method cannot avoid the occurrence of assembly errors
[0011] In summary, there are still bottlenecks in the data analysis of current metagenomics, and the specific performance is as follows: (1) High-throughput metagenomics detection is sensitive, but a single assembly method often causes too many false positives in the detection results and poor specificity, It cannot meet the needs of identification methods with high specificity requirements; (2) The existing metagenomic data analysis methods have poo

Method used

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  • Analysis method for detecting microorganisms by using metagenome or metatranscriptome
  • Analysis method for detecting microorganisms by using metagenome or metatranscriptome

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Experimental program
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Effect test

Embodiment 1

[0097] Embodiment 1 A kind of microbial detection and identification analysis method based on metagenomic sequencing

[0098] 1. Obtain nucleic acid from the sample

[0099] Process such as figure 1 As shown, standard specimens were collected, and RNA extraction and DNA extraction were performed respectively according to requirements (when it is expected to detect that DNA is a sample of genetic material, extract DNA; when it is expected to detect that RNA is a sample of genetic material, extract RNA). DNA nucleic acid was extracted using QIAamp cador Pathogen Mini Kit (54104, giagen), and RNA nucleic acid was extracted using miRNeasy Serum / Plasma Kit (217184, giagen). After the nucleic acid is extracted, the quality of the nucleic acid will be tested. If the quality of the nucleic acid does not meet the quality control standards (Table 1), the nucleic acid needs to be re-extracted.

[0100] After the nucleic acid is extracted from the sample, the DNA is fragmented using ult...

Embodiment 2

[0131] Embodiment 2 A kind of microbial detection and identification analysis method based on metagenomic sequencing

[0132] 1. Experimental method

[0133] 1. Sample preparation

[0134] Mixed sample Mix1 is configured. Mix1 is cultivated, concentration determined, mixed and identified. The titer added to the sample is 3.2×10 8 TCID 50 / mL of human parainfluenza virus 2, 3.2×10 7 TCID 50 / mL of human parainfluenza virus 1, 6.3×10 5 TCID 50 / mL of human respiratory syncytial virus type B, 3.2×10 8 TCID 50 / mL of human respiratory syncytial virus type A mixed. Mix1 spiked with human interference: HeLa cells at a concentration of 2.5×10 5 pieces / ml.

[0135] Mix1 reference substance was diluted according to the concentration gradient (stock solution, 1:10 1 Dilution, 1:10 2 Dilution, 1:10 3 Dilution) to test 4 samples (named Mix1-0, Mix1-1, Mix1-2 and Mix1-3), and the amount of sequencing data is 13.6M reads. Then, carry out data volume gradient analysis with stoc...

Embodiment 3

[0154] Embodiment 3 method scope of application detection

[0155] 1. Experimental method

[0156] 1. Sample preparation

[0157] Four types of standard substances are used to detect the detection rate of the present invention to medium and low concentration target microorganisms. The four types of standard products are divided into: RNA virus standard products, DNA virus standard products, bacterial standard products, and fungal standard products.

[0158] RNA virus standard in 2.5×10 5 / mL Hela cells as matrix, add 3.2×10 2 TCID 50 Respiratory syncytial virus B / L, 3.2×10 3 TCID 50 Parainfluenza virus type 1 (PIV1) per L, 3.2×10 3 TCID 50 / L parainfluenza virus type 2 (PIV2) three kinds of RNA viruses to make RNA virus standard.

[0159] DNA virus standard in 2.5×10 5 / mL Hela as matrix, add 3.8×10 3 TCID 50 / L adenovirus C.

[0160] Bacterial standards are 2.5×10 5 / mL Hela cells as matrix, add 4.9×10 3 CFU / mL of Enterococcus faecium, 7.8×10 3 Escherichia col...

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Abstract

The invention discloses an analysis method for detecting microorganisms by using a metagenome or a metatranscriptome. The analysis method comprises the following steps: performing data quality controlon sequencing off-line data, and performing filtering to obtain high-quality data; performing assembly and double-end splicing (single end is not spliced) in a parallel manner, comparing the data obtained by assembly and splicing to a microorganism reference database based on a bwa algorithm, and identifying microorganism species and abundance information thereof in a sample; filtering low-quality results and then integrating analysis results. The method is wide in application range, various types of microorganisms can be detected, and the method is compatible with various mainstream sequencing platforms, and gives consideration to the characteristics of single-ended and double-ended sequencing data and long-read and short-read sequence data; and microorganism species and abundance thereof in a sample from various types of sequencing data can be accurately detected. The method provided by the invention can effectively reduce false positive, overcome the difficulty that most microorganisms cannot be cultured, and accurately, quickly and comprehensively detect microorganisms in various types of samples.

Description

technical field [0001] The invention relates to the technical field of biological detection, and more specifically, relates to an analysis method for detecting microorganisms using a metagenome or a metatranscriptome. Background technique [0002] Microorganisms widely exist in nature, most of which are single-celled organisms. Microorganisms usually include viruses, bacteria, fungi, protozoa, and some algae. The vast majority of microorganisms are beneficial to humans, animals and plants, and are beneficial to industry, agriculture, and pharmaceutical production, but they also have a side that is harmful to humans, such as mildew and deterioration of food and industrial and agricultural products, and contamination of animal and plant cells or pure cultures of microorganisms in laboratories. , the pollution of miscellaneous bacteria in the fermentation industry; animals and plants are infected by pathogenic microorganisms and suffer from various infectious diseases, etc. B...

Claims

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Application Information

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IPC IPC(8): G16B30/10G16B40/00
CPCG16B30/10G16B40/00
Inventor 龚浩何雪莹余旻斐陈杰
Owner 湖南赛哲医学检验所有限公司
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