A library construction method, kit and sequencing method for metagenomic sequencing

A metagenomic and kit technology, applied in the field of sequencing, can solve serious problems in identification, increase the number of PCR cycles, and increase the bias of amplification, so as to reduce the initial amount of library construction, reduce the bias of PCR amplification, The effect of improving the success rate of building a database

Active Publication Date: 2020-06-30
BEIJING SIMCERE MEDICAL LAB CO LTD +2
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AI Technical Summary

Problems solved by technology

The modified process allows some samples to successfully build a library, but some samples that have gone through the host removal process fail in PCR, and the number of PCR cycles is increased, which further aggravates the bias of amplification, resulting in serious problems in the identification of some bacteria with high GC content. The problem is that the abundance ratio of bacteria is seriously out of balance

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  • A library construction method, kit and sequencing method for metagenomic sequencing
  • A library construction method, kit and sequencing method for metagenomic sequencing
  • A library construction method, kit and sequencing method for metagenomic sequencing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 The official library construction method of the SQK-RPB004 kit

[0045] Using the zymo standard as the sample, build the library according to the official procedure provided by the SQK-RPB004 kit, in which the initial amount of sample DNA is 1ng and 0.25ng (3μL), the FRM fragmentation mixture is 1μL, and the RLB rapid barcode primer is 1μL , and the annealing temperatures were 56°C and 51°C, respectively (see Table 1). After completing 14 cycles according to the official procedure, the nucleic acid concentration of the PCR products was all <1ng / μL (Table 2), which was far from meeting the requirements of the machine (according to the official instructions, the total nucleic acid amount in the mixed library stage needs to be at least 100ng, and considering the purification loss , the mixing amount of a single sample in the mixed library stage should not be less than 200ng, that is, based on a 50μL system, the concentration should not be less than 4ng / μL).

[0...

Embodiment 2

[0050] Example 2 Grady database construction method

[0051] 1. Build the library according to the Grady method, in which 7.5 μL of DNA template is mixed with 2.5 μL of FRM fragmentation mixture, the number of cycles is increased to 25 cycles, and the extension time is shortened to 4 minutes. Still using 1ng and 0.25ng of DNA template as the initial amount, the amount of RLB rapid barcode primer used was 1 μL, and 56°C and 51°C were tried respectively (see Table 3 for specific PCR conditions). The results of library construction showed that after 25 cycles, the amount of nucleic acid in the PCR product met the requirements for the machine (see Table 4).

[0052] Table 3. SQK-RPB004Grady process trial PCR conditions for Zymo standards

[0053]

[0054] Table 4. 25-cycle PCR product results of the SQK-RPB004Grady process for Zymo standards

[0055]

[0056] 2. According to the official procedure, the library is mixed with the computer, and the bioinformatics analysis is ...

Embodiment 3

[0063] Embodiment 3 library system optimization (RLB primers and PCR reaction cycle number)

[0064] On the basis of Example 2, optimize the PCR system for RLB rapid barcode primers and the number of amplification cycles in order to improve the success rate of library construction: increase the amount of RLB primers incorporated to 2 μL, and reduce the number of PCR amplification cycles to 14 and 20 to examine whether the requirements for library construction can be met (see Table 7 for specific PCR conditions). The results showed that when the number of cycles was 14, the amount of nucleic acid in the PCR product could not be used on the machine (see Table 8). And when the number of cycles is 20, the amount of nucleic acid of the PCR product meets the requirements of the machine (see Table 9). Therefore, compared with the results of 1 μL of RLB rapid barcode primers, 2 μL of RLB primers (20 cycles) is the optimized amplification condition.

[0065] Table 7. SQK-RPB004Grady ...

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Abstract

The invention relates to a library establishment method for metagenome sequencing, a kit and a sequencing method. The library establishment method comprises the steps of fragmentation of a DNA template, PCR amplification, connector addition and the like. The primer application quantity of PCR amplification is 1.5-2.5 muL / 50muL in system, the circulation number is 18-22, the annealing temperature is 50-60 DEG C, and DNA polymerase with non-biased GC is selected. According to the method, by optimizing a library establishment system, the initial number of library establishment DNA templates is lowered, the library establishment success rate is increased, the PCR amplification bias is lowered, and the technical problems are solved that for an existing library establishment method, the demand for the initial number of the DNA templates is high, the amplification bias easily occurs, and the strain abundance is severely out-of-balance. The method is more beneficial to precise detection of themetagenome.

Description

technical field [0001] The present invention relates to the field of sequencing, in particular to a library construction method, a kit and a sequencing method for metagenomic sequencing. Background technique [0002] Compared with the traditional culture identification method, metagenomic sequencing identification has the advantages of short identification period and low requirements on the technical level of operators and identification personnel. Metagenome sequencing overcomes the disadvantages of identifying many microorganisms that cannot be cultivated, and is increasingly used in microbial identification, especially the identification of unknown pathogenic microorganisms. The current mainstream metagenomic sequencing methods are mainly high-throughput sequencing methods, including next-generation sequencing and third-generation sequencing. Among them, the third-generation sequencing, such as nanopore sequencing, is currently a relatively fast and convenient metagenomi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C40B50/06C12Q1/6869C12Q1/6806
Inventor 张烨程晨周水莲
Owner BEIJING SIMCERE MEDICAL LAB CO LTD
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