Library establishment method for metagenome sequencing, kit and sequencing method

A metagenomics and kit technology, applied in the field of sequencing, can solve serious problems in identification, increase the number of PCR cycles, aggravate amplification bias, etc., reduce the initial amount of library construction, reduce PCR amplification bias, The effect of improving the success rate of database construction

Active Publication Date: 2019-05-21
BEIJING SIMCERE MEDICAL LAB CO LTD +2
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AI Technical Summary

Benefits of technology

This patented technology improves upon existing techniques used during genetic analysis such as qPCR or other nucleic acid-based assays that require multiple steps before obtaining results from each reaction. By doing this, these new technologies improve accuracy while reducing errors associated with contamination between different reactions. Additionally, they provide better control over how well various types of bacteria live alongside human beings than previously possible due to their ability to hide within certain structures inside cells. Overall, this innovation enhances precision medicine and enables improved healthcare outcomes based on accurate diagnosis of diseases caused by specific organism populations (microbial communities).

Problems solved by technology

This patented technology proposes improving upon existing techniques like quick pyrosequencers while reducing their complexity. It involves developing new types of molecular probes called metabolites, specifically ribonucleotides, nucleosids, peptide sequences, proteins, enzyme complexes, etc., that allow for faster and easier detection of small amounts of organism from large specimens compared to previous technologies. These advancements make metallurgytic sequential testing possible without requiring expensive instrumentations at all levels.

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  • Library establishment method for metagenome sequencing, kit and sequencing method
  • Library establishment method for metagenome sequencing, kit and sequencing method
  • Library establishment method for metagenome sequencing, kit and sequencing method

Examples

Experimental program
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Embodiment 1

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

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

[0046] Table 1. SQK-RPB...

Embodiment 2

[0050] Example 2 Grady library construction method

[0051] 1. Build the library according to the Grady method, in which 7.5μL DNA template and 2.5μL FRM fragmentation mixture are mixed, the number of cycles is increased to 25 cycles, and the extension time is shortened to 4mins. Still taking 1ng and 0.25ng DNA template as the starting amount, the RLB fast barcode primer dosage is 1μL, try 56°C and 51°C respectively (see Table 3 for specific PCR conditions). The result of library construction shows that after 25 cycles, the amount of nucleic acid of PCR product meets the computer requirements (see Table 4).

[0052] Table 3. SQK-RPB004Grady process of Zymo standard product tried PCR conditions

[0053]

[0054] Table 4. Results of 25-cycle PCR products of the SQK-RPB004Grady process of Zymo standard products

[0055]

[0056] 2. Follow the official process to mix the library on the computer and perform bioinformatics analysis according to the standard process. However, the sequencin...

Embodiment 3

[0063] Example 3 Optimization of library construction system (RLB primers and PCR reaction cycles)

[0064] On the basis of Example 2, the PCR system was optimized for RLB rapid barcode primers and the number of amplification cycles 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, check whether the database can be built (see Table 7 for specific PCR conditions). The results show that when the number of cycles is 14, the amount of nucleic acid of the PCR product cannot be used on the machine (see Table 8). When the number of cycles is 20, the amount of nucleic acid in the PCR product meets the computer requirements (see Table 9). Therefore, comparing the results of the 1μL dosage of RLB fast barcode primer, 2μL RLB primer (20 cycles) is the optimized amplification condition.

[0065] Table 7. SQK-RPB004Grady process improvement of Zymo standard products try ...

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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.

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Claims

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Application Information

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Owner BEIJING SIMCERE MEDICAL LAB CO LTD
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