Metagenome library construction method and kit for nanopore sequencing platform

Abstract

The invention relates to a macrogene library building method of a nanopore sequencing platform and a kit thereof. By optimizing and adjusting the final connection and PCR amplification in the construction of the library, the library construction method and its kit of the present invention effectively reduce the initial amount of library construction, improve the read length and quality of sequencing data, and compress the library construction process. And sequencing time, saving the cost of sequencing, and at the same time improving the sensitivity of the whole process, meeting the clinical requirements for infection detection, suitable for popularization and application.

Description

technical field

[0001] The invention relates to the field of sequencing, in particular to a method for constructing a macrogene library based on a nanopore sequencing platform and a kit thereof.

[0002] technical background

[0003] Infectious diseases are an important cause of human disease and death. Among respiratory infections, in 2016, lower respiratory infections caused at least 3 million deaths globally. However, determining the etiology of lower respiratory tract infection remains challenging due to methodological limitations. Traditional methods for diagnosing lower respiratory tract infections include culture and serological tests, which are insensitive and time-consuming. Statistically, only 38% of adult pneumonia patients with imaging evidence could successfully identify the causative agent. The overuse of broad-spectrum antibiotics has made identification of pathogens more difficult. For each patient, the slow diagnostic process leads to inappropriate empiri...

Examples

Embodiment 1

[0105] Embodiment 1 performance optimization and parameter adjustment experiment

[0106] The present invention uses ONT's PCR barcode kit (SQK-PBK004) as the basis for optimization experiments.

[0107] The present invention first selected a DNA nucleic acid standard product ZymoBIOMICS produced by Zymo Research Company TM Microbial Community DNA Standard (CatalogNos.D6305) is used as the input for verification. It is composed of 8 kinds of bacteria and 2 kinds of fungi that are clinically very common in precise quantitative proportions. The overall GC content is widely distributed (15%-85%, as follows Table, DNA Standard Composition (Catalog Nos.D6305 (Zymo Research, Product Manual)). It is a good standard for studying microbiome.

[0108] Table 1. ZymoBIOMICS TM Microbial Community DNA Standard strain information

[0109]

[0110] 1) Optimal screening of end repair and ligase

[0111] Considering the high requirements for sample size, experimental accuracy, and time...

Embodiment 2

[0124] Embodiment 2 establishes the method system of the present invention

[0125] Based on the optimization experiment in Example 1, the library construction method system of the present invention was obtained, and the method system was based on the PCR barcode kit (SQK-PBK004) of ONT.

[0126] 1. End repair of DNA fragments

[0127] 1 Add the finishing system to the 0.2mL PCR tube:

[0128]

[0129] Note: The total amount of sample extraction is supplemented with 100ng, and 45μL is put into it.

[0130] 2 Gently blow and mix with a pipette (do not shake and mix), collect the reaction solution to the bottom of the tube after a short centrifugation, and place the PCR tube on the PCR machine for the following reactions:

[0131] temperature time 20℃ 10min 65℃ 10min

[0132] 2. Joint connection:

[0133] 1 After the reaction is over, add the adapter connection system to the PCR tube:

[0134]

[0135] 2 Mix by blowing with a pipette, place...

Embodiment 3

[0161] The preparation of embodiment 3 kits

[0162] According to the process system determined in Example 2, we assembled this protocol into two library preparation kits as shown in the table below. In view of the difference in storage temperature of the kits, we prepared them into two packaged kits, which need to be stored separately. Under the conditions of -20℃±5℃ and 2~8℃.

[0163] Kit 1 contains the following components (stored at -20°C±5°C):

[0164]

[0165]

[0166] Kit 2 contains the following components (stored at 2-8°C):

[0167]

technical field

[0001] The invention relates to the field of sequencing, in particular to a method for constructing a macrogene library based on a nanopore sequencing platform and a kit thereof.

[0002] technical background

[0003] Infectious diseases are an important cause of human disease and death. Among respiratory infections, in 2016, lower respiratory infections caused at least 3 million deaths globally. However, determining the etiology of lower respiratory tract infection remains challenging due to methodological limitations. Traditional methods for diagnosing lower respiratory tract infections include culture and serological tests, which are insensitive and time-consuming. Statistically, only 38% of adult pneumonia patients with imaging evidence could successfully identify the causative agent. The overuse of broad-spectrum antibiotics has made identification of pathogens more difficult. For each patient, the slow diagnostic process leads to inappropriate empiri...

Examples

Embodiment 1

[0105] Embodiment 1 performance optimization and parameter adjustment experiment

[0106] The present invention uses ONT's PCR barcode kit (SQK-PBK004) as the basis for optimization experiments.

[0107] The present invention first selected a DNA nucleic acid standard product ZymoBIOMICS produced by Zymo Research Company TM Microbial Community DNA Standard (CatalogNos.D6305) is used as the input for verification. It is composed of 8 kinds of bacteria and 2 kinds of fungi that are clinically very common in precise quantitative proportions. The overall GC content is widely distributed (15%-85%, as follows Table, DNA Standard Composition (Catalog Nos.D6305 (Zymo Research, Product Manual)). It is a good standard for studying microbiome.

[0108] Table 1. ZymoBIOMICS TM Microbial Community DNA Standard strain information

[0109]

[0110] 1) Optimal screening of end repair and ligase

[0111] Considering the high requirements for sample size, experimental accuracy, and time...

Embodiment 2

[0124] Embodiment 2 establishes the method system of the present invention

[0125] Based on the optimization experiment in Example 1, the library construction method system of the present invention was obtained, and the method system was based on the PCR barcode kit (SQK-PBK004) of ONT.

[0126] 1. End repair of DNA fragments

[0127] 1 Add the finishing system to the 0.2mL PCR tube:

[0128]

[0129] Note: The total amount of sample extraction is supplemented with 100ng, and 45μL is put into it.

[0130] 2 Gently blow and mix with a pipette (do not shake and mix), collect the reaction solution to the bottom of the tube after a short centrifugation, and place the PCR tube on the PCR machine for the following reactions:

[0131] temperature time 20℃ 10min 65℃ 10min

[0132] 2. Joint connection:

[0133] 1 After the reaction is over, add the adapter connection system to the PCR tube:

[0134]

[0135] 2 Mix by blowing with a pipette, place...

Embodiment 3

[0161] The preparation of embodiment 3 kits

[0162] According to the process system determined in Example 2, we assembled this protocol into two library preparation kits as shown in the table below. In view of the difference in storage temperature of the kits, we prepared them into two packaged kits, which need to be stored separately. Under the conditions of -20℃±5℃ and 2~8℃.

[0163] Kit 1 contains the following components (stored at -20°C±5°C):

[0164]

[0165]

[0166] Kit 2 contains the following components (stored at 2-8°C):

[0167]

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