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GB/T 38481-2020 English PDF (GBT38481-2020)

GB/T 38481-2020 English PDF (GBT38481-2020)

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GB/T 38481-2020: Determination of ultralow-frequency mutations for microorganisms - Duplex sequencing

This Standard specifies the method for the determination of ultralow-frequency mutations for microorganisms by duplex sequencing. This Standard is applicable to the determination of ultralow-frequency mutations in microbial genomes or gene fragments.
GB/T 38481-2020
GB
NATIONAL STANDARD OF THE
PEOPLE REPUBLIC OF CHINA
ICS 07.080
A 21
GB 38481-2020
Determination of Ultralow-Frequency
Mutations for Microorganisms ?€? Duplex Sequencing
ISSUED ON: NOVEMBER 19, 2020
IMPLEMENTED ON: NOVEMBER 19, 2020
Issued by: State Administration for Market Regulation;
Standardization Administration of PRC.
Table of Contents
Foreword ... 3
1 Scope ... 4
2 Normative References ... 4
3 Terms and Definitions ... 4
4 Principle ... 5
5 Reagents ... 5
6 Apparatus and Utensil ... 6
7 Preparation of the Sample ... 6
8 Synthesis of Linker Barcode Label ... 6
9 Connection and Amplification of the Linker Barcode Label and the DNA of the Sample to be Tested ... 7
10 High-throughput Sequencing ... 8
11 Analysis of the Results ... 8
12 Calculation and Presentation of the Results ... 8
Determination of Ultralow-Frequency
Mutations for Microorganisms ?€? Duplex Sequencing
1 Scope
This Standard specifies the method for the determination of ultralow-frequency mutations for microorganisms by duplex sequencing.
This Standard is applicable to the determination of ultralow-frequency mutations in microbial genomes or gene fragments.
2 Normative References
The following documents are essential to the application of this document. For the dated documents, only the versions with the dates indicated are applicable to this document; for the undated documents, only the latest version (including all the amendments) is applicable to this document.
GB/T 6682 Water for Analytical Laboratory Use - Specification and Test Methods 3 Terms and Definitions
For the purpose of this document, the following terms and definitions apply. 3.1 Ultralow-frequency mutations
Chemical, physical or biological factors lead to permanent changes of microbial DNA with a frequency of less than 1??10-5.
3.2 Barcode label
Use the nucleotide fragments connected to both sides of the DNA fragment to be tested to mark the DNA of the same amplified family and assist in the confirmation of mutation sites.
3.3 Complementary tag family
All DNA fragments with complementary barcode labels at both ends.
5.7 DNA analysis kit.
5.8 Tris-ethylenediaminetetraacetic acid (Tris-EDTA) buffer solution, pH 8.0. Prepared by 10mmol/L analytical pure Tris and 0.1mmol/L EDTA, adjust the pH value to 8.0. 6 Apparatus and Utensil
6.1 PCR thermal cycler.
6.2 Magnetic separator.
6.3 High-throughput sequencer.
6.4 Nucleic acid analysis system.
6.5 Electronic balance: the accuracy is 0.01g.
7 Preparation of the Sample
7.1 DNA extraction
Use microbial genome or plasmid extraction kit to perform genomic or plasmid DNA extraction operation on the sample to be tested; and finally use 50??L of sterile double- distilled water for DNA elution. The extracted DNA sample is used to detect the concentration and purity of nucleic acid by a nucleic acid quantifier.
7.2 DNA fragmentation and end repair
Use the high-throughput gene sequencing library building kit to fragment the DNA of the sample to be tested to 100bp~1000bp, and repair the end to the Tail-A. 8 Synthesis of Linker Barcode Label
8.1 Annealing
Take 100??L of each of the two linker chain oligonucleotide fragments with a concentration of 100??mol/L for annealing; heat at 95??C for 5min, then cool down naturally, and stand for 1h.
8.2 Extension
The product obtained in 8.1 is mixed with deoxyribonucleoside triphosphate mixture (dNTPs) and Klenow enzyme according to the instructions of the reagents; divide them Use the sorted product as the template and the linker chain amplification primer sequence as the primer; 1 amol DNA sample amount per 200,000 readings was used for PCR amplification. The amplification program is set according to the requirements of the selected high-fidelity DNA polymerase.
10 High-throughput Sequencing
Use a high-throughput sequencing kit to perform high-throughput sequencing on the amplified PCR products; and the number of sequencing bases for each sample to be tested is set to be greater than or equal to 10Gb.
11 Analysis of the Results
Clean the high-throughput sequencing data; and then perform data analysis according to the following procedures:
a) Complementary label classification based on random labels is performed; each family needs to contain two-way complementary sequences. For the
complementary tag family size distribution after excluding 1, the maximum peak shall be distributed between 6 and 12.
b) Only when the double-stranded homologous sequence recognizes the mutation at the same site shall it be regarded as the real mutation site.
12 Calculation and Presentation of the Results
12.1 Calculation of the results
The mutation rate is calculated according to Formula (1):
Where:
M - mutation rate;
Nm - the number of mutation sites of double-stranded homologous sequence; Nt - the total number of sequenced nucleotides without tags.
12.2 Presentation of results

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