GB/T 38481-2020.Determination of ultralow-frequency mutations for microorganisms-Duplex sequencing.
1 Scope
GB/T 38481 specifies the method for determining ultralow-frequency mutations of microorganisms by duplex sequencing.
GB/T 38481 is applicable to the determination of ultralow-frequency mutations in microbial genomes or gene fragments.
2 Normative references
The following documents are indispensable for the application of this document. For any dated referenced document, only the dated version applies to this document. For any undated referenced document, its latest version (including all amendments) applies to this document.
GB/T 6682 Specifications and test methods for water for analytical laboratories
3 Terms and definitions
The following terms and definitions apply to this document.
3.1
Ultralow-frequency mutations
Permanent changes in microbial DNA caused by chemical, physical or biological factors with a frequency lower than 1X 10-.
3.2
Barcode label
Nucleotide fragments attached to both sides of the DNA fragment to be tested, used to mark the DNA of the same amplification family. Assist in the confirmation of mutation sites.
3.3
Complementary tag family
All DNA fragments with complementary barcode tags at both ends.
3.4
?? Double-strand consensus sequences; DCSs
Double-strand DNA sequences with the same barcode tag, including the reverse complementary strand.
4 Principle
A 12 nt random and complementary double-stranded nucleotide barcode tag is attached to both ends of the DNA fragment to be tested, and then all sequences with barcode tags are subjected to double high-throughput sequencing. The 12 nt random complementary tag is used to exclude mutations introduced during the preparation of the sequencing library, thereby accurately detecting the mutation site and mutation rate.
5 Reagents
Unless otherwise specified, only analytical reagents are used.
5.1 Water. GB/T 6682, Grade 1.
This standard specifies the method for determining ultra-low frequency mutations in microorganisms by double sequencing. This standard is applicable to the determination of ultra-low frequency mutations in microbial genomes or gene fragments.

ICS07.080
National Standard of the People's Republic of China
GB/T38481—2020
Determination of ultralow-frequency mutations for microorganisms-Duplex sequencing
Published on 2020-11-19
State Administration for Market Regulation
National Administration of Standardization
Published
Implementation on 2020-11-19
This standard was drafted in accordance with the rules given in GB/T1.1-2009. This standard was proposed and managed by the China National Institute of Standardization. GB/T38481—2020
Drafting organizations of this standard: Tsinghua University, Luoyang Huaqing Tianmu Biotechnology Co., Ltd., Jianghan University, China National Institute of Standardization. The main drafters of this standard: Zhang Du, Xing Xinhui, Li Mei, Jian Xingjin, Wang Liyan, Guo Xiaojie, Zhang Lele, Peng Hai, Ma Aijin, I
1 Scope
Determination of ultra-low frequency mutations in microorganisms
Double sequencing method
This standard specifies the method for determining ultra-low frequency mutations in microorganisms by double sequencing. This standard applies to the determination of ultra-low frequency mutations in microbial genomes or gene fragments. Normative references
GB/T38481—2020
The following documents are indispensable for the application of this document. For all dated references, only the dated version applies to this document. For all undated references, the latest version (including all amendments) applies to this document. GB/T6682 Specifications and test methods for water for analytical laboratories Terms and definitions
The following terms and definitions apply to this document. 3.1
Ultralow-frequency mutations
Ultralow-frequency mutations Chemical, physical or biological factors cause permanent changes in microbial DNA with a frequency lower than 1X10-5. 3.2
Barcode label
barcode label
Nucleic acid fragments attached to both sides of the DNA fragment to be tested, used to mark the DNA of the same amplification family and assist in the confirmation of mutation sites.
Complementary tag family
complementarytagfamily
All DNA fragments with complementary barcode labels at both ends. 3.4
Double-strand consensus sequences
Double-strand consensus sequences; DCSsDouble-strand DNA sequences with the same barcode label, including the reverse complementary strand. 4 Principle
Attach a 12nt random and complementary double-stranded nucleotide barcode tag to both ends of the DNA fragment to be tested, and then perform double high-throughput sequencing on all sequences with barcode tags. The 12nt random complementary tag is used to exclude mutations introduced during the preparation of the sequencing library, thereby accurately detecting the mutation site and mutation rate. 6
Reagents
Unless otherwise specified, only analytical reagents are used. 5.1 Water. GB/T6682, Grade 1.
5.2 Linker chain tag sequence:
GB/T38481—2020
a) 5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3'; b) 5'-TCTTCTACAGTCANNNNNNNNNNNNAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC-3' N is A, T, C or G, and the tag fragment consists of 12 random bases. Dissolve the oligonucleotide in Tris-EDTA buffer (5.8) or pure water to a final concentration of 100 μmol/L and store it at -20°C.
5.3 Primer sequence for linker chain amplification:
a) 5'-AATGATACGGCGACCACCGAG-3'; b) 5'-CAAGCAGAAGACGGCATACGAGATXXXXXXGTGACTGGAGTTCAGACGTGTGC-3'. X is the library tag sequence designed according to sample requirements. Dissolve the oligonucleotide in Tris-EDTA buffer (5.8) or pure water to a final concentration of 20μmol/L and store at -20℃. 5.4 Gene extraction kit.
5.5 High-throughput sequencing library construction kit.
5 DNA sorting magnetic bead kit.
DNA analysis kit.
5.71
Tris-EDTA buffer, pH 8.0. Prepared with 10mmol/L analytical grade Tris and 0.1mmol/L EDTA, adjust the pH value to 8.0.6 Instruments and apparatus
PCR amplification instrument.
2 Magnetic separator.
3 High-throughput sequencer.
6.4 Wood
Nucleic acid analysis system.
5 Electronic balance: accuracy of 0.01g.
7 Sample preparation
DNA extraction
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Use microbial genome or plasmid extraction kit to extract genome or plasmid DNA from the sample to be tested, and finally use 50μL sterile double distilled water to elute the DNA. The extracted DNA samples were tested for nucleic acid concentration and purity using a nucleic acid quantifier. 7.2
DNA fragmentation and end repair
The DNA of the sample to be tested was fragmented to 100 bp to 1000 bp using a high-throughput gene sequencing library construction kit, and the ends were repaired to A tails.
8 Synthesis of adapter barcode tags
8.1 Annealing
Take 100 μL of each of the two adapter chain oligonucleotide fragments with a concentration of 100 umol/L for annealing, heat at 95°C for 5 min, cool naturally, and let stand for 1 h.
2 Extension
Mix the product obtained in 8.1 with deoxyribonucleoside triphosphate mixture (dNTPs) and Klenow enzyme according to the reagent instructions, divide it into two 0.2mL PCR tubes and incubate at 37℃ for 1h. 2
8.3 DNA recovery
Precipitate DNA with anhydrous ethanol and add 200μL pure water to dissolve it. 8.4 Enzyme digestion
GB/T38481—2020
Digest the DNA product with restriction endonuclease HpyCH4Ⅲ. Divide the mixture into 4 0.2mL PCR tubes and incubate at 37℃ for 16h.
8.5 Purification
Add and evenly mix 50uL of 3mol/L sodium acetate solution (pH5.2), and the final volume is 550uL. Divide the mixed solution equally into 1.5mL cold centrifuge tubes, 275μL per tube, and add 625μL of room temperature anhydrous ethanol to each tube. Mix thoroughly and centrifuge at a speed greater than 10000r/min at 4℃ for 30min. After removing the supernatant, add 1mL of 75% (volume fraction) ethanol to each tube. Mix thoroughly and immediately centrifuge at a speed greater than 10000r/min at 4℃ for 30min.
After removing the supernatant, invert the test tube on a paper towel for 10min to dry, then place it upright for 5min. Add 100uL of Tris-EDTA buffer to the product of the previous step and mix well. Collect all the test tubes together, the final volume is 200μL, the final concentration is about 50μmol/L, and store in a refrigerator at -20℃. 9. Connecting and amplifying the barcode label of the adapter and the DNA of the sample to be tested 9.1 Connecting
Mix the complementary barcode label with the DNA fragmentation and end repair product at a total concentration of 20:1, connect them using T4 DNA ligase, and incubate at 25℃ for 15min.
2 Sorting
Sorting the obtained products using a DNA sorting magnetic bead kit to obtain DNA fragments with a length of 200bp to 900bp, and quantifying the DNA concentration using a DNA kit.
9.3PCR amplification
Using the sorted product as template and the adapter chain amplification primer sequence as primer, 1amol DNA sample was taken for PCR amplification for every 200,000 reads. The amplification program was set according to the requirements of the selected high-fidelity DNA polymerase. 10
High-throughput sequencing
The amplified PCR products were subjected to high-throughput sequencing using a high-throughput sequencing kit. The number of sequencing bases for each sample to be tested was set to be greater than or equal to 10 Gb.
11Result analysis
The high-throughput sequencing data was cleaned and then analyzed according to the following procedures:Complementary tags were classified according to random tags, and each family must contain bidirectional complementary sequences.Complementary tag family size distributiona)
After excluding 1, the maximum peak distribution should be between 6 and 12. b) Double-stranded homologous sequences that recognize mutations at the same site are considered true mutation sites. GB/T38481—2020
Result calculation and expression
12.1
Result calculation
The mutation rate is calculated according to formula (1):
Wherein:
——Mutation rate;
N.——Number of mutation sites in double-stranded homologous sequences; N,——Total number of sequenced nucleotides without labels. 12.2
Result expression
The result is expressed as the mutation rate M of the sample to be tested. SZAC
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