Comparison of amplicon sequencing and metagenomic sequencing strategies in MPXV whole-genome sequencing testing
10.3969/j.issn.1002-2694.2024.00.138
- VernacularTitle:扩增子与宏基因组策略在猴痘病毒全基因组测序考核中的应用比较
- Author:
Zhi-Miao HUANG
1
;
Yu-Wei WENG
;
Wei CHEN
;
Li-Bin YOU
;
Jin-Zhang WANG
;
Ting-Ting YU
;
Qi LIN
Author Information
1. 福建省疾病预防控制中心,福建省人兽共患病研究重点实验室,福州 350012
- Keywords:
Mpox virus;
whole-genome sequencing;
amplicon sequencing;
metagenomic sequencing
- From:
Chinese Journal of Zoonoses
2024;40(10):944-949
- CountryChina
- Language:Chinese
-
Abstract:
The implementation of amplicon sequencing and metagenomic sequencing methods in the whole-genome sequen-cing for MPXV testing was compared,to provide a technical reference for sequencing,tracing,and epidemic prevention and control of MPXV.For amplicon sequencing,targeted amplification of the viral whole genome was performed on MPXV DNA,and was followed by next-generation sequencing of the amplification products.For metagenomic sequencing,next-generation sequencing was performed directly on MPXV DNA.After the sequences were obtained,software such as CLC and IGV were used to analyze the effective data percentage,sequencing depth,and whole-genome sequencing coverage under different sequen-cing depths for both sequencing methods,to evaluate sequencing quality.Nextclade was used to analyze virus typing,muta-tions,and deletions.Subsequently,the similarity and completeness of sequences obtained through both sequencing methods were further compared.On the basis of mapping to the refer-ence sequence of strain MPXV-M5312_HM12_Rivers(Gen-Bank number NC_063383.1),the percentage effective data obtained from amplicon sequencing and metagenomic sequen-cing was 99.72％and 7.54％,respectively,with a sequencing depth range of 0× to 334 839 ×,and 44 × to 1 000 ×.On the basis of a sequencing depth of 10 ×,the site coverage of the above was 90.3％and 100％,respectively.IGV was used to validate the whole-genome coverage under different sequencing depths.The depth coverage of whole-genome sites for metagenomic sequencing was uniform,whereas that of the whole-genome sites for amplicon sequencing was uneven and significantly differed.Virus typing and sequence similarity analysis indicated that the viral sequences obtained with the two sequencing methods all belonged to the Ⅱb B.1 lineage of MPXV.Comparison with the reference sequence indicated that metagenomic sequencing identified 73 nucleotide mutation sites,whereas amplicon sequen-cing identified 68 mutation sites.Further analysis demonstrated that seven common mutation sites of Ⅱb B.1 were not detected in the amplicon sequencing,and two false positive private mutation sites were identified.Amplicon or metagenomic sequencing methods thus can be flexibly used in MPXV virus whole-genome sequencing.Amplicon sequencing yields more effective data,whereas metagenomic sequencing provides better uniformity of coverage and sequence accuracy.This study provides a prelimi-nary understanding of the efficacy of each method and may serve as a technical reference for improving the success rate of whole-genome sequencing of MPXV.
