Establishment of a high-throughput sequencing platform for the whole genome of Chikungunya virus based on a multiplex-PCR method
10.3760/cma.j.cn112150-20231114-00339
- VernacularTitle:一种基于多重PCR的基孔肯雅病毒全基因组高通量测序方法的建立
- Author:
Wenzhe SU
1
;
Yan LI
;
Weizhi LU
;
Huaping XIE
;
Kuibiao LI
;
Biao DI
;
Kai NIE
;
Huanyu WANG
;
Zhoubin ZHANG
;
Songtao XU
Author Information
1. 广州市疾病预防控制中心病毒免疫部，广州 510440
- Keywords:
Chikungunya virus;
Whole genome sequencing;
Multiplex-PCR;
High throughput sequencing
- From:
Chinese Journal of Preventive Medicine
2024;58(4):489-496
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To establish a rapid pipeline for whole genome sequencing of Chikungunya virus (CHIKV) by combining imbricated multiplex-PCR amplification and Illumina high-throughput sequencing platform.Methods:The primary reference sequences of CHIKV were downloaded from the National Center for Biotechnology Information (NCBI) database, covering all genotypes of CHIKV. After multiple alignments using the Mafft software and phylogenetic analysis, the 20 CHIKV references were selected for primer design. The Primal Scheme tool and Geneious Prime software were used to design, evaluate and optimize the primer panel. Finally, seven CHIKV-positive samples were involved in the validation of the primer panel.Results:All the amplicons of the designed panel were generated successfully. The consensuses generated from the mapping results could cover 100.00% of the coding region of the CHIKV genome when the Ct-value of the sample was less than 33, as the percentage would decrease to 99.38% when the Ct-value reached 35. The mapping percentage could be increased by 5.70%-25.43% when using the stepwise correction mapping strategy.Conclusion:The multiplex-PCR amplification method for CHIKV whole genome sequencing is relatively simple and convenient, which only requires two tubes of PCR amplification and performs well on CHIKV-positive clinical samples with different concentration levels of virus.
