Establishment of a high-throughput sequencing method for the whole genome of human adenovirus 3 based on multiplex PCR
10.3760/cma.j.cn112866-20230731-00012
- VernacularTitle:基于多重PCR的HAdV-3全基因组高通量测序方法的建立
- Author:
Qi LIN
1
;
Zhimiao HUANG
;
Yuwei WENG
;
Wenxiang HE
;
Libin YOU
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:
Human adenovirus 3;
Multiplex PCR;
Whole genome sequence;
High-throughput sequencing
- From:
Chinese Journal of Experimental and Clinical Virology
2023;37(5):530-536
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To improve the efficiency and success rate of human adenovirus 3 (HAdV-3) whole genome sequencing, a high-throughput sequencing method for the whole genome of HAdV-3 based on multiplex PCR was established.Methods:Multiplex PCR primers suitable for the whole genome amplification of HAdV-3 were designed. The whole genome sequence of HAdV-3 was amplified by multiplex PCR, and the specificity of the amplification product was preliminarily verified by agarose gel electrophoresis. High-throughput sequencing of the multiplex PCR products was performed using Illumina second-generation sequencing. After obtaining the sequence, software such as CLC and IGV were used to analyze the effective data amount, average sequencing depth, and whole genome coverage obtained by high-throughput sequencing, then the sequencing quality was evaluated. Based on the whole genome sequencing result, a phylogenetic tree was constructed to confirm the virus type and analyze homology of the sequences, and then the accuracy of this method was evaluated.Results:A total of 70 (35 pairs) multiplex PCR amplification primers for the whole genome of HAdV-3 were designed, with amplicon size of approximately 1 200 bp. And the expected whole genome coverage could reach 99.8% (with a total genome length of approximately 35 240 bp). Agarose gel electrophoresis analysis showed that the size of the multiplex PCR products was consistent with expectations, and the amplification specificity was high. The high-throughput sequencing result showed that the whole genome sequences obtained by this method were complete and intact, and the genome coverage was consistent with expectations. Sequence quality analysis showed that the high-throughput sequencing method based on multiplex PCR could obtain more effective data and greater sequencing depth, resulting in more uniform whole genome coverage. Phylogenetic analysis showed that the evolutionary typing result of viral DNA sequenced after multiplex PCR amplification were consistent with those of viral DNA sequenced directly and had high homology, indicating that the multiplex PCR method had high amplification fidelity and the results obtained in combination with high-throughput sequencing were accurate.Conclusions:A high-throughput sequencing method for the whole genome of HAdV-3 based on multiplex PCR was established in this study successfully. This method could improve the efficiency and success rate of HAdV-3 whole genome sequencing, aiming to provide better technical support and reference for HAdV-3 pathogen surveillance and epidemic source-tracing based on whole genome sequencing.
