Low-host double MDA workflow for uncultured ASFV positive blood and serum sample sequencing

Chengjun Zhang; Tangyu Cheng; Dongfan Li; Xuexiang Yu; Fangzhou Chen; Qigai He

doi:10.3389/fvets.2022.936781

Low-host double MDA workflow for uncultured ASFV positive blood and serum sample sequencing

Front Vet Sci. 2022 Sep 20:9:936781. doi: 10.3389/fvets.2022.936781. eCollection 2022.

Authors

Chengjun Zhang^{1

2}, Tangyu Cheng^{1

2}, Dongfan Li^{2

3}, Xuexiang Yu^{2

3}, Fangzhou Chen^{1

2

3}, Qigai He^{1

2

3}

Affiliations

¹ State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China.
² College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.
³ The Cooperative Innovation Center for Sustainable Pig Production, Huazhong Agricultural University, Wuhan, China.

Abstract

African swine fever (ASF) is a highly lethal and contagious disease caused by African swine fever virus (ASFV). Whole-genome sequencing of ASFV is necessary to study its mutation, recombination, and trace its transmission. Uncultured samples have a considerable amount of background DNA, which causes waste of sequencing throughput, storage space, and computing resources. Sequencing methods attempted for uncultured samples have various drawbacks. In this study, we improved C18 spacer MDA (Multiple Displacement Amplification)-combined host DNA exhaustion strategy to remove background DNA and fit NGS and TGS sequencing. Using this workflow, we successfully sequenced two uncultured ASFV positive samples. The results show that this method can significantly reduce the percentage of background DNA. We also developed software that can perform real-time base call and analyses in set intervals of ASFV TGS sequencing reads on a cloud server.

Keywords: African swine fever; genome sequencing; nanopore sequencing; next generation sequencing (NGS); workflow.