Generation of High-Quality African Swine Fever Virus Complete Genome from Field Samples by Next-Generation Sequencing

Abstract

African swine fever (ASF) is a lethal contagious viral disease of domestic pigs and wild boars caused by the African swine fever virus (ASFV). The pandemic spread of ASF has caused severe effects on the global pig industry. Whole-genome sequencing provides crucial information for virus strain characterization, epidemiology analysis and vaccine development. Here, we evaluated the performance of next-generation sequencing (NGS) in generating ASFV genome sequences from clinical samples. Thirty-four ASFV-positive field samples including spleen, lymph node, lung, liver and blood with a range of Ct values from 14.73 to 25.95 were sequenced. For different tissue samples collected from the same sick pigs, the proportion of ASFV reads obtained from the spleen samples was 3.69–9.86 times higher than other tissues. For the high-viral-load spleen samples (Ct < 20), a minimum of a 99.8% breadth of ≥10× coverage was revealed for all the samples. For the spleen samples with Ct ≥ 20, 6/12 samples had a minimum of a 99.8% breadth of ≥10× coverage. A high average depth of sequencing coverage was also achieved from the blood samples. According to our results, high-quality ASFV whole-genome sequences could be obtained from the spleen or blood samples with Ct < 20. The high-quality ASFV genome sequence generated in this study was further used for the high-resolution phylogenetic analysis of the ASFV genomes in the early stage of the ASF epidemic in China. Our study demonstrates that NGS may act as a useful tool for efficient ASFV genome characterization, providing valuable information for disease control.

Rapid Detection and Quick Characterization of African Swine Fever Virus Using the VolTRAX Automated Library Preparation Platform

“…As previously described, no normalization steps were performed, in order to simulate samples processed in the field. The samples used for this experiment showed Ct values between 17.45 and 19.75 (Table 3), which ensured obtaining a good quality sequence for ASFV, as described by Shi et al [20]. ASFV frozen whole-blood sample and fresh whole-blood sample from a pig experimentally infected with ASFV Georgia 2007/1 were processed for library preparation with VolTRAX and sequenced on Nanopore next-generation sequencers to evaluate the consistency of library preparation when frozen samples are used.…”

“…As previously described, no normalization steps were performed, in order to simulate samples processed in the field. The samples used for this experiment showed Ct values between 17.45 and 19.75 (Table 3), which ensured obtaining a good quality sequence for ASFV, as described by Shi et al [20]. Concentrations obtained from the extractions ranged from 21 to 72 ng/µL (Table 3).…”