Abstract
Coronaviruses are of major importance for both animal and human health. With the emergence of novel coronaviruses such as SARS and MERS, the need for fast genome characterisation is ever so important. Further, in order to understand the influence of quasispecies of these viruses in relation to biology, techniques for deep-sequence and full-length viral genome analysis are needed. In the present study, we compared the efficiency of two sequence-independent approaches [sequence-independent single primer amplification (SISPA) and single primer isothermal amplification (SPIA, represented by the Ovation kit)] coupled with high-throughput sequencing to generate the full-length genome of bovine coronavirus (BCoV) from a nasal swab. Both methods achieved high genome coverage (100% for SPIA and 99% for SISPA), however, there was a clear difference in the percentage of reads that mapped to BCoV. While approximately 45% of the Ovation reads mapped to BCoV (sequence depth of 169–284 944), only 0.07% of the SISPA reads (sequence depth of 0–249) mapped to the reference genome. Although BCoV was the focus of the study we also identified a bovine rhinitis B virus (BRBV) in the data sets. The trend for this virus was similar to that observed for BCoV regarding Ovation vs. SISPA, but with fewer sequences mapping to BRBV due to a lower amount of this virus. In summary, the SPIA approach used in this study produced coverage of the entire BCoV (high copy number) and BRBV (low copy number) and a high sequence/genome depth compared to SISPA. Although this is a limited study, the results indicate that the Ovation method could be a preferred approach for full genome sequencing if a low copy number of viral RNA is expected and if high sequence depth is desired.
Highlights
Generation sequencing (NGS) has become a valuable tool in virology studies and has the power to generate whole genome sequences (WGS) from small amounts of virus, rapidly and at a relatively low cost [1]
Mapping of the reads to the bovine coronavirus (BCoV) reference revealed a clear difference between the datasets originating from the two amplification methods, as only 0.07% of the reads from the sequence-independent single primer amplification (SISPA) datasets mapped to BCoV compared to 42–47% of the reads from the Ovation datasets (Table 1)
Regarding the non-BCoV sequences, for both methods the majority (87–89%) could be annotated through blastx as eukaryotic and 10–13% were of bacterial origin
Summary
Generation sequencing (NGS) has become a valuable tool in virology studies and has the power to generate whole genome sequences (WGS) from small amounts of virus, rapidly and at a relatively low cost [1]. Sequence-independent methods are attractive in that they allow sequencing of highly divergent viruses, but efficient reduction of background nucleic acids is required in order to get good coverage and depth (number of times each nucleotide is sequenced) of the viral genomes [3]. While standard sequencing misses allelic variants with a frequency below 20% in a population [4], NGS can detect less abundant variants depending on sequencing depth. This provides an increased opportunity for viral metagenomics (study of the total viral community), for epidemiologic surveillance of viruses, and for obtaining insight into viral evolution and fitness. It is important to have techniques that can rapidly sequence new emerging coronaviruses and those that investigate the quasispecies population in order to understand the biology of these important viruses
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