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Research article - Peer-reviewed, 2017

Single primer isothermal amplification (SPIA) combined with next generation sequencing provides complete bovine coronavirus genome coverage and higher sequence depth compared to sequence-independent single primer amplification (SISPA)

Myrmel, Mette; Oma, Veslemoy; Khatri, Mamata; Hansen, Hanne H.; Stokstad, Maria; Berg, Mikael; Blomstrom, Anne-Lie


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.

Published in

2017, Volume: 12, number: 11, article number: e0187780