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)

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.

Nanopore metagenomic sequencing of influenza virus directly from respiratory samples: diagnosis, drug resistance and nosocomial transmission

“…The lower sensitivity for enterovirus and coronavirus could be due to low viral titres in these samples, although we are not able to confirm this as the BioFire® RP2 assay is a non-quantitative test. Another possibility is that the SISPA method is less sensitive for certain viruses [26]. Moreover, no influenza B virus reads are present in our 90 influenza-positive samples, congruent with the global low level of influenza B virus during the 2018/19 season.…”

A benchmarking study of SARS-CoV-2 whole-genome sequencing protocols using COVID-19 patient samples

“…The protocol P4 is based on single primer isothermal amplification technology (SPIA, NuGEN) coupled with the high-throughput sequencing, which can also generate a full-length SARS-CoV-2 genome. The SPIA has been shown to generate the full-length genomes for HIV, West Nile virus, and bovine coronavirus etc 33,34 . However, for samples with low viral input (< 1k copies), we observed that only 0.003% of the SPIA reads could map to the viral genome, suggesting that P4 might not be ideal if a low copy number of SARS-CoV-2 within sample is expected.…”

“…The protocol P4 is based on single primer isothermal amplification technology (SPIA, NuGEN) coupled with the high-throughput sequencing, which can also generate a full-length SARS-CoV-2 genome. The SPIA has been shown to generate the full-length genomes for HIV, West Nile virus, and bovine coronavirus etc 33,34 .…”

High Throughput Sequencing for the Detection and Characterization of RNA Viruses

“…The common end sequence allows one strand of the double-stranded primer to be used in repeated rounds of annealing, extension and denaturation in the presence of a high-fidelity polymerase. SISPA has been used for the discovery of new viral agents, particularly in the veterinary field (Moser et al, 2016 ; Chrzastek et al, 2017 ; Myrmel et al, 2017 ; Cholleti et al, 2018 ; Zhao et al, 2018a ).…”

“…Once extension of the primer is complete, the RNA is cleaved and digested by RNase H and the entire process is repeated, producing multiple copies of the amplification product ( Figure 2 ). SPIA requires high input amounts of total RNA, which can be challenging when dealing with clinical samples but has been successfully used for detection of bovine coronavirus (Hrdlickova et al, 2017 ; Myrmel et al, 2017 ).…”