A universal, single primer amplification protocol (R-SPA) to perform whole genome sequencing of segmented dsRNA reoviruses

Abstract

BackgroundThe Reoviridae family represents the largest family of double-stranded RNA (dsRNA) viruses, and the members have been isolated from a wide range of mammals, birds, reptiles, fishes, insects, plants. Orthoreoviruses, one of the 15 recognized genera in the Reoviridae family, can infect humans and nearly all mammals, and birds. Genomic characterization of reoviruses has not been adopted on a large-scale due to the complexity of obtaining sequences for all 10 segments.ResultsIn this study, we developed a time-efficient, and practical method to enrich reovirus sequencing reads from isolates that allowed for full genome recovery using single-primer amplification method coupled with next generation sequencing. We refer to this protocol as reovirus-Single Primer Amplification (R-SPA). Our results demonstrated that most of the genes were covered with at least 500 reads per base space. Furthermore, R-SPA covered both 5’ and 3’ end of each reovirus genes.ConclusionA universal and fast amplification protocol that yields double-stranded cDNA in sufficient abundance and facilitates and expedites the whole genome sequencing of reoviruses was presented in this study.

Optimizing the Conditions for Whole-Genome Sequencing of Avian Reoviruses

“…For this reason, only the strategies that included the R-SPA step were investigated in our subsequent experiments. The results obtained by applying only R-SPA to the samples (Figure 4, average of 14% ARV reads in a sample) were consistent with the results reported by Chrzastek et al [13]. However, we observed that R-SPA seemed to work better for the vaccine strain S1133 (~45% ARV reads) than it did for the clinical isolates (<11% ARV reads).…”

“…The amplification of the ARV genetic content in a sample can be conducted via PCR amplification of the viral genome before NGS [ 24 , 25 ] or via target capture NGS [ 26 ]. While we are unaware of an available target capture NGS scheme with ARV-specific probes, early this year, Chrzastek and collaborators reported a single primer amplification assay (R-SPA) that significantly improved the recovery of ARV mapping reads after sequencing [ 13 ]. This approach was evaluated in the experiments documented herein, alone and in combination with the other ARV purification methods mentioned above.…”

“…We first hypothesized that S1133 (vaccine strain and reference genome) was most probably among the group of strains used to design the R-SPA oligos, and therefore, we observed a better performance with this isolate. To test this hypothesis, we compared the sequence homology of R-SPA primer R8N [ 13 ] with the ARV whole genomes obtained in this study, and we observed that the 5′ end of the ARV fragments (where R8N aligns) is very conserved, and therefore, it is very unlikely that the observed differences in the R-SPA performance were caused by different primer affinities between the ARV strains. Another potential explanation resides in the fact that although all the isolates were expanded in LMH cells for the same period of time, S1133 is cell-culture-adapted, so it could be disproportionately expanded compared to the other clinical isolates, yielding a higher viral titer, and therefore, more viral genomes for PCR amplification to act upon.…”

“…The single primer amplification protocol (R-SPA) described by Chrzastek et al [ 13 ] was used to produce ARV cDNA for whole-genome sequencing. Briefly, SuperScript IV reverse transcriptase (ThermoFisher Scientific, Waltham, MA, USA, catalog number 18090010) was used to convert ARV RNA into cDNA.…”

“…Generally researchers opt for sequencing a small number of ARV isolates per sequencing run, hoping to obtain enough viral reads for good coverage [ 12 ]. Egaña-Labrin and collaborators introduced a host rRNA depletion step with a Terminator TM 5′-Phosphate-Dependent Exonuclease degradation step (the degradation of RNAs without a 5′ triphosphate cap) before converting the ARV RNA genome into cDNA [ 2 ], while Chrzastek et al [ 13 ] used a reovirus single primer amplification (R-SPA) approach to enrich ARV cDNA. There are no reports showing if these intermediate steps significantly reduced host contamination and/or enriched for ARV-mapping reads after sequencing.…”