A viral metagenomic protocol for nanopore sequencing of group A rotavirus.

Aim: Development of an unbiased methodology using Oxford Nanopore Technology (ONT) sequencing to obtain whole-genome sequences (WGS) of Rotavirus A (RVA) from clinical samples.

Methods: 157 RVA qRT-PCR positive faecal samples were enriched by virus-like particle (VLP) purification and host nuclease digestion to enhance the detection of viral nucleic acids and cDNA generated as per the NetoVIR protocol. ONT sequencing was then performed using the ONT Native Barcoding kit (SQK-LSK-109) on the GridION platform. Data was basecalled, demultiplexed and assembled into near complete RVA genomes. The accuracy and quality of the obtained sequences was assessed by comparing to Sanger sequencing and RVA reference genomes.

Results: The developed protocol generated 146 near-complete RVA WGS out of the 157 RVA-positive clinical samples. The quality of the assembled genomes was assessed by comparison against publicly-available sequences with results showing 98.76 % ± 0.03 % similarity and > 90 % genome coverage. A concordance assessment was performed comparing the identity of partial RVA VP7 and VP4 segments obtained by Sanger sequencing (n = 51) against corresponding nanopore sequences which demonstrated an overall identity of 100.0 % ± 0.02 %.

Conclusions: The nanopore protocol generated both high quality and accurate RVA WGS extracted from faecal samples. This protocol can be extended to other viral agents in other sample types.