Viral Metagenomics utilising SMART-9N Amplification v1

Following on from the original SMART-9N (Switch Mechanism at the 5′ End of RNA Templates) protocol utilised for viral metagenomics, this protocol offers an optimised approach to improve genome representation in diverse microbial communities and also incorporate DNA viruses. This protocol is also adapted from the "Rapid metagenomic sequencing for surveillance of bacterial, fungal and viral pathogens using SQK-RPB114.24" protocol published by Oxford Nanopore Technologies (https://nanoporetech.com/document/rapid-sequencing-metagenomics-sqk-rpb114-2). The protocol utilises a shotgun approach using random 9N priming to reverse transcribe RNA and PCR-amplify RNA present. Furthermore, as the random priming is independent of the poly(A) tail of RNA, DNA is also annealed and amplified (similarly to as described in https://doi.org/10.1016/j.meegid.2015.03.018). Therefore, it is ideal for viral metagenomics to enrich for the viruses present in the sample often obscured by high host backgrounds. Optimisation Implemented: Host depletion has been moved before the total nucleic acid extraction, removing extracellular DNA present in the sample and allowing DNA viruses to be processed through the extraction. Total nucleic acid extraction utilising column purification has been replaced by a magnetic bead extraction. Primer concentrations have been adjusted to reflect the conditions providing the greatest yield and genome coverage. An unmodified PCR primer is utilised in place of the ONT RLB barcoding primers to ensure sufficient amplification for library preparation that the barcoding primers do not provide. We have found that utilising the barcoding primers result in 10x less yield post-PCR in comparison to the unmodified primer.