1. Overview of the proposed standard operating procedure (SOP) for rapid next-generation sequencing library preparation and inactivation of ssRNA+ viruses.
Overview of the proposed standard operating procedure (SOP) for rapid next-generation sequencing library preparation and inactivation of ssRNA+ viruses. (A) Stepwise overview of the SOP. A detailed protocol is provided in Text S1 in the supplemental material. Steps in the pink box denote work performed in a biosafety level 3 and/or 4 (BSL-3/4) laboratory. Steps in the blue box denote work that can be performed in a BSL-2 laboratory. The asterisk in step 1 indicates that for nonselect agent pathogens (e.g., West Nile virus), extracted RNA may be moved to BSL-2 for library construction. Step 1, generating cDNA and SISPA, utilizes a primer with a random hexamer coupled to a unique barcode (BC-N6). SISPA stands for sequence-independent single-primer amplification. (B) The BC-N6 primer is used for both generating single-stranded cDNA from input RNA and generating double-stranded DNA by randomly priming the synthesized cDNA. A PCR step using primers only encoding the barcode sequence with either three or four random nucleotides (3N/4N) at the 5′ end simultaneously amplifies and uniquely identifies (barcodes) a sample. (C) Representative gel image that displays products of the SOP obtained from serial dilutions of genomic human rhinovirus 16 (HRV-16) virion RNA. At high-input RNA amounts, a smear between 200 bp and 1,000 bp is visible. This signal intensity diminishes as the starting material is diluted. (D) Summary of diverse types of starting material which can feed into the SOP. Samples enriched for virus-specific sequence (e.g., virion stocks) can directly proceed to the SOP. For samples that contain a majority of host nucleic acid, the use of upstream procedures to enrich for virus-specific signal (e.g., rRNA depletion or mRNA enhancement) is recommended.
Lindsey A. Moser et al. mSystems 2016; doi: /mSystems