Next-generation sequencing of dsRNA is greatly improved by treatment with the inexpensive denaturing reagent DMSO

Abstract

dsRNA is the genetic material of important viruses and a key component of RNA interference-based immunity in eukaryotes. Previous studies have noted difficulties in determining the sequence of dsRNA molecules that have affected studies of immune function and estimates of viral diversity in nature. DMSO has been used to denature dsRNA prior to the reverse-transcription stage to improve reverse transcriptase PCR and Sanger sequencing. We systematically tested the utility of DMSO to improve the sequencing yield of a dsRNA virus (Φ6) in a short-read next-generation sequencing platform. DMSO treatment improved sequencing read recovery by over two orders of magnitude, even when RNA and cDNA concentrations were below the limit of detection. We also tested the effects of DMSO on a mock eukaryotic viral community and found that dsRNA virus reads increased with DMSO treatment. Furthermore, we provide evidence that DMSO treatment does not adversely affect recovery of reads from a ssRNA viral genome (influenza A/California/07/2009). We suggest that up to 50 % DMSO treatment be used prior to cDNA synthesis when samples of interest are composed of or may contain dsRNA.

Unveiling Viruses Associated with Gastroenteritis Using a Metagenomics Approach

“…Immunochromatographic tests for RVA diagnosis have been a useful tool to provide quick and cost-effective tests for routine clinical analyses at the expense of low sensitivity when compared to molecular methods such as RT-PCR [ 75 , 76 ]. The low number of reads obtained within the pools might be explained by three important facts: (a) the samples could have low levels of virus, since all were negative in the commonly used test; (b) individual patient samples were pooled, diluting the number of viral reads present in positive patients; (c) dsRNA viruses have been shown to be difficult to detect using current NGS techniques [ 77 ].…”

“…Similarly to HAdV, the small number of obtained reads could be explained by an inefficient tagging of double-stranded genomes during the retro-transcription or sequenase steps resulting in low coverage and the small number of reads assigned to this viral family. Specific protocols to increase the sensitivity towards double-stranded genomes (dsDNA and dsRNA) might need to be applied to explore the viral diversity within these genomic conformations [ 77 ].…”

Deep Impact of Random Amplification and Library Construction Methods on Viral Metagenomics Results

“…Therefore, the improved detection of the dsRNA virus is due to the higher temperature used in the denaturation step before reverse-transcription. The use of the denaturing reagent DMSO prior to cDNA synthesis is also known to increase the dsRNA virus reads [ 37 ].…”

Absolute quantitation of individual SARS-CoV-2 RNA molecules: a new paradigm for infection dynamics and variant differences

“…However, their detection by RT-qPCR or sequencing is hampered by cDNA library protocols that employ oligo(dT) selection and by primer binding to double stranded RNA (dsRNA) structures (Ramanan et al, 2016;Sethna et al, 1991). To detect negative sense viral RNAs, we denatured dsRNA complexes through either formamide, DMSO or sodium hydroxide treatment (Singer et al, 2021;Wilcox et al, 2019). The combination of DMSO with heat treatment resulted in a loss of anti-dsRNA J2 signal, while maintaining cell integrity, suggesting a disruption of dsRNA hybrids (Figure S2A).…”