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

Alexander H Wilcox,Samuel L Díaz-Muñoz,Eric Delwart

doi:10.1099/mgen.0.000315

Alexander H Wilcox, Samuel L Díaz-Muñoz + Show 1 more

Open Access

https://doi.org/10.1099/mgen.0.000315

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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.

Highlights

RNA is a ubiquitous biological molecule involved in transcription and translation, which serves as the genetic material of a large number of important viruses
This work investigated four questions regarding the effect of DMSO treatment on next-generation sequencing. (i) Does DMSO treatment improve recovery of double- stranded form of RNA (dsRNA) reads and at what concentration? (ii) Does DMSO affect read coverage and accuracy of a viral genome? (iii) Is the effect of DMSO independent of RNA concentration? (iv) Does DMSO treatment negatively affect the recovery of ssRNA genomes? Our results suggest that treatment with a high concentration of DMSO greatly increases the number of reads generated when sequencing dsRNA with no effect on read accuracy, without adversely affecting sequencing of ssRNA virus genomes on the Illumina short-read sequencing platform
For dsRNA, the raw quantity of starting material may not be as important as the efficiency of cDNA synthesis, a fact that should accounted for when preparing quality-c ontrol thresholds before next-generation sequencing

Summary

Introduction

RNA is a ubiquitous biological molecule involved in transcription and translation, which serves as the genetic material of a large number of important viruses. The double- stranded form of RNA (dsRNA) is believed to be less abundant in nature, but is a crucial component of a number of biological systems It has a central role in the RNA interference system [1], which modulates innate immunity in plants and animals, and serves as a replicative intermediate of (+) ssRNA viruses, while being present in dsDNA and (−) ssRNA infections [2]. It serves as the genetic material of a number of virus lineages DsRNA virus lineages may be under-r epresented and dsRNAs involved in immunity may be underestimated

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