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. 2019 Nov;5(11):e000315.
doi: 10.1099/mgen.0.000315.

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

Alexander H Wilcox  1 , Eric Delwart  2   3 , Samuel L Díaz-Muñoz  1   4
Affiliations

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

Alexander H Wilcox et al. Microb Genom. 2019 Nov.

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.

Keywords: dsRNA; dsRNAseq; innate immunity; metagenomics; sociovirology; viral sequencing.

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Conflict of interest statement

The authors declare that there are no conflicts of interest.

Figures

Fig. 1.
Fig. 1.
DMSO treatment does not affect sequencing read coverage across the ssRNA influenza genome. The read depth at each position in the influenza genome under varying concentrations of DMSO is shown. Note that 8000 is the maximum read depth supported by the SAM/BAM file format, so some peaks in the 0 % DMSO plots have been truncated.
Fig. 2.
Fig. 2.
DMSO treatment greatly increases sequence coverage of the dsRNA Φ6 genome. Read depth at each position in the Φ6 genome under varying concentrations of DMSO is shown. There were insufficient reads to generate a plot for 0 % DMSO.
Fig. 3.
Fig. 3.
DMSO treatment has a greater effect than RNA concentration on generating dsRNA reads using next-generation sequencing. The percentages of sequencing reads mapping to the Φ6 reference genome are shown with no treatment (Untreated), concentration (Concentrated), nuclease treatment (Nuclease), nuclease treatment and concentration (Concentrated Nuclease), nuclease and DMSO treatments and concentration (Concentrated Nucelase DMSO), and nuclease and DMSO treatments (Nuclease DMSO).
Fig. 4.
Fig. 4.
DMSO treatment does not adversely affect sequencing error rates in influenza (a) or Φ6 (b). The R package ShadowRegression estimates reference-free error rates (inset) based on a transform of the slope of read counts and their ‘shadows’ (main plot line graphs).
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