Next-generation sequencing of double stranded RNA is dramatically improved by treatment with an inexpensive reagent

Double stranded RNA (dsRNA) serves as the genome of important viruses and is a key component of RNA interference-based immunity in eukaryotes. DMSO has been used to denature dsRNA prior to reverse transcription stage to improve RT-PCR and Sanger sequencing. We systematically tested the utility of DMSO to improve sequencing yield of a dsRNA virus (Φ6) in a short-read next generation sequencing platform. We found that DMSO treatment improved sequencing read recovery by over two orders of magnitude, even when RNA and cDNA concentrations were below the limit of detection. Furthermore, we provide evidence that this treatment does not adversely affect recovery of reads from single-stranded RNA viral genomes (influenza A/California/07/2009). We suggest that 50% DMSO treatment is used prior to cDNA synthesis when samples of interest are or may contain dsRNA.These libraries were created from a premade reagent containing a mixture of dsRNA, ssRNA, and DNA viruses, treated with varying concentrations of DMSO before cDNA synthesis.

双链RNA（double stranded RNA, dsRNA）既是多种重要病毒的基因组，同时也是真核生物中基于RNA干扰的免疫应答的关键组成部分。二甲基亚砜（dimethyl sulfoxide, DMSO）常被用于在逆转录步骤前对dsRNA进行变性处理，以提升逆转录聚合酶链式反应（reverse transcription-polymerase chain reaction, RT-PCR）与桑格测序（Sanger sequencing）的实验效果。
本研究系统评估了DMSO在短读长下一代测序平台中提升双链RNA病毒Φ6测序产出率的效用。
研究结果表明，即便在RNA与互补脱氧核糖核酸（complementary DNA, cDNA）浓度低于检测限的情况下，DMSO处理仍可使测序读段回收率提升两个数量级以上。
此外，本研究还证实，该处理不会对单链RNA病毒基因组（influenza A/California/07/2009）的读段回收率产生不利影响。
我们建议，当待分析样本含有或可能含有dsRNA时，应在cDNA合成前采用50%浓度的DMSO进行处理。
本研究中的测序文库均源自一款预制试剂，该试剂包含dsRNA、单链RNA（single-stranded RNA, ssRNA）与DNA病毒的混合物，并在cDNA合成前使用不同浓度的DMSO完成处理。