A reverse-transcription/RNase H based protocol for depletion of mosquito ribosomal RNA facilitates viral intrahost evolution analysis, transcriptomics and pathogen discovery.

Abstract

Studies aimed at identifying novel viral sequences or assessing intrahost viral variation require sufficient sequencing coverage to assemble contigs and make accurate variant calling at low frequencies. Many samples come from host tissues where ribosomal RNA represents more than 90% of total RNA preparations, making unbiased sequencing of viral samples inefficient and highly expensive, as many reads will be wasted on cellular RNAs. In the presence of this amount of ribosomal RNA, it is difficult to achieve sufficient sequencing depth to perform analyses such as variant calling, haplotype prediction, virus population analyses, virus discovery or transcriptomic profiling. Many methods for depleting unwanted RNA or enriching RNA of interest have been devised, including poly-A selection, RNase H based specific depletion, duplex-specific nuclease treatment and hybrid capture selection, among others. Although these methods can be efficient, they either cannot be used for some viruses (i.e. non-polyadenylated viruses), have been optimized for use in a single species, or have the potential to introduce bias. In this study, we describe a novel approach that uses an RNaseH possessing reverse transcriptase coupled with selective probes for ribosomal RNA designed to work broadly for three medically relevant mosquito genera; Aedes, Anopheles, and Culex. We demonstrate significant depletion of rRNA using multiple assessment techniques from a variety of sample types, including whole mosquitoes and mosquito midgut contents from FTA cards. To demonstrate the utility of our approach, we describe novel insect-specific virus genomes from numerous species of field collected mosquitoes that underwent rRNA depletion, thereby facilitating their detection. The protocol is straightforward, relatively low-cost and requires only common laboratory reagents and the design of several small oligonucleotides specific to the species of interest. This approach can be adapted for use with other organisms with relative ease, thus potentially aiding virus population genetics analyses, virus discovery and transcriptomic profiling in both laboratory and field samples.