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

Abstract

22Studies aimed at identifying novel viral sequences or assessing intrahost viral variation require sufficient 23 sequencing coverage to assemble contigs and make accurate variant calling at low frequencies. Many 24 samples come from host tissues where ribosomal RNA represents more than 90% of total RNA 25 preparations, making unbiased sequencing of viral samples inefficient and highly expensive, as many 26 reads will be wasted on cellular RNAs. In the presence of this amount of ribosomal RNA, it is difficult to 27 achieve sufficient sequencing depth to perform analyses such as variant calling, haplotype prediction, 28 virus population analyses, virus discovery or transcriptomic profiling. Many methods for depleting 29 unwanted RNA or enriching RNA of interest have been devised, including poly-A selection, RNase H 30 based specific depletion, duplex-specific nuclease treatment and hybrid capture selection, among 31 others. Although these methods can be efficient, they either cannot be used for some viruses (i.e. non-32 polyadenylated viruses), have been optimized for use in a single species, or have the potential to 33 introduce bias. In this study, we describe a novel approach that uses an RNaseH possessing reverse 34 transcriptase coupled with selective probes for ribosomal RNA designed to work broadly for three 35 medically relevant mosquito genera; Aedes, Anopheles, and Culex. We demonstrate significant depletion 36 of rRNA using multiple assessment techniques from a variety of sample types, including whole 37 mosquitoes and mosquito midgut contents from FTA cards. To demonstrate the utility of our approach, 38 we describe novel insect-specific virus genomes from numerous species of field collected mosquitoes 39 that underwent rRNA depletion, thereby facilitating their detection. The protocol is straightforward, 40 relatively low-cost and requires only common laboratory reagents and the design of several small 41 oligonucleotides specific to the species of interest. This approach can be adapted for use with other 42 organisms with relative ease, thus potentially aiding virus population genetics analyses, virus discovery 43 and transcriptomic profiling in both laboratory and field samples. 44 RNA:cDNA hybrid. The samples were then digested with DNase I (NEB) to remove the cDNA and residual 108 oligos. The RNA was then purified using RNAClean XP beads at a 1.8x ratio. 109

Sensitivity and specificity of metatranscriptomics as an arbovirus surveillance tool

“…An alternate way to increase the number of viral sequences is by depleting the mosquito RNA, generally by targeting highly abundant ribosomal RNA (rRNA). A variety of rRNA depletion kits are available, however, these are not specific to mosquitoes and so custom probes based on mosquito rRNA sequences need to be generated 20,21 .…”

Non-target RNA depletion strategy to improve sensitivity of next-generation sequencing for the detection of RNA viruses in poultry

“…This level of host reduction is comparable to those observed in other host systems using similar methods with fold enrichment from 5-10× following removal of host and carrier RNA via RNase H-based protocols. 9,11,12,27 Sensitivity to virus was dramatically improved using the custom probe sets compared to untreated controls and both commercial kits. This was true for detection of NDV, which predominated in OPs and CLs from Tanzania, infectious bronchitis virus (Avian coronavirus), and picornaviruses, which were abundant in choanal or lung tissues, and infectious bursal disease virus, which was present in some bursal tissues (Suppl.…”

“…This level of host reduction is comparable to those observed in other host systems using similar methods with fold enrichment from 5–10× following removal of host and carrier RNA via RNase H–based protocols. 9,11,12,27…”

“…A second and perhaps more promising approach to increase sensitivity of NGS for pathogen detection is to deplete abundant, non-target sequences. 9,27 For many sample types, a large portion of the bulk RNA or DNA extract is dominated by a few host sequences or non-target bacterial reads. To enrich extracts, biotinylated baits can be used to pull out non-target sequences, or standard oligonucleotide baits can be added to generate DNA-RNA hybrids that are selectively degraded enzymatically.…”

Decoding rRNA sequences for improved metagenomics of sylvatic mosquito species

“…Due to their length and their abundance, they are a sink for precious next generation sequencing reads, decreasing the sensitivity of pathogen detection unless depleted during library preparation. Yet the most common rRNA depletion protocols require prior knowledge of rRNA sequences of the species of interest as they involve hybridizing antisense oligos to the rRNA molecules prior to removal by ribonucleases (Fauver et al, 2019; Phelps et al, 2021) or by bead capture (Kukutla et al, 2013). Presently, reference sequences for rRNAs are limited to only a handful of species from three genera: Aedes, Culex , and Anopheles (Ruzzante et al, 2019).…”