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

Joseph R. Fauver, Shamima Akter, Aldo Ivan Ortega Morales, William C. Black, Americo D. Rodriguez, Mark D. Stenglein, Gregory D. Ebel, James Weger-Lucarelli

Research output: Contribution to journalArticlepeer-review

17 Scopus citations

Abstract

Identifying novel viruses or assessing viral variation by NGS requires high sequencing coverage. More than 90% of total RNA is ribosomal (rRNA), making variant calling, virus discovery or transcriptomic profiling difficult. Current methods to increase informative reads suffer from drawbacks, either they cannot be used for some viruses, are optimized for a single species, or introduce bias. We describe a two-part approach combining reverse-transcription to create RNA/DNA hybrids which are then degraded with RNaseH/DNase sequentially that works for three medically relevant mosquito genera; Aedes, Anopheles, and Culex. We demonstrate depletion of rRNA from different samples, including whole mosquitoes and midgut contents from FTA cards. We describe novel insect-specific virus genomes from field collected mosquitoes. The protocol requires only common laboratory reagents and small oligonucleotides specific to rRNA. This approach can be adapted for other organisms, aiding virus diversity analyses, virus discovery and transcriptomics in both laboratory and field samples.

Original languageEnglish (US)
Pages (from-to)181-197
Number of pages17
JournalVirology
Volume528
DOIs
StatePublished - Feb 2019
Externally publishedYes

Keywords

  • Arbovirus
  • Insect-specific viruses
  • Mosquito-borne viruses
  • Next-generation sequencing
  • Ribosomal depletion
  • Transcriptomics
  • Virus discovery

ASJC Scopus subject areas

  • Virology

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