Details

Autor(en) / Beteiligte

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

Titel

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

Ist Teil von

• Virology (New York, N.Y.), 2019-02, Vol.528, p.181-197

Ort / Verlag

United States: Elsevier Inc

Erscheinungsjahr

2019

Quelle

MEDLINE

Beschreibungen/Notizen

• 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. •rRNA can make up most of the RNA in metagenomic sequencing from mosquitoes.•Short DNA probes bind rRNA and create DNA/RNA hybrids after reverse transcription.•RNase H and DNase treatment will selectively degrade rRNA and subsequent cDNA.•This method increases NGS reads to viruses, bacteria, and mosquito transcripts.