Selective ablation of 3' RNA ends and processive RTs facilitate direct cDNA sequencing of full-length host cell and viral transcripts

Christian M. Gallardo, Anh Viet T. Nguyen, Andrew L. Routh, Bruce E. Torbett

Research output: Contribution to journalArticlepeer-review

4 Scopus citations

Abstract

Alternative splicing (AS) is necessary for viral proliferation in host cells and a critical regulatory component of viral gene expression. Conventional RNA-seq approaches provide incomplete coverage of AS due to their short read lengths and are susceptible to biases and artifacts introduced in prevailing library preparation methodologies. Moreover, viral splicing studies are often conducted separately from host cell transcriptome analysis, precluding an assessment of the viral manipulation of host splicing machinery. To address current limitations, we developed a quantitative full-length direct cDNA sequencing strategy to simultaneously profile viral and host cell transcripts. This nanopore-based approach couples processive reverse transcriptases with a novel one-step chemical ablation of 3' RNA ends (termed CASPR), which decreases ribosomal RNA reads and enriches polyadenylated coding sequences. We extensively validate our approach using synthetic reference transcripts and show that CASPR doubles the breadth of coverage per transcript and increases detection of long transcripts (>4 kb), while being functionally equivalent to PolyA+ selection for transcript quantification. We used our approach to interrogate host cell and HIV-1 transcript dynamics during viral reactivation and identified novel putative HIV-1 host factors containing exon skipping or novel intron retentions and delineated the HIV-1 transcriptional state associated with these differentially regulated host factors.

Original languageEnglish (US)
Article numbere98
JournalNucleic acids research
Volume50
Issue number17
DOIs
StatePublished - Sep 23 2022

ASJC Scopus subject areas

  • Genetics

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