Skip to main content
bioRxiv
  • Home
  • About
  • Submit
  • ALERTS / RSS
Advanced Search
New Results

Selective Ablation of 3’ RNA ends and Processive RTs Facilitate Direct cDNA Sequencing of Full-length Host Cell and Viral Transcripts

View ORCID ProfileChristian M. Gallardo, Anh-Viet T. Nguyen, View ORCID ProfileAndrew L. Routh, View ORCID ProfileBruce E. Torbett
doi: https://doi.org/10.1101/2022.01.27.478099
Christian M. Gallardo
1Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA, USA
2Center for Immunity and Immunotherapies, Seattle Children’s Research Institute, Seattle WA, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Christian M. Gallardo
Anh-Viet T. Nguyen
1Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Andrew L. Routh
3Department of Biochemistry and Molecular Biology, Sealy Center for Structural Biology, University of Texas Medical Branch, Galveston, TX, USA
4Sealy Center for Structural Biology, University of Texas Medical Branch, Galveston, TX, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Andrew L. Routh
Bruce E. Torbett
1Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA, USA
2Center for Immunity and Immunotherapies, Seattle Children’s Research Institute, Seattle WA, USA
5Institute for Stem Cell & Regenerative Medicine, University of Washington, Seattle, WA, USA
6Department of Laboratory Medicine and Pathology, University of Washington School of Medicine, Seattle, WA, USA
7Department of Pediatrics, University of Washington School of Medicine, Seattle, WA, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Bruce E. Torbett
  • For correspondence: bertorbet@uw.edu
  • Abstract
  • Full Text
  • Info/History
  • Metrics
  • Preview PDF
Loading

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 for poly-adenylated coding sequences. We extensively validate our approach using synthetic reference transcripts and show CASPR doubles the breadth of coverage per transcript and increases detection of long transcripts (>4kb), 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.

Competing Interest Statement

CMG and BET are listed as inventors on a provisional patent filed by Seattle Children's Research Institute related to the CASPR methodology.

Footnotes

  • - Added cross-validation to existing Illumina-sequenced dataset using same J-Lat 10.6 clone and TNF-alpha induction conditions. - Added qPCR validation of a subset of DGE hits - Added new figure to illustrate assay and bioninformatic components of sequencing pipeline

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
Back to top
PreviousNext
Posted May 04, 2022.
Download PDF
Email

Thank you for your interest in spreading the word about bioRxiv.

NOTE: Your email address is requested solely to identify you as the sender of this article.

Enter multiple addresses on separate lines or separate them with commas.
Selective Ablation of 3’ RNA ends and Processive RTs Facilitate Direct cDNA Sequencing of Full-length Host Cell and Viral Transcripts
(Your Name) has forwarded a page to you from bioRxiv
(Your Name) thought you would like to see this page from the bioRxiv website.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
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
bioRxiv 2022.01.27.478099; doi: https://doi.org/10.1101/2022.01.27.478099
Digg logo Reddit logo Twitter logo Facebook logo Google logo LinkedIn logo Mendeley logo
Citation Tools
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
bioRxiv 2022.01.27.478099; doi: https://doi.org/10.1101/2022.01.27.478099

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
  • Tweet Widget
  • Facebook Like
  • Google Plus One

Subject Area

  • Genomics
Subject Areas
All Articles
  • Animal Behavior and Cognition (3575)
  • Biochemistry (7520)
  • Bioengineering (5479)
  • Bioinformatics (20677)
  • Biophysics (10258)
  • Cancer Biology (7931)
  • Cell Biology (11583)
  • Clinical Trials (138)
  • Developmental Biology (6563)
  • Ecology (10136)
  • Epidemiology (2065)
  • Evolutionary Biology (13540)
  • Genetics (9498)
  • Genomics (12788)
  • Immunology (7872)
  • Microbiology (19451)
  • Molecular Biology (7614)
  • Neuroscience (41875)
  • Paleontology (306)
  • Pathology (1252)
  • Pharmacology and Toxicology (2179)
  • Physiology (3249)
  • Plant Biology (7007)
  • Scientific Communication and Education (1291)
  • Synthetic Biology (1942)
  • Systems Biology (5406)
  • Zoology (1107)