Error baseline rates of five sample preparation methods used to characterize RNA virus populations

Jeffrey R. Kugelman, Michael R Wiley, Elyse R. Nagle, Daniel Reyes, Brad P. Pfeffer, Jens H. Kuhn, Mariano Sanchez-Lockhart, Gustavo F. Palacios

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

Individual RNA viruses typically occur as populations of genomes that differ slightly from each other due to mutations introduced by the error-prone viral polymerase. Understanding the variability of RNA virus genome populations is critical for understanding virus evolution because individual mutant genomes may gain evolutionary selective advantages and give rise to dominant subpopulations, possibly even leading to the emergence of viruses resistant to medical countermeasures. Reverse transcription of virus genome populations followed by next-generation sequencing is the only available method to characterize variation for RNA viruses. However, both steps may lead to the introduction of artificial mutations, thereby skewing the data. To better understand how such errors are introduced during sample preparation, we determined and compared error baseline rates of five different sample preparation methods by analyzing in vitro transcribed Ebola virus RNA from an artificial plasmid- based system. These methods included: shotgun sequencing from plasmid DNA or in vitro transcribed RNA as a basic "no amplification" method, amplicon sequencing from the plasmid DNA or in vitro transcribed RNA as a "targeted" amplification method, sequenceindependent single-primer amplification (SISPA) as a "random" amplification method, rolling circle reverse transcription sequencing (CirSeq) as an advanced "no amplification" method, and Illumina TruSeq RNA Access as a "targeted" enrichment method. The measured error frequencies indicate that RNA Access offers the best tradeoff between sensitivity and sample preparation error (1.4-5) of all compared methods.

Original languageEnglish (US)
Article numbere0171333
JournalPLoS One
Volume12
Issue number2
DOIs
StatePublished - Feb 1 2017
Externally publishedYes

Fingerprint

RNA Viruses
Viruses
RNA
Amplification
Population
Genes
sampling
Genome
Plasmids
plasmids
reverse transcription
Transcription
genome
methodology
DNA Sequence Analysis
viruses
Reverse Transcription
Ebolavirus
mutation
RNA viruses

ASJC Scopus subject areas

  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

Cite this

Kugelman, J. R., Wiley, M. R., Nagle, E. R., Reyes, D., Pfeffer, B. P., Kuhn, J. H., ... Palacios, G. F. (2017). Error baseline rates of five sample preparation methods used to characterize RNA virus populations. PLoS One, 12(2), [e0171333]. https://doi.org/10.1371/journal.pone.0171333

Error baseline rates of five sample preparation methods used to characterize RNA virus populations. / Kugelman, Jeffrey R.; Wiley, Michael R; Nagle, Elyse R.; Reyes, Daniel; Pfeffer, Brad P.; Kuhn, Jens H.; Sanchez-Lockhart, Mariano; Palacios, Gustavo F.

In: PLoS One, Vol. 12, No. 2, e0171333, 01.02.2017.

Research output: Contribution to journalArticle

Kugelman, Jeffrey R. ; Wiley, Michael R ; Nagle, Elyse R. ; Reyes, Daniel ; Pfeffer, Brad P. ; Kuhn, Jens H. ; Sanchez-Lockhart, Mariano ; Palacios, Gustavo F. / Error baseline rates of five sample preparation methods used to characterize RNA virus populations. In: PLoS One. 2017 ; Vol. 12, No. 2.
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