Error baseline rates of five sample preparation methods used to characterize RNA virus populations.
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, sequence-independent 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.
Center for Genome Sciences, United States Army Medical Research Institute of Infectious Diseases (USAMRIID), Fort Detrick, Frederick, Maryland, United States of America.
Jeffrey R
JR
Kugelman
Center for Genome Sciences, United States Army Medical Research Institute of Infectious Diseases (USAMRIID), Fort Detrick, Frederick, Maryland, United States of America.
Michael R
MR
Wiley
Center for Genome Sciences, United States Army Medical Research Institute of Infectious Diseases (USAMRIID), Fort Detrick, Frederick, Maryland, United States of America.
Elyse R
ER
Nagle
Center for Genome Sciences, United States Army Medical Research Institute of Infectious Diseases (USAMRIID), Fort Detrick, Frederick, Maryland, United States of America.
Daniel
D
Reyes
Center for Genome Sciences, United States Army Medical Research Institute of Infectious Diseases (USAMRIID), Fort Detrick, Frederick, Maryland, United States of America.
Brad P
BP
Pfeffer
Integrated Research Facility at Fort Detrick (IRF-Frederick), National Institute of Allergy and Infectious Diseases, National Institutes of Health, Fort Detrick, Frederick, Maryland, United States of America.
Jens H
JH
Kuhn
Center for Genome Sciences, United States Army Medical Research Institute of Infectious Diseases (USAMRIID), Fort Detrick, Frederick, Maryland, United States of America.
Mariano
M
Sanchez-Lockhart
Center for Genome Sciences, United States Army Medical Research Institute of Infectious Diseases (USAMRIID), Fort Detrick, Frederick, Maryland, United States of America.
Gustavo F
GF
Palacios
D004252:DNA Mutational Analysis / Q000379:methods / Q000592:standards; D003951:Diagnostic Errors; D016679:Genome, Viral; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods / Q000592:standards; D006801:Humans; D020641:Polymorphism, Single Nucleotide; D012328:RNA Viruses / Q000235:genetics*; D012367:RNA, Viral / Q000032:analysis* / Q000235:genetics; D012107:Research Design; D017423:Sequence Analysis, RNA / Q000379:methods / Q000592:standards; D013048:Specimen Handling / Q000379:methods* / Q000592:standards
D012367:RNA, Viral