A general method to eliminate laboratory induced recombinants during massive, parallel sequencing of cDNA library.

Caryll Waugh,Miles Davenport,Andrew Grimm,Deborah Cromer,Johnson Mak,Simon Mallal,Abha Chopra

doi:10.1186/s12985-015-0280-x

Abstract

BackgroundMassive, parallel sequencing is a potent tool for dissecting the regulation of biological processes by revealing the dynamics of the cellular RNA profile under different conditions. Similarly, massive, parallel sequencing can be used to reveal the complexity of viral quasispecies that are often found in the RNA virus infected host. However, the production of cDNA libraries for next-generation sequencing (NGS) necessitates the reverse transcription of RNA into cDNA and the amplification of the cDNA template using PCR, which may introduce artefact in the form of phantom nucleic acids species that can bias the composition and interpretation of original RNA profiles.MethodUsing HIV as a model we have characterised the major sources of error during the conversion of viral RNA to cDNA, namely excess RNA template and the RNaseH activity of the polymerase enzyme, reverse transcriptase. In addition we have analysed the effect of PCR cycle on detection of recombinants and assessed the contribution of transfection of highly similar plasmid DNA to the formation of recombinant species during the production of our control viruses.ResultsWe have identified RNA template concentrations, RNaseH activity of reverse transcriptase, and PCR conditions as key parameters that must be carefully optimised to minimise chimeric artefacts.ConclusionsUsing our optimised RT-PCR conditions, in combination with our modified PCR amplification procedure, we have developed a reliable technique for accurate determination of RNA species using NGS technology.

Highlights

Massive, parallel sequencing is a potent tool for dissecting the regulation of biological processes by revealing the dynamics of the cellular RNA profile under different conditions
Using our optimised reverse transcription PCR (RT-PCR) conditions, in combination with our modified PCR amplification procedure, we have developed a reliable technique for accurate determination of RNA species using next-generation sequencing (NGS) technology
We have shown that homologous recombination during transfection, sometimes perceived as a source of experimental bias during virus production [8,9,10,11], is a negligible source of error and the effect of laboratory manipulations on estimates of mutation rate can be readily managed with appropriate rigorous experimental procedure

Summary

Introduction

Parallel sequencing is a potent tool for dissecting the regulation of biological processes by revealing the dynamics of the cellular RNA profile under different conditions. Massive, parallel sequencing can be used to reveal the complexity of viral quasispecies that are often found in the RNA virus infected host. The massive capacity of generation sequencing (NGS) technologies is revolutionising transcriptome analysis and can be used to assess the complexity of viral diversity in highly mutable, genomes of RNA viruses that cause disease in humans, such as human immunodeficiency virus-1 (HIV-1). Several papers have addressed the issue of minimising PCR artefacts [1,2,3] and there is growing interest in optimising RT-PCR techniques to enable accurate analysis of patient RNA virus load and early detection of the emergence of drug resistant quasi-species [3,4,5,6]

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