Reverse transcriptase enzyme and priming strategy affect quantification and diversity of environmental transcripts

Abstract

<jats:title>Summary</jats:title><jats:p>RT-Q-PCR, and RT-PCR amplicon sequencing, provide a convenient, target-specific, high-sensitivity approach for gene expression studies and are widely used in environmental microbiology. Yet, the effectiveness and reproducibility of the reverse transcription step has not been evaluated. Therefore, we tested a combination of four commercial reverse transcriptases with two priming techniques to faithfully transcribe <jats:italic>16S rRNA</jats:italic> and <jats:italic>amoA</jats:italic> transcripts from marine sediments. Both enzyme and priming strategy greatly affected quantification of the exact same target with differences of up to 600-fold. Furthermore, the choice of RT system significantly changed the communities recovered. For <jats:italic>16S rRNA</jats:italic>, both enzyme and priming had a significant effect with enzyme having a stronger impact than priming. Inversely, for <jats:italic>amoA</jats:italic> only the change in priming strategy resulted in significant differences between the same sample. Specifically, more OTUs and better coverage of <jats:italic>amoA</jats:italic> transcripts diversity were obtained with GS priming indicating this approach was better at recovering the diversity of <jats:italic>amoA</jats:italic> transcripts. Moreover, sequencing of RNA mock communities revealed that, even though transcript α diversities (<jats:italic>i.e.</jats:italic> OTU counts within a sample) can be biased by the RT, the comparison of β diversities (<jats:italic>i.e.</jats:italic> differences in OTU counts between samples) is reliable as those biases are reproducible between environments.</jats:p><jats:sec><jats:title>Originality-Significance Statement</jats:title><jats:p>Is the complementary DNA (cDNA) produced after Reverse Transcription (RT) a faithful representation of the starting RNA? This is a fundamental and important question for transcriptomic-based studies in environmental microbiology that aim to quantify and/or examine the diversity of transcripts via RT approaches. Yet little is known about the reliability and reproducibility of this step. Here, we evaluated the effect of the two main components of the RT reaction – the retro transcriptase enzyme and priming strategy (gene specific vs random priming), on the quantification and diversity of cDNA. We found that both have a significant impact. We further provide evidence to enable informed choices as to the enzyme and priming combinations to improve the performance of RT-PCR approaches. Taken together, this work will improve the reliability and reproducibility of transcript-based studies in environmental microbiology.</jats:p></jats:sec>