Comparison of direct cDNA and PCR-cDNA Nanopore sequencing ofEscherichia coliisolates
Authors: Rodger G , Lipworth S , Barrett L , Oakley S , Crook DW , Eyre DW , Stoesser N 
Published: January 2024

2.AbstractWhole-transcriptome (long-read) RNA sequencing (Oxford Nanopore Technologies, ONT) holds promise for agnostic analysis of differential gene expression (DGE) in pathogenic bacteria, including for antimicrobial resistance genes (ARGs). However, direct cDNA ONT sequencing requires large concentrations of polyadenylated mRNA, and amplification protocols may introduce technical bias. Here we evaluated the impact of direct cDNA and cDNA PCR-based ONT sequencing on transcriptomic analysis of clinicalEscherichia coli. FourE. colibloodstream infection-associated isolates (n=2 biological replicates/isolate) were sequenced using the ONT Direct cDNA Sequencing SQK-DCS109 and PCR-cDNA Barcoding SQK-PCB111.24 kits. Biological and technical replicates were distributed over 8 flow cells using 16 barcodes to minimise batch/barcoding bias. Reads were mapped to a transcript reference and transcript abundance quantified afterin silicodepletion of low abundance and rRNA genes. We found there were strong correlations between read counts using both kits and when restricting the analysis to include only ARGs. We highlighted correlations were weaker for genes with a higher GC content. Read lengths were longer for the direct cDNA kit compared to the PCR-cDNA kit whereas total yield was higher for the PCR-cDNA kit. In this small but methodologically rigorous evaluation of biological and technical replicates of isolates sequenced with the direct cDNA and PCR-cDNA ONT sequencing kits, we demonstrated that PCR-based amplification substantially improves yield with largely unbiased assessment of core gene and ARG expression. However, users of PCR-based kits should be aware of a small risk of technical bias which appears greater for genes with an unusually high (>52%)/low (<44%) GC-content.3.Impact statementRNA sequencing allows quantification of RNA within a biological sample providing information on the expression of genes at a particular time. This helps understand the expression of antimicrobial resistance genes (ARGs). In RNA-Seq experimental workflows extra steps of reverse transcription may be needed to generate more stable cDNA to allow for amplification by PCR if starting RNA input was low. Two current methods of long-read RNA sequencing include direct cDNA and PCR-cDNA based sequencing (Oxford Nanopore Technologies, ONT). However, few studies have compared these two methods of RNA-sequencing using clinical bacterial isolates. We therefore undertook a study to compare both kits using a methodological balanced design of biological and technical replicates ofE. coli. Our study showed that direct cDNA and PCR-cDNA sequencing is highly reproducible between biological and technicalE. colireplicates with very small differences in gene expression signatures generated between kits. The PCR-cDNA kit generates increased sequencing yield but a smaller proportion of mappable reads, the generation of shorter reads of lower quality and some PCR-associated bias. PCR-based amplification greatly increased sequencing yield of core genes and ARGs, however there may be a small risk of PCR-bias in genes that have a higher GC content.4.Data summaryThe transcript reads of the four sequencedEscherichia colistrains have been deposited in the Figshare, DOI: 10.6084/m9.figshare.25044051.The authors confirm all supporting data (available in Figshare), code (available at:https://github.com/samlipworth/rna_methods) and protocols have been provided within the article or through supplementary data files.