Journal article
Comparison of direct cDNA and PCR-cDNA Nanopore sequencing of RNA from Escherichia coli isolates
- Abstract:
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Whole-transcriptome (long-read) RNA sequencing (Oxford Nanopore Technologies, ONT) holds promise for reference-agnostic analysis of differential gene expression 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 clinical Escherichia coli. Four E. coli bloodstream infection-associated isolates (n=2 biological replicates per 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 eight flow cells using 16 barcodes to minimize batch/barcoding bias. Reads were mapped to a transcript reference and transcript abundance was quantified after in silico depletion 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 that 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.
- Publication status:
- Published
- Peer review status:
- Peer reviewed
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- Files:
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(Preview, Version of record, pdf, 1.1MB, Terms of use)
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- Publisher copy:
- 10.1099/mgen.0.001296
Authors
- Funder identifier:
- https://ror.org/0187kwz08
- Grant:
- NIHR200915
- Funder identifier:
- https://ror.org/00k27zs44
- Publisher:
- Microbiology Society
- Journal:
- Microbial Genomics More from this journal
- Volume:
- 10
- Issue:
- 10
- Article number:
- 001296
- Publication date:
- 2024-10-25
- Acceptance date:
- 2024-08-29
- DOI:
- EISSN:
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2057-5858
- Pmid:
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39453698
- Language:
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English
- Keywords:
- Pubs id:
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2044685
- Local pid:
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pubs:2044685
- Deposit date:
-
2025-01-09
Terms of use
- Copyright holder:
- Rodger et al.
- Copyright date:
- 2024
- Rights statement:
- © 2024 The Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.
- Licence:
- CC Attribution (CC BY)
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