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The long and the short of it: unlocking nanopore long-read RNA sequencing data with short-read differential expression analysis tools

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journal contribution
posted on 28.07.2021, 03:44 by Xueyi Dong, Luyi Tian, Quentin GouilQuentin Gouil, Hasaru Kariyawasam, Shian Su, Ricardo De Paoli-Iseppi, Yair David Joseph Prawer, Michael B Clark, Kelsey Breslin, Megan Iminitoff, Marnie E Blewitt, Charity W Law, Matthew E Ritchie
Abstract Application of Oxford Nanopore Technologies’ long-read sequencing platform to transcriptomic analysis is increasing in popularity. However, such analysis can be challenging due to the high sequence error and small library sizes, which decreases quantification accuracy and reduces power for statistical testing. Here, we report the analysis of two nanopore RNA-seq datasets with the goal of obtaining gene- and isoform-level differential expression information. A dataset of synthetic, spliced, spike-in RNAs (‘sequins’) as well as a mouse neural stem cell dataset from samples with a null mutation of the epigenetic regulator Smchd1 was analysed using a mix of long-read specific tools for preprocessing together with established short-read RNA-seq methods for downstream analysis. We used limma-voom to perform differential gene expression analysis, and the novel FLAMES pipeline to perform isoform identification and quantification, followed by DRIMSeq and limma-diffSplice (with stageR) to perform differential transcript usage analysis. We compared results from the sequins dataset to the ground truth, and results of the mouse dataset to a previous short-read study on equivalent samples. Overall, our work shows that transcriptomic analysis of long-read nanopore data using long-read specific preprocessing methods together with short-read differential expression methods and software that are already in wide use can yield meaningful results.

History

Publication Date

09/04/2021

Journal

NAR Genomics and Bioinformatics

Volume

3

Issue

2

Pagination

(p. lqab028)

Publisher

Oxford University Press (OUP)

ISSN

2631-9268

Rights Statement

The Author reserves all moral rights over the deposited text and must be credited if any re-use occurs. Documents deposited in OPAL are the Open Access versions of outputs published elsewhere. Changes resulting from the publishing process may therefore not be reflected in this document. The final published version may be obtained via the publisher’s DOI. Please note that additional copyright and access restrictions may apply to the published version.

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