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Identifying differential isoform abundance with RATs: a universal tool and a warning

View ORCID ProfileKimon Froussios, View ORCID ProfileKira Mourão, View ORCID ProfileGordon G. Simpson, View ORCID ProfileGeoffrey J. Barton, View ORCID ProfileNick J. Schurch
doi: https://doi.org/10.1101/132761
Kimon Froussios
1Division of Computational Biology, School of Life Sciences, University of Dundee, Dundee, UK
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Kira Mourão
1Division of Computational Biology, School of Life Sciences, University of Dundee, Dundee, UK
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Gordon G. Simpson
2Centre for Gene Regulation & Expression, School of Life Sciences, University of Dundee, Dundee, UK
3Division of Plant Sciences, School of Life Sciences, University of Dundee, Dundee, UK
4Cell and Molecular Sciences, James Hutton Institute, Invergowrie, Dundee, UK
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Geoffrey J. Barton
1Division of Computational Biology, School of Life Sciences, University of Dundee, Dundee, UK
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Nick J. Schurch
1Division of Computational Biology, School of Life Sciences, University of Dundee, Dundee, UK
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Abstract

Motivation The biological importance of changes in gene and transcript expression is well recognised and is reflected by the wide variety of tools available to characterise these changes. Regulation via Differential Transcript Usage (DTU) is emerging as an important phenomenon. Several tools exist for the detection of DTU from read alignment or assembly data, but options for detection of DTU from alignment-free quantifications are limited.

Results We present an R package named RATs – (Relative Abundance of Transcripts) – that identifies DTU transcriptome-wide directly from transcript abundance estimations. RATs is agnostic to quantification methods and exploits bootstrapped quantifications, if available, to inform the significance of detected DTU events. RATs contextualises the DTU results and shows good False Discovery performance (median FDR ≤0.05) at all replication levels. We applied RATs to a human RNA-seq dataset associated with idiopathic pulmonary fibrosis with three DTU events validated by qRT-PCR. RATs found all three genes exhibited statistically significant changes in isoform proportions based on Ensembl v60 annotations, but the DTU for two were not reliably reproduced across bootstrapped quantifications. RATs also identified 500 novel DTU events that are enriched for eleven GO terms related to regulation of the response to stimulus, regulation of immune system processes, and symbiosis/parasitism. Repeating this analysis with the Ensembl v87 annotation showed the isoform abundance profiles of two of the three validated DTU genes changed radically. RATs identified 414 novel DTU events that are enriched for five GO terms, none of which are in common with those previously identified. Only 141 of the DTU evens are common between the two analyses, and only 8 are among the 248 reported by the original study. Furthermore, the original qRT-PCR probes no longer match uniquely to their original transcripts, calling into question the interpretation of these data. We suggest parallel full-length isoform sequencing, annotation pre-filtering and sequencing of the transcripts captured by qRT-PCR primers as possible ways to improve the validation of RNA-seq results in future experiments.

Availability The package is available through Github at https://github.com/bartongroup/Rats.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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Posted May 02, 2017.
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Identifying differential isoform abundance with RATs: a universal tool and a warning
Kimon Froussios, Kira Mourão, Gordon G. Simpson, Geoffrey J. Barton, Nick J. Schurch
bioRxiv 132761; doi: https://doi.org/10.1101/132761
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Identifying differential isoform abundance with RATs: a universal tool and a warning
Kimon Froussios, Kira Mourão, Gordon G. Simpson, Geoffrey J. Barton, Nick J. Schurch
bioRxiv 132761; doi: https://doi.org/10.1101/132761

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