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TVscript Summary
TVscript is a new tool that allows for the removal of transcripts within a reference sets that are associated with high levels of intra-condition read count variation following read mapping. It can be used to test the effects of such transcripts on down stream analysis e.g. on the identification of differentially expressed genes.

Publication
(publication under review)
1. Lobo D, Linheiro R, Godinho R, Archer JP (2022) On taming the effect of transcript level intra-condition count variation during differential expression analysis: A story of dogs, foxes and wolves. PLOS ONE 17(9): e0274591. (view)

Abstract
The evolution of RNA-seq technologies has yielded datasets of high scientific value that are often generated as condition associated biological replicates within differential expression studies. As the number of replicates increase, so to does confidence in identifying differentially expressed transcripts. With rapidly expanding RNA-seq data archives there is opportunity to augment replicate numbers when conditions of interest overlap at an inter-study level. Despite correction procedures for estimating transcript abundance, a remaining source of error is transcript level intra-condition count variation; as partially indicated by the disjointed results between differential expression analysis tools. Such variation is amplified at an inter-study level. Here, we present TVscript, a tool that removes reference-based transcripts associated with intra-condition variation above specified thresholds. With this tool we explore the effects of removing transcripts associated with varying degrees of intra-condition variation on differential expression analysis. This is done in relation to inter- and intra-study datasets representing brain samples of dogs, wolves and foxes (wolves vs. dogs and aggressive vs. tame foxes). We demonstrate that 20% of the transcripts identified as being differentially expressed are associated with high levels of intra-condition variation. This is an over-representation relative to the reference set. As transcripts harbouring such variation are removed from the reference prior to differential expression analysis a discordance from 26 to 40% in the lists of differentially expressed transcripts is observed when compared to those obtained using the non-filtered reference. For our data, the removal of transcripts possessing intra-condition variation values within (and above) the 97th and 95th percentiles, for wolves vs. dogs and aggressive vs. tame foxes, maximized the detection of differentially expressed transcripts as a result of alterations within gene-wise dispersion estimates. Through this analysis the support for seven genes with potential for being involved with selection for tameness is provided. TVscript is available at: https://sourceforge.net/projects/tvscript/.

Conference(s)
(1) A poster, presented by Diana Lobo at ENBE, is available here.
(2) A talk, presented by Diana Lobo at 8th European Congress of Mammalogy is available here.

Getting Started
See the [Quick Start] guide for running TVscript along with an explaination of parameter options.

Funding
This work was funded by the project NORTE-01-0246-FEDER-000063, supported by Norte Portugal Regional Operational Programme (NORTE2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and by research funding from the projects under the references PTDC/BIA-EVF/29115/2017, PTDC/BIA-EVF/2460/2014 and POCI-01-0145-FEDER-029115 co-funded by Operational Competitiveness and Internationalization Program, Portugal 2020 and the European Union via the European Regional Development Fund (ERDF) and by National Funds through FCT. DL, RG were supported by FCT (PD/BD/132403/2017 to DL, contract under DL57/2016 to RG) and JA was supported by Funds through FCT under the references POCI-01-0145-FEDER-029115 and PTDC/BIA-EVL/29115/2017. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. FCT and NORTH2020 url’s: https://www.fct.pt/ and https://www.norte2020.pt.

Related Software
1. CStone
2. CSReadGen
3. CView
4. ChimSim
5. TVScript < (current location)

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