Introduction

The production of multiple transcript isoforms from one gene is a major source of transcriptome complexity. RNA-Seq experiments, in which transcripts are converted to cDNA and sequenced, allow the resolution and quantification of alternative transcript isoforms. However, methods to analyze splicing are underdeveloped and errors resulting in incorrect splicing calls occur in every experiment.We used RNA-Seq data to develop sequencing and aligner error models. By applying these error models to known input from simulations, we found that errors result from false alignment to minor splice motifs and antisense stands, shifted junction positions, paralog joining, and repeat induced gaps. By using a series of quantitative and qualitative filters, we eliminated diagnosed errors in the simulation, and applied this to RNA-Seq data from Drosophila melanogaster heads. We used high-confidence junction detections to specifically interrogate local splicing differences between transcripts. This method out-performed commonly used RNA-seq methods to identify known alternative splicing events in the Drosophila sex determination pathway. We describe a flexible software package to perform these tasks called Splicing Analysis Kit (Spanki), available at http://www.cbcb.umd.edu/software/spanki.Splice-junction centric analysis of RNA-Seq data provides advantages in specificity for detection of alternative splicing. Our software provides tools to better understand error profiles in RNA-Seq data and improve inference from this new technology. The splice-junction centric approach that this software enables will provide more accurate estimates of differentially regulated splicing than current tools.

Publications

  1. Design of RNA splicing analysis null models for post hoc filtering of Drosophila head RNA-Seq data with the splicing analysis kit (Spanki).
    Cite this
    Sturgill D, Malone JH, Sun X, Smith HE, Rabinow L, Samson ML, Oliver B, 2013-11-01 - BMC bioinformatics

Credits

  1. David Sturgill
    Developer

    National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health

  2. John H Malone
    Developer

  3. Xia Sun
    Developer

  4. Harold E Smith
    Developer

  5. Leonard Rabinow
    Developer

  6. Marie-Laure Samson
    Developer

  7. Brian Oliver
    Investigator

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Summary
AccessionBT006529
Tool TypeApplication
Category
PlatformsLinux/Unix
Technologies
User InterfaceTerminal Command Line
Download Count0
Submitted ByBrian Oliver