[Useq-users] Usage guide for Extended RNASeq Analysis in USeq

[David N. <Dav...@hc...>]

Hello Folks,

I wrote up some instructions for how to take advantage of the reworked RNA-Seq apps in Useq.  The approach here is different than what most other RNA-Seq analysis entail.  Alignments are made to genome fastas plus splice junctions that, when needed, span multiple exons.  This includes all known splice junctions as well as all theoretical splice junction combinations.  Thus junction matches are biased toward aligning to known annotation instead of the closest partial match as Tophat and other seed and extend algorithms employ.  This avoids a common problem of creating apparent novel exons inside intronic regions. As before, I'm leaning heavily on Anders' DESeq package for  calling differential expression since it controls for overdispersion in the count data with a negative binomial test.  Gone are the days of not running biological replicas.  3-4 are mandatory for each condition.

http://useq.sourceforge.net/usageRNASeq.html

This is included in the Documentation folder in the Useq download from SourceForge.

There are three key improvements with this reworking:

 1.  It supports any length of sequence reads, the prior version was designed to work with 36bp reads, 50bp and 101 bp reads would often only partially align to splice junctions.
 2.  It uses BAM alignments as the working raw data format enabling collection and analysis of the underlying sequence data as well as the structure of the splice junction matches to better call differential splicing.  The use of BAM files also enables users to take advantage of external apps for analysis and visualization.
 3.  Depth of coverage tracks now accurately map splice junction reads across multiple exons.

I'll be building support for these apps into the RNASeq wrapper and depreciating the old methods.

-cheers, D

--
David Austin Nix, PhD
Research Assistant Professor in Oncological Sciences
Bioinformatics Shared Resource
Huntsman Cancer Institute and the University of Utah
2000 Circle of Hope, Salt Lake City, UT 84112
(801) 587-4611
Room HCI 3165
dav...@hc...
http://bioserver.hci.utah.edu