Merging RNA-seq replicates from Tophat

Sometimes, you might want to present your replicates as one consensus sequencing track on viewers like IGV or IGB.

To merge bam files : Use samtools merge.
To merge junctions.bed files : Use the tbedjuncs and combineJuncs scripts at seqscripts

cat junctions_file1.bed junction_file_2.bed junction_file_3.bed | tbed2juncs | combineJuncs > combined.junctions.bed

Ref: http://seqanswers.com/forums/showthread.php?t=3381

SAM file directionality

Understanding direction flags and how to extract them

• http://biobeat.wordpress.com/2013/03/14/separating-bam-files-to-forward-and-reverse-strand-files/
• http://biobeat.wordpress.com/2013/04/29/directional-rna-seq-part-1-extract-strand-information-from-sam-file/

 Paired-end specific discussion:

• http://ppotato.wordpress.com/2010/08/25/samtool-bitwise-flag-paired-reads/
• http://www.novocraft.com/wiki/tiki-index.php?page=Extracting+unmapped+reads+from+a+BAM+file+produced+by+novoalign

Useful samflag utility
http://picard.sourceforge.net/explain-flags.html

MISO sashimi plot (RNA-seq alternative splicing)

This is a modified python script from the MISO package for plotting alternative splicing events.

Modifications from the source:
1) Normalized junction counts (assumming that the junction reads are proportional to total read coverage)
2) Proper plotting of isoforms (lines do not extend beyond final exon/utr if one isoform is shorter than the other)