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shenkers / isoscm

Transcript assembly tool using multiple change-point inference to improve 3'UTR annotation
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Mapping with star - XS flag #9

Closed adomingues closed 9 years ago

adomingues commented 9 years ago

I have mapped alignments with STAR (stranded data, dUTP, PE101), that lack the XS flag. I guess that is reason why assembly step fails:

java -Xmx6g -jar ~/bin/IsoSCM-2.0.9.jar assemble -bam WT.bam -base WT -s reverse_forward
2015-06-23T12:44:03,657 [main] INFO  executable.IsoSCM - Starting assemble ...
2015-06-23T12:44:04,257 [main] INFO  executable.IsoSCM - tabulating splice junctions...
Exception in thread "main" java.lang.NullPointerException
        at processing.FindSpliceJunctions.tabulateSpliceJunctions(FindSpliceJunctions.java:144)
        at executable.IsoSCM.main(IsoSCM.java:221)

Is the lack of XS flag the issue? Have you tested isoscm with STAR alignments?

Cheers.

shenkers commented 9 years ago

Yes, absence of the XS flag would cause an error, although people have used STAR with IsoSCM before. I believe there is a flag you can set when using STAR to add the XS flag. This thread provides the details of how to do that:

http://seqanswers.com/forums/archive/index.php/t-30905.html

Let me know if you run into any more problems.

Best, Sol

cyrilcros commented 9 years ago

The STAR manual has a compatibility with Cufflinks section:

4.2.3 Compatibility with Cufflinks Cu ffdiff. For unstranded RNA-seq data, Cufflinks/Cuff diff require spliced alignments with XS strand attribute, which STAR will generate with --outSAMstrandField intronMotif option. As required, the XS strand attribute will be generated for all alignments that contain splice junctions. The spliced alignments that have unde ned strand (i.e. containing only non-canonical unannotated junctions) will be suppressed. If you have stranded RNA-seq data, you do not need to use any speci c STAR options. Instead, you need to run Cufflinks with the library option --library-type options.> For example, cufflinks ... --library-type fr-firststrand should be used for the standard dUTP protocol, including Illumina's stranded Tru-Seq. >This option has to be used only for Cufflinks runs and not for STAR runs. In addition, it is recommended to remove the non-canonical junctions for Cufflinks runs using --outFilterIntronMotifs RemoveNoncanonical.