Re: [Inchworm-users] XS tags in non-strand-specific blat alignments

[Brian H. <bh...@br...>]

Oh, I should also mention that I haven't had good luck with getting
cufflinks to run on non-TopHat-generated alignments...

There are some additional fields that are also required in the sam output,
such as NM:i:\d+ and NH:i:\d+  (something like that... pulling from my
inaccurate memory), that seem to be essential for cufflinks to run on these
files.

If you want to use cufflinks for expression analysis, I definitely recommend
sticking with TopHat as the aligner for now.

If you want to try out RSEM (one of my personal favorites right now), you
could give it a whirl using the built-in bowtie alignments to unspliced
transcript sequences.

best,

-brian


On Fri, Oct 14, 2011 at 3:47 PM, Brian Haas <bh...@br...>wrote:

> Hi Thomas,
>
> I'm glad you're having mostly good luck with these tools, and able to tweak
> them as needed.
>
> I've been slowly moving the inchworm and related developments over to the
>
>    http://trinity.sf.net
>
> site, and there's a new alignment wrapper under
>
>     util/alignReads.pl
>
> that now supercedes the old blat alignment wrapper.  With the above, you'd
> use
>
>    --aligner BLAT
>
> If you want to give it a whirl, pull all the code from SVN directly, since
> this stuff has been pretty fluid lately.
>  svn co
> https://trinityrnaseq.svn.sourceforge.net/svnroot/trinityrnaseq/trunktrinityrnaseq
>
> I don't remember what the blat system does in the context of
> non-strand-specific data, but it sounds like we'll need to add the XS
> attribute there.  I'll have to investigate this further.  I'll check into
> the other issues you describe, since they sound very familiar.
>
>
> thanks!
>
> -brian
>
>
>
>
> On Fri, Oct 14, 2011 at 3:22 PM, Thomas Sandmann <tom...@go...>wrote:
>
>> Dear Brian,
>>
>> I have used your blat alignment pipeline to map non-strand-specific
>> 2x100 bp RNASeq HiSeq reads to a reference genome.
>>
>> The sequence headers contained spaces, so I modified your perl scripts
>> "fastQ_to_fastA.pl" and "fastQ_to_tab.pl" to split the headers on the
>> first space and use only the first element as fasta header.
>>
>> Now, I would like to use cufflinks to assemble transcripts.
>> Unfortunately, the "XS" tags seem to be missing from my .bam file. Is
>> this expected ? Does the blat pipeline only add these for stranded reads ?
>>
>> Also, I would like to combine two .bam output files that were mapped to
>> the same reference genome in separate blat alignment runs. Is the
>> "samtools sort" command suitable for use with the output from blat
>> alignments / input into cufflinks ?
>>
>> Thanks a ton for providing such amazing tools to the community !
>> Thomas
>>
>>
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>
>
>
> --
> --
> Brian J. Haas
> Manager, Genome Annotation Research and Development
> The Broad Institute
> http://broad.mit.edu/~bhaas
>
>
>
>
>


-- 
-- 
Brian J. Haas
Manager, Genome Annotation Research and Development
The Broad Institute
http://broad.mit.edu/~bhaas