tophat - cannot view sam output in samtools tview

[lmilne]

Tophat (v1.0.11) generates a SAM format file, but when I try to generate a BAM file from it to use in samtools tview I get:

samtools view -b -S -o accepted_hits.bam accepted_hits.sam
[samopen] no @SQ lines in the header.
[sam_read1] missing header? Abort!

Anyone got ideas on how to get round this?

[sdriscoll]

I have this same issue. The samtools instructions explain what is going on but I'm still not all the way to a solution myself. The @SQ line is a single header line from the SAM format specification (from the samtools site - they have a PDF) that identifies the reference sequence and the length of that sequence. In their example they specify a single chromosome and the length is that chromosome's length - but I'm not sure how to extend that to a SAM file from Tophat that has the entire genome in it.

http://samtools.sourceforge.net/samtools.shtml

According to the 'view' command description the -S option tells samtools that the @SQ lines aren't included but when you use -S you also have to specify -t (Bowtie's site doesn't mention this). The -t option is used to specify a reference file in fasta format. I'm guessing that might be something like a single chromosome's FASTA file but again I don't understand how to extend that to the entire genome. Or maybe you can't make a BAM for the entire genome? I'm not sure...but I'm getting closer.

[kch]

I was able to resolve this issue by following the second set of instructions from here: http://sourceforge.net/apps/mediawik..._SAM_to_BAM.3F

How to convert SAM to BAM?

...

samtools faidx ref.fa
samtools view -bt ref.fa.fai aln.sam > aln.bam

Hope this helps