STAR Output Sam Files

[mtiwaridros]

Hello

I am using STAR2.3.0e for alignments.
Are the output sam files (Aligned.out..sam) already sorted and can be used directly with htseq-count?
Or do I need to convert them into bam, sort the bam, and use the sorted bam for htseq-count?

Any help is appreciated.

Thanks.
M

[WhatsOEver]

No, they aren't. You need to run something like

samtools view -bS yourStarOutput.sam | samtools sort - yourSortedStarOutput

[mtiwaridros]

Hi

Thanks for the reply.
However, if I run:

samtools view -bS sample1.sam | samtools sort - sample1_sorted

I get the error message:

[bam_header_read] EOF marker is absent. The input is probably truncated.
[samopen] SAM header is present: 1870 sequences.

Although the conversion is working.

On the other hand, if I just convert the sam file to bam file using:

samtools view -bS sample1.sam > sample1

And then sort it using:

samtools sort sample1.bam sample1_sorted.bam

Then everything appears to work fine, and there is no error message.
I have checked my sam files and they have headers, and seem to be fine.

Is this a bug with samtools or am I missing something?

Thanks.
M

[WhatsOEver]

The End Of File marker just tells samtools that the file ends at this point. It can be missing or unrecognizable because of different reasons (eg. moving files between linux and windows or it was deleted). If you know that your file isnt't truncated, you can ignore this warning (Its not an error! ). The conversion is not influenced by that.

[mtiwaridros]

But then why I don't get the error if I run the commands separately?

[WhatsOEver]

I think that the sort command throws the warning and that you don't get it by splitting the command is because you write to a new file using view. This way you get a file with a proper EOF marker set at the end.
You could try to set an EOF manually in your original file (Ctrl+D under Linux, no idea under Windows) and see if that prevents the warning.

[GenoMax]

See #11 in this post for Peter's solution: http://seqanswers.com/forums/showthread.php?t=19228

[dpryan]

Some versions of samtools incorrectly show the EOF message when you pipe a file into them (at least that's the case with the sort command). What's supposed to happen is that it keeps track of how it's reading the file and then doesn't check for the EOF if you use a pipe (since you can't seek to the end), but at some point that code got changed. I think this works correctly in the current (1.0) version.

[alexdobin]

Hello

I am using STAR2.3.0e for alignments.
Are the output sam files (Aligned.out..sam) already sorted and can be used directly with htseq-count?
Or do I need to convert them into bam, sort the bam, and use the sorted bam for htseq-count?

Any help is appreciated.

Thanks.
M

STAR .sam files do not need to be sorted for ht-seq, you can simply convert them to .bam and feed the .bam to ht-seq.
I would strongly recommend switching to a newer version of STAR from: https://github.com/alexdobin/STAR/releases
Then you can get the BAM file, or - if you need - coordinate-sorted BAM directly from STAR:
--outSAMtype BAM Unsorted
OR
--outSAMtype BAM SortedByCoordinate
OR BOTH
--outSAMtype BAM Unsorted SortedByCoordinate

Cheers
Alex

[mtiwaridros]

Thank you all for the replies. The issue is clear now.

Best
M