Can I use htseq --samout file for subsequent annotation?

[heso]

Hi,
So briefly what I'm doing is that:
-->I'm annotating my smallRNA sequencing data using htseq-count.
-->I'm using original.sam to annotate miRNAs and using -o to get the out.sam
-->From that out.sam I can grep out the 'no_feature' reads/rows.

The question:
Maybe a stupid idea but is there any way I could use this out_no_feature.sam file for another round of htseq annotationagainst e.g. rRNA.gtf?

As the out_no_feature.sam does not have a header, is there any way to reheader it or use the header from the original.sam file? Also, I guess I need to remove the last column "XF:Z:__no_feature" from the out_no_feature.sam

Thanks ahead for comments and help

[Michael.Ante]

Hi Heso,

You can use RSeQC's bam2fq.py to make a fastq file out of your "out_no_feature.sam".
In order to get the header into your files, use:

samtools view -SH original.sam > header.txt

Then use something like:

cat header.txt out_no_feature.sam | samtools view -bSh - > out_no_feature.bam

. In case it was not sorted according to the position, you may need to include a samtools sort into the pipe.
The "XF:Z"-field does not influence the downstream analysis. But since you have only alignments with this field will not give you any further information.

Cheers,
Michael

[heso]

Hi Heso,

You can use RSeQC's bam2fq.py to make a fastq file out of your "out_no_feature.sam".
In order to get the header into your files, use:

samtools view -SH original.sam > header.txt

Then use something like:

cat header.txt out_no_feature.sam | samtools view -bSh - > out_no_feature.bam

. In case it was not sorted according to the position, you may need to include a samtools sort into the pipe.
The "XF:Z"-field does not influence the downstream analysis. But since you have only alignments with this field will not give you any further information.

Cheers,
Michael

Thanks Michael, it worked perfectly!!

/Helena