Error With Samtools

Question

[Sameet]

I recently tried to use bowtie2 and samtools to process some paired end data. I used bowtie2-build to index my reference genome, and used this index to align the paired end data. Once the alignment was over, i got the following on my STDOUT:

7659592 reads; of these:
    7659592 (100.00%) were paired; of these:
    136410 (1.78%) aligned concordantly 0 times
    6927190 (90.44%) aligned concordantly exactly 1 time
    595992 (7.78%) aligned concordantly >1 times
98.22% overall alignment rate

With this message, i assumed that the alignment is complete. I had used the -S option to obtain sam output file. So i tried to use samtools to sort and index the output file, but every time i get EOF not found error.

Please advise. Sameet

Answer 1

[swbarnes2]

samtools sort works on .bam files, not .sam files, that might be your problem.

[Sameet]

Actually, when i try to convert the sam file to a bam file, i still get the same error.

[Philipp Bayer]

Have you used the -S flag on samtools, too? You have to tell samtools that you are using sam-files, too, like this:

samtools view -S bla.sam

or

samtools view -Sb in.sam out to convert to a bam-file called out.bam

[Sameet]

Hi, thanks for the post. I figured it out. Its correct, the samtools requires a bam file!! I managed to get what i wanted. Thank you all for your help.

Answer 2

[Bilby]

If your sam file has no SQ header you'll need to create a reference index in samtools first with: samtools faidx <refgenome.fa>