Sorting SAM output from Bowtie

[DrD2009]

Hello everyone,

I have an easy one for you today. I have SAM output created from Bowtie that I want to use with Cufflinks, but Cufflinks only accepts SAM files that have been sorted. I've tried using SAM tools to sort, but it seems like I have to convert the SAM into a BAM in order to be able to sort it. Is that correct? Then won't I have to convert it back to SAM in order for use with Cufflinks?


Thanks again,
Brandon

[RockChalkJayhawk]

The cufflinks manual suggests:

sort -k 3,3 -k 4,4n hits.sam > hits.sam.sorted

Will this work?

Hello everyone,

I have an easy one for you today. I have SAM output created from Bowtie that I want to use with Cufflinks, but Cufflinks only accepts SAM files that have been sorted. I've tried using SAM tools to sort, but it seems like I have to convert the SAM into a BAM in order to be able to sort it. Is that correct? Then won't I have to convert it back to SAM in order for use with Cufflinks?


Thanks again,
Brandon

[genbio64]

You could also sort using samtools.

[DrD2009]

@RockChalkJayhawk
Thanks. That seems to work although I have no idea what that code represents.

@genbio64
Do I have to convert the SAM into a BAM to sort? That's what samtools seems to want.

[RockChalkJayhawk]

@RockChalkJayhawk
Thanks. That seems to work although I have no idea what that code represents.

@genbio64
Do I have to convert the SAM into a BAM to sort? That's what samtools seems to want.

The code just means to sort on column 3, then by column 4(numerically) of the hits.sam file and print to hits.sam.sorted

[DrD2009]

Ah. Well it worked perfectly. I guess I don't have to deal with samtools for now afterall.

Thanks again.

[shoncho]

If I am not using tophat but want to use some other resource, such as Bowtie, is there a way to sort the sequences into useable size files without using SAM tools?

- Shoncho

[rgregor]

Cufflinks
Error: this SAM file doesn't appear to be correctly sorted!
current hit is at scaffold_737:1311, last one was at scaffold_736:2605

But "scaffold_736" is before "scaffold_737" and the sorting is correct? Is this a bug?

best regards,
Gregor

[krobison]

Yes, you must convert from sam to bam to sort. The current cufflinks can use BAM

To sort your sam

# will create myfile.sorted.bam
samtools view -uS myfile.sam | samtools sort - myfile.sorted

[adarob]

@rgregor,

Are you using a reference GTF? If so, it must be sorted in the same order OR your SAM file must include a header that lists the scaffolds in the correct order.