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-   -   SAM file after Bowtie is messed up (http://seqanswers.com/forums/showthread.php?t=63754)

Morgane_AUS 10-26-2015 04:27 PM

SAM file after Bowtie is messed up
 
Hi,



I have chip-seq data from E. coli (51 bp). I mapped my fastq file to my reference genome (custom build) using Bowtie in Galaxy. In the SAM output file, some rows have the sequence in the quality score columns, and the quality scores in the OPT column. Some rows are fine.



Anyone would know what is causing it and how to fix that ?



Thanks

dpryan 10-27-2015 12:11 AM

Are you sure that's actually the case? It's incredibly more likely that you're just miscounting the columns.

Morgane_AUS 10-27-2015 02:30 PM

See step 14

http://galaxy-qld.genome.edu.au/u/m-...ple-2-chip-seq

GenoMax 10-27-2015 04:15 PM

Have you set the file format to "fastqsanger" for your original data files (I can't tell from the history you shared). Here is how you would do it: https://wiki.galaxyproject.org/Suppo...ognize_dataset Then you should not have to groom your data. If this is recent data correct so it should already be in sanger fastq format.

It appears that part of illumina fastq header (1:N:0:18) is missing from the reads that appear to have an alignment (at least that is what it looks like in the web page).

Morgane_AUS 10-27-2015 05:26 PM

Hi,

thank you for looking at my data.

I have tried without grooming, just changing data type (my reads are illumina 1.9 encoding) and I have the exact same result.

The illumina fastq header (1:N:0:18) is present for all reads in the fastq file.

I have tried galaxy GVL instance and galaxy main. Same results.

I don't have this problem when I use BWA mapping. But it's better to use Bowtie for E. coli reads since BWA looks for intron so better used for eukaryotes is that right ?

blancha 10-27-2015 10:56 PM

Quote:

I don't have this problem when I use BWA mapping. But it's better to use Bowtie for E. coli reads since BWA looks for intron so better used for eukaryotes is that right ?
No, BWA, like Bowtie does not take into account the introns.
Only splice-junction aware aligners, like TopHat and STAR do, in which case you have to provide them with the genome annotation indicating the location of the junctions.
TopHat actually delegates the alignment to Bowtie1 or 2, and only handles the splicing.

In the link to the Galaxy instance that you posted, you are using a version of Bowtie that dates back to 2010, version 0.12.7. It's not clear from your post if you've already tried this, but the first troubleshooting step I would take would be to upgrade to a more modern version of Bowtie. There is a long list of bugs that have been fixed in Bowtie since 2010.

Morgane_AUS 10-28-2015 02:32 PM

Thanks for that ! That's really helpful.

I didn't check which version of Bowtie I was using thinking that the Galaxy main instance would display the most up to date version. I will have a look at that.

Thanks a lot.


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