Tophat mapping in Galaxy

[guzhi100]

Hi all,

After mapping, I used IGV to have a look at the mapping. There are a lot of mapped reads without pair reads. Should I keep these reads? or Is this a problem for cufflinks analysis?

What I tried is:
1. BAM to SAM
2. Filter SAM: set Read mapped in a proper pair to Yes.
The result is that only 1/5 reads were left.

Can anybody tell me if this operation is proper??
How do you normally optimize the mapping rerults from Tophat?
Which considerations should I take into account?
Looking forward to your reply.
Jiwen

[jxu666]

which tool did you use to filter SAM? Thanks,

[yingzhang]

If you have ever run samtools flagstat on Tophat output (accepted_hits.bam), you will always see a fairly low percentage of mapped reads properly paired. This is NOT because Tophat can't pair reads, but because TopHat WRONGLY assigns the flag for pairing. I can't recall the exact detail now, but I roughly remember that if a read pair covers intron, or one read from a pair is spliced (this is the part I am not sure), then the read(s) will be assigned as "NOT properly paired".

So the "correct" way to check the percentage of reads paired is to generate a transcriptome sequence and map reads to it to check the percentage of mapped reads properly paired.

By the way, picard tools (FixMateInformation) might be able to fix the pairing issue, however, I never tried it.