Crazy unmapped reads

[caiosuz]

I don't know if I am understanding the correct meaning of the reads index in sam files.

This information is present in the Flags description:

'Next, we have the cases when only one read in a pair is mapped.
69 - 0001000101 - First read in pair. This read is unmapped but its mate is mapped.
133 - 0010000101 - 2nd in pair. Read unmapped but mate is mapped.'

Soooo, does it means that If I have a read with 133 or 69, its paired read can't be present in the unmapped reads file, ok?
I am assuming that reads with the same index (in this case "M03092:8:000000000-AG2GN:1:2117:2591:14346") are paired. Am I correct? If I am wrong I understood what happened but I'd like to know what are these lines with same index.

Following this line of thought (same index, paired reads), why are there so many lines of my unmapped paired reads like this?

M03092:8:000000000-AG2GN:1:2117:2591:14346 69
M03092:8:000000000-AG2GN:1:2117:2591:14346 133

Can anyone explain what's going on with these reads?
Could the reason be a wrong configuration of TopHat or the Bowtie version specified?

[offspring]

It's a bug in TopHat (all versions); it doesn't set the 0x8 bit ("next segment in the template unmapped") when both reads are unmapped.

This is one of the issues TopHat-Recondition fixes (https://bmcbioinformatics.biomedcent...859-016-1058-x , https://github.com/cbrueffer/tophat-recondition).

[caiosuz]

Thanks a lot, offspring.
I will re-run my mapping with this new issue!
=D

[offspring]

No Problem! You don't need to re-run the mapping though. TopHat-Recondition is a post-processor; you just pass it your TopHat output directory and it writes a corrected unmapped file. But maybe that's what you meant