Can Cuffdiff treat paired-end and single-end reads at the same time?

[zun]

Hi there,

I run cuffdiff with two paired-end reads samples and 2 single-end reads samples at the same time the other day(after running tophat toward each samples), and got results which seemed normal and correct with no error messages.

As I read cufflinks, cuffcompare, cuffdiff manuals and didn't find any statements that say they can't treat single-end and paired-end samples together, so I think my results are reliable(I don't know how accurate they are, though).

But I just want to know other RNA-seq users' opinions over cuffdiff's treatment with single/paired-end reads together.

Have anyone done the same analysis before?
I would be very happy if anyone would reply to me and give some advises!

zun

[zun]

Hi there,
I am now wondering how I can treat pair-end reads as two single-end read, and integrate them together when doing cufflinks.

I got a low mapping rate(40%) after running tophat to my pair-end reads, but when I checked log files(ex. logs/bowtie.left_kept_reads.fixmap.log), the rate was more than 75%!! I think it is because this high rate was just a result from Bowtie, after screening the left and right reads which are not mapped on the same chromosome and disposing these.

Then, I come to think whether I can separate pair-end reads to single-end ones , run tophat for each of them and finally run cufflinks for them together....

What do you think of this idea?
Does anyone have other idea on this ?

[zorph]

hello out there,
I too was also wondering whether the cufflinks calculations would be correct if I merged a single read data set with a paired-end read data set and ran cufflinks on the merge file?

Would everything be ok? or would the numbers be off?

Thanks!

[zun]

Hello again,

Now Tophat2 was released and has a new option called "--report-discordant-pair-alignments", which allows mate pairs to map to different chromosomes.
So my problems was solved described above, in fact my mapping rate improved up to 70% from 40%! thanks!

BUT, on the other hand, when it comes to FPKM calculation, how are the mate pairs which are mapped on different chromosomes treated in Cufflinks??
Is there any new calibration to it? or these are treated as single read?
I don't know whether I can trust the FPKM resulted from this option....

I will be appreciated any help!

zun