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Old 05-15-2012, 04:03 AM   #1
sphil
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Default TopHat vs Tophat2

Hey,

since bowtie2 is released also tophat2 is released. The question is: How does data structure change using tophat2?
I got a fully automated pipeline using tophat (bowtie) which also provides some downstream analysis. Changing that to tophat2 will it affect the downstream data structure which means are there any other file formats used for output in tophat2?

Thanks for help, i can't find anything on the internet about that...


best,

phil
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Old 05-15-2012, 03:26 PM   #2
sdriscoll
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Default

tophat2 still generates an output folder containing accepted_hits.bam, junctions.bed, etc. The call to the command might be a little different if you're using any customizations to the call but the output of it is the same as tophat(1).

you could always run it and find out...
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Old 05-15-2012, 05:33 PM   #3
vkartha
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Default Tophat2 -r option and alignment results

Hey guys,
I have paired end reads on RNASeq data and I wanted to test run Tophat2 to map these reads for a given sample using 5 different -r values(that is the mean inner distance between paired ends) to see how it would affect the alignment.

I'm not exactly sure what I should look at once the alignment is over to figure out the so called 'alignment results'.

Tophat2 gives out a log folder along with the accepted_hits.bam, junction.bed files etc. in which I looked at two files namely:

bowtie.left_kept_reads.fixmap.log
bowtie.right_kept_reads.fixmap.log

These give summaries on the alignment from each end such as:

22630038 reads; of these:
22630038 (100.00%) were unpaired; of these:
2447457 (10.82%) aligned 0 times
15049769 (66.50%) aligned exactly 1 time
5132812 (22.68%) aligned >1 times
89.18% overall alignment rate

Now I notice there are also log files namely:

bowtie.right_kept_reads_seg1.fixmap.log
bowtie.right_kept_reads_seg2.fixmap.log
bowtie.right_kept_reads_seg3.fixmap.log
bowtie.right_kept_reads_seg4.fixmap.log

(And the same 4 for the left kept reads)

I don't understand what these files are or how they are different from the file I mentioned earlier (fixmap.log file)

I'm not sure if I'm looking at the correct log file, and if the fixmap.log file is indeed the right one to look at, I noticed that the alignment percentage is the SAME (i.e. 89%) for ALL -r options (I tried -50, -16, -33, 0, 50) using the same std deviation value. I just cannot understand why!!! - Does this mean that the -r option doesn't make a big difference in the alignment???

Hope to hear back from you guys soon. Thanks a lot.
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Old 12-04-2012, 06:03 PM   #4
maize
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Default tophat2

Wondering for the same question? Any update?
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Old 12-04-2012, 06:50 PM   #5
sdriscoll
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Since Tophat handles pairing up the left and right side reads there is no bowtie log that tells you how many total fragments were aligned. If I were doing this test I would probably be interested in the -r setting's impact on the number of successful properly paired fragments aligned. I think simply running 'samtools flagstat accepted_hits.bam' is going to be good enough for getting that count. There will be a count of total properly paired alignments...divide that by 2 for total fragments. If you count the number of reads you have in your FASTQ files then you'll have your percent aligned.

You could count those like so...

gunzip -c reads.gz | awk 'END {print NR/4}'
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