[bpb9]

How to use?

Short update: the script posted earlier in this thread has seen some updates (Python 3 compatibility, better argument handling etc) and is now called tophat_recondition.

It can be found here: https://github.com/cbrueffer/tophat-recondition

Does this program need to be installed in the same directory as the unmapped bams that need to be reconditioned? I can't seem to get the program to work. I have python and pysam installed, but the program doesn't run.

wget https://github.com/cbrueffer/tophat-...ive/master.zip
unzip master
set PATH to /home/user/tophat_recondition/tophat-recondition-master
set PATH to /cluster/software/python2.7.6/bin
#Try tophat-recondition
tophat-recondition.py -hv /home/scratch/user/Flowcell1/Raw_PE/sampleID/sampleBAM /home/scratch/user/Flowcell1/Raw_PE/sampleID/

[offspring]

Does this program need to be installed in the same directory as the unmapped bams that need to be reconditioned? I can't seem to get the program to work. I have python and pysam installed, but the program doesn't run.

wget https://github.com/cbrueffer/tophat-...ive/master.zip
unzip master
set PATH to /home/user/tophat_recondition/tophat-recondition-master
set PATH to /cluster/software/python2.7.6/bin
#Try tophat-recondition
tophat-recondition.py -hv /home/scratch/user/Flowcell1/Raw_PE/sampleID/sampleBAM /home/scratch/user/Flowcell1/Raw_PE/sampleID/

Try without -hv, those are optional parameters that will terminate the program (i.e., -h print help and exit, -v print version and exit).
The script can be run from whereever you like, as long as python and pysam can be found.

[bpb9]

So it got it to run if I stayed in the same directory as tophat-recondition.py and removed -hv command, and pointed it to the tophat output directories. I think the reason why is because on the cluster, HTseq is installed under one version of python, and pysam under another. If I use the version of python in my local directory, it runs. It produced an output file unmapped_fixup.bam. However when I run samtools flagstat on the unmapped.bam and the unmapped_fixup.bam, the flagstat summaries are the same…

samtools flagstat unmapped_fixup.bam
8903498 + 2048970 in total (QC-passed reads + QC-failed reads)
0 + 0 duplicates
0 + 0 mapped (0.00%:0.00%)
8903498 + 2048970 paired in sequencing
4380074 + 1096160 read1
4523424 + 952810 read2
0 + 0 properly paired (0.00%:0.00%)
0 + 0 with itself and mate mapped
0 + 0 singletons (0.00%:0.00%)
0 + 0 with mate mapped to a different chr
0 + 0 with mate mapped to a different chr (mapQ>=5)



samtools flagstat unmapped.bam
8903498 + 2048970 in total (QC-passed reads + QC-failed reads)
0 + 0 duplicates
0 + 0 mapped (0.00%:0.00%)
8903498 + 2048970 paired in sequencing
4380074 + 1096160 read1
4523424 + 952810 read2
0 + 0 properly paired (0.00%:0.00%)
0 + 0 with itself and mate mapped
0 + 0 singletons (0.00%:0.00%)
0 + 0 with mate mapped to a different chr
0 + 0 with mate mapped to a different chr (mapQ>=5)


somehow I just can't believe that none of the unmapped reads are duplicates…could this be a consequence of me running tophat with the --no-mix option turned on?

[offspring]

Sounds like it might have an effect, I don't have experience with that option myself. To see the effect of the script itself, try running the following command on unmapped.bam and unmapped_fixup.bam:

samtools view -f 0x8 unmapped.bam

On the original file it should not print anything, but on the fixed file you should get output.

[bpb9]

You are correct, there is indeed output for unmapped_fixup.bam but not unmapped.bam.

I still think it's really weird that there are no duplicates in the unmapped file, but that must be a separate issue.

[offspring]

Yes, this is seperate indeed. The point of tophat-recondition is to fix issues that prevent unmapped.bam from being used by downstream tools (i.e., to make it possible to merge accepted_hits.bam and unmapped.bam, and feed the merged file to a QC software).

[offspring]

I've just release version 0.3 of tophat-recondition (https://github.com/cbrueffer/tophat-recondition), and it may fix the "Mapped mate should have mate reference name" problem id0 was seeing.

The details are in TopHat bug #16, https://github.com/infphilo/tophat/issues/16 and tophat-recondition bug #1 https://github.com/cbrueffer/tophat-...ition/issues/1.