Entering edit mode
7.0 years ago
rebeccakurtz62
•
0
I am really new to RNA sequencing, and am having some issues. My htseq count matrices seemed to be off for a few of my samples. I did some back tracing, and I believe it is because a few of my tophat files are actually inaccurate. They should all be about the same size, but tophat2, tophat3, and tophat4 are smaller then the rest. I'm not sure why. I took a screenshot of the size of some of the files.
I would like to add a side note to this question of yours for future readers.
Things can go wrong and it's not always your fault: with this, I mean that if you run 4 mapping processes in a row on the same machine and they run out of memory, quite possibly all the exceeding ones will be killed.
How to solve this?
More than solving, I would suggest you to run commands like this:
This way, you create a file with the standard error (if something goes wrong, you'll see it there), and a file with the time (if something ends too soon, you'll notice it in the time file).
For example, on which machine (number of CPUs, RAM) did you run Tophat, and how many threads did you assign to each process?
You will have to be more precise than that to get more specific help.
That being said, you should know that Tophat is no longer the "advisable" tool for RNA-seq analysis. The software is deprecated/ in low maintenance and should be replaced by HISAT2, StringTie and ballgown. See this paper: Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown. (If you can't get access to that publication, let me know and I'll -cough- help you.) There are also other alternatives, including alignment with STAR and bbmap,...
I also strongly suggest you to -get help- if you don't have access. That paper is really the state of the art.