Hello,
I have a few questions about running my paired-end reads through Tophat (using Galaxy).
1) From what I've read on this forum, it sounds like paired end reads have to be properly mate-matched (that is, each pair must have a mate, and be in the same order, in the R1 and R2 files) in order for Tophat to map the mates properly. My question is, if I save any remaining unpaired mates after QC in a separate file, and run them through Tophat separately from the paired end reads, how can I then join the single-end and paired-end data together for analysis in Cufflinks?
2) How do I determine the standard deviation of distances between my mate-pairs? All I've got to work off of is a graph of the size distributions, which range from about 200 to about 1000 (average ~300). I want to ensure that Tophat is still able to successfully map those larger fragments.
3) What is the shortest fragment length that it is reasonable to try and map? I noticed that the default Tophat setting on galaxy is to map a minimum read segment length of 25. So I'm wondering if this is a good cutoff for minimum length of read to keep after QC.
Any thoughts or suggestions are greatly appreciated
I have a few questions about running my paired-end reads through Tophat (using Galaxy).
1) From what I've read on this forum, it sounds like paired end reads have to be properly mate-matched (that is, each pair must have a mate, and be in the same order, in the R1 and R2 files) in order for Tophat to map the mates properly. My question is, if I save any remaining unpaired mates after QC in a separate file, and run them through Tophat separately from the paired end reads, how can I then join the single-end and paired-end data together for analysis in Cufflinks?
2) How do I determine the standard deviation of distances between my mate-pairs? All I've got to work off of is a graph of the size distributions, which range from about 200 to about 1000 (average ~300). I want to ensure that Tophat is still able to successfully map those larger fragments.
3) What is the shortest fragment length that it is reasonable to try and map? I noticed that the default Tophat setting on galaxy is to map a minimum read segment length of 25. So I'm wondering if this is a good cutoff for minimum length of read to keep after QC.
Any thoughts or suggestions are greatly appreciated
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