Hi,
I am using tophat 2.4.0.1 and have come across two main issues.
cheers
Ashok
I am using tophat 2.4.0.1 and have come across two main issues.
- One is that even for uniquely mapped read-pairs some of the alignments are missing their quality scores i.e. quality scores are set to "*" as for secondary alignments. But I have checked and these reads are primary alignments. Any insight in this regard will be appreciated
- While aligning trimmed reads tophat fails and this behavior is kind of random. I finally figured out that this is due to the fact that I have trimmed my reads (pe 50, minimum length 30 bp) and Bowtie2 throws and error when tophat tries its segment mapping. This is mainly because the default is 25 "--segment-length Each read is cut up into segments, each at least this long. These segments are mapped independently. The default is 25." It ends up trying to match the other 5 bases when the read is trimmed. Bowtie2 specifically fails here as the number of mismatches in the seed is specified to be 2. One fix is to manually run Bowtie2 with the no mismatches in seed option, the other is to change the tophat segment-length option to at least half the size of your smallest read length
- Has anyone encountered this behavior and any clues as to how this affects the analysis in general? I would much appreciate any comments or insights in this regard
cheers
Ashok
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