Tophat 1.4 Output (Accepted_Hits.Bam)

Question

[thecuriousbiologist]

I have read the Tophat 1.4 manual, however I do not seem to understand the outputs very well.

I used a reference genome for my alignment with Tophat and I got a file called "accepted_hits.bam"

This file's cigar strings don't contain any soft clips (S). Can someone explain me why this is happening ?

Also, this was for the reference genome. If I wanted to align to a junction database, should I just use the junction reference instead of the genome reference ? Or does Tophat align to junctions automatically ?

Very confused with the documentation.

Answer 1

[Ido Tamir]

• tophat splits the reads, but still requires everything to match. You could have mismatches in the beginning or end, but these would be scored as M in the CIGAR string. You have to look in the MD tag for mismatches. Old tophat versions dont have the optional MD tag. You can generate it with samtools calmd.

• You always need a reference sequence. You can use a junction file or a GTF file in addition to this. Tophat automatically tries to align reads that can not be mapped completely by aligning them in a split fashion, thus discovering junctions. You can turn this off, modify it with parameters or supply your own junctions. But you should also have a junctions.bed file in your output and see some reads with a CIGAR like 20M500N50M.

[thecuriousbiologist]

Can the 500N part of the CIGAR contain soft clips ?

[karl.stamm]

500N is a gap tophat is marking an intron