How to run Tophat with annotation file

[masylichu]

Hi, i want to map the single-end short reads to the genome.

first, i map the reads to the genome with bowtie.

second, for the unaligned reads, i want to map it across the annotation file. i download the GTF file from UCSC.

tophat -G ~/GFF3/ce6-sangerGene.gtf /ws190_index/C.elegans.ws190.dna unaligned.txt
but failed.

could anyone help to solve this problem?

[simonandrews]

Well the basic options you've set look OK. You're going to need to show us the errors you got so we can see what failed.

One thing to note is that tophat is very picky about the exact formatting of the GTF files it will accept. I've tried a few GTF files from Ensembl and about half of them required some manual editing before tophat would accept them. You can test your GTF file by running the gtf_juncs program which comes with tophat. You simply run:

gtf_juncs [your gtf file]

You may see some (or possibly lots of) warnings, but if you then see output like:

X 54671454 54675019 +
X 54675146 54676441 +
X 54676552 54686385 +
X 54686464 54689586 +
X 54689694 54690560 +

..then your GTF file is OK. If you get an error like:

Error: duplicate GFF ID 'ENSMUST00000127664' (or exons too far apart)!

Then your GTF file won't process and tophat will abort if you try to use it (but I don't think it shows you the error you'll see from running gtf_juncs).

Hope this helps

[masylichu]

This is the output of Tophat.

[Wed Sep 7 10:52:44 2011] Beginning TopHat run (v1.3.1)
-----------------------------------------------
[Wed Sep 7 10:52:44 2011] Preparing output location ./tophat_out/
[Wed Sep 7 10:52:44 2011] Checking for Bowtie index files
[Wed Sep 7 10:52:44 2011] Checking for reference FASTA file
[Wed Sep 7 10:52:44 2011] Checking for Bowtie
Bowtie version: 0.12.7.0
[Wed Sep 7 10:52:44 2011] Checking for Samtools
Samtools Version: 0.1.8
[Wed Sep 7 10:52:44 2011] Generating SAM header for /home/wgf/bowtie/ws190_index/C.elegans.ws190.dna
[Wed Sep 7 10:52:45 2011] Preparing reads
format: fasta
[Wed Sep 7 10:52:45 2011] Reading known junctions from GTF file
Left reads: min. length=33, count=2951216
Warning: you have only one segment per read
we strongly recommend that you decrease --segment-length to about half the read length because TopHat will work better with multiple segments
[Wed Sep 7 10:53:46 2011] Mapping left_kept_reads against C.elegans.ws190.dna with Bowtie
[Wed Sep 7 11:08:01 2011] Processing bowtie hits
[Wed Sep 7 11:08:35 2011] Retrieving sequences for splices
[Wed Sep 7 11:08:57 2011] Indexing splices
Warning: Empty input file
Error: No unambiguous stretches of characters in the input. Aborting...
Command: /home/wgf/bin/bowtie-build ./tophat_out/tmp/segment_juncs.fa ./tophat_out/tmp/segment_juncs
[FAILED]
Error: Splice sequence indexing failed with err =1