Go Back   SEQanswers > Bioinformatics > Bioinformatics

Similar Threads
Thread Thread Starter Forum Replies Last Post
tophat with/without annotation,and cufflink with annotation? louis7781x Bioinformatics 19 04-05-2013 08:09 AM
Problem with agreement between cow annotation/build is screwing up my cufflinks run mmcgo002 Bioinformatics 1 12-01-2011 08:06 PM
Run cufflinks with or without annotation? reut Bioinformatics 7 06-01-2011 02:01 PM
prep_reads file in Tophat run shows a different number of reads gen2prot Bioinformatics 7 03-24-2011 02:50 AM
Split accepted_hits.bam file after Tophat run? hong_sunwoo Bioinformatics 6 10-18-2010 01:06 AM

Thread Tools
Old 09-05-2011, 12:04 AM   #1
Location: Beijing, China

Join Date: Oct 2010
Posts: 30
Default How to run Tophat with annotation file

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?
masylichu is offline   Reply With Quote
Old 09-06-2011, 12:26 AM   #2
Simon Andrews
Location: Babraham Inst, Cambridge, UK

Join Date: May 2009
Posts: 871

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
simonandrews is offline   Reply With Quote
Old 09-06-2011, 08:25 PM   #3
Location: Beijing, China

Join Date: Oct 2010
Posts: 30

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:
[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
Error: Splice sequence indexing failed with err =1
masylichu is offline   Reply With Quote

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off

All times are GMT -8. The time now is 05:55 PM.

Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2021, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO