SEQanswers

Go Back   SEQanswers > Applications Forums > RNA Sequencing



Similar Threads
Thread Thread Starter Forum Replies Last Post
General question about TopHat mhayes Bioinformatics 1 02-03-2012 02:00 PM
question about tophat camelbbs Bioinformatics 7 07-07-2011 06:24 PM
a question about tophat and cufflinks camelbbs Bioinformatics 0 06-27-2011 09:21 AM
Simple Tophat question kwatts59 RNA Sequencing 2 06-02-2011 07:31 PM
One question about Tophat fgh1124 RNA Sequencing 0 12-15-2010 04:42 PM

Reply
 
Thread Tools
Old 03-11-2011, 01:19 AM   #1
lei
Member
 
Location: germany

Join Date: Mar 2010
Posts: 14
Default a question about tophat

I am using tophap with solid single-end data.
the command line is :
tophat --color --quals -p 8 -o $outdir --library-type fr-secondstrand -G $gtffile hg19 $reads $qual

it reports the error message:
Error: you must set the mean inner distance between mates with -r

but -r is only for paired-end data, so I don't know what should I do.
lei is offline   Reply With Quote
Old 03-11-2011, 01:43 AM   #2
seqguy
Junior Member
 
Location: SK

Join Date: Oct 2010
Posts: 8
Default

Dear Lie

You have to rearrange tophat parameters to run it successfully. Tyr the below mentioned command:

./tophat -G <path to .gtf file> --color --quals <path to index folder/index prefix> <path to .csfasta file> <path to .qual file>
seqguy is offline   Reply With Quote
Old 03-13-2011, 10:54 AM   #3
lei
Member
 
Location: germany

Join Date: Mar 2010
Posts: 14
Default

Dear seqguy
Thanks a lot!
I runned the command in your order. It started to run, but reported another error:
IOError: [Errno 2] No such file or directory: '/home/lei/solid/mec1_f3/tmp/left_kept_reads_missing.fq'
But I can find the file left_kept_reads_missing.fq in the tmp directory.
So do you have any idea what's going on this time?
lei is offline   Reply With Quote
Old 03-14-2011, 03:40 AM   #4
seqguy
Junior Member
 
Location: SK

Join Date: Oct 2010
Posts: 8
Default

Could you please let me know the configuration of the system you are using to run this pipeline, the organism genome size and also if you are using root user or not.
seqguy is offline   Reply With Quote
Old 03-14-2011, 04:19 AM   #5
lei
Member
 
Location: germany

Join Date: Mar 2010
Posts: 14
Default

it was running on a Server, the CPU is 24x 2.3 GHz, the Memory is 80 GB, and the OS is suse11.0.
I used the human genome from UCSC.
I'm not the root user.
lei is offline   Reply With Quote
Old 03-14-2011, 04:23 AM   #6
lei
Member
 
Location: germany

Join Date: Mar 2010
Posts: 14
Default

It's strange that this time, I can run it with my previous parameters' order. But still report the same error message: IOError: [Errno 2] No such file or directory: '/home/lei/solid/mec1_f3/tmp/left_kept_reads_missing.fq'
lei is offline   Reply With Quote
Old 03-14-2011, 04:29 AM   #7
seqguy
Junior Member
 
Location: SK

Join Date: Oct 2010
Posts: 8
Default

Have you created the bowtie index of your reference genome with -C option?
seqguy is offline   Reply With Quote
Old 03-14-2011, 04:39 AM   #8
lei
Member
 
Location: germany

Join Date: Mar 2010
Posts: 14
Default

sure, I runned tophat before with illumina data, everything was fine. those error just happened when I use solid data.
lei is offline   Reply With Quote
Old 03-16-2011, 03:03 AM   #9
seqguy
Junior Member
 
Location: SK

Join Date: Oct 2010
Posts: 8
Default

1) Check if your GTF and reference genome files are having same naming convention for chromosomes.
2) Reference genome index should be in colorspace.
3) Check if .csfasta and .qual files are having same counts.
seqguy is offline   Reply With Quote
Old 03-17-2011, 02:22 AM   #10
lei
Member
 
Location: germany

Join Date: Mar 2010
Posts: 14
Default

yes, 1),2),3) are all fine.
1) it's possible that the genome fasta file has "chr1" etc. whereas the GTF file has "1" etc. but I ran cuffdiff from the same author successfully with the same GTF file and the original genome from which the bowtie index was built is the same as the one I used there.
3) the alignments I ran would never have worked if there had been a different number of reads in the csfasta and .qual files.

Maybe I need to pose my question again to make it clear:

The first time I run the tophat using qsub. It reports the error:
Error: you must set the mean inner distance between mates with -r

Then, I rearrange the parameters, test it in a local machine with both parameters' orders mentioned above, get another error:
IOError: [Errno 2] No such file or directory: '/home/lei/solid/mec1_f3/tmp/left_kept_reads_missing.fq'

Now, I test it using qsub again, get the same error again:
Error: you must set the mean inner distance between mates with -r
lei is offline   Reply With Quote
Old 03-19-2011, 12:06 AM   #11
seqguy
Junior Member
 
Location: SK

Join Date: Oct 2010
Posts: 8
Default

could you please share the exact command you are using to run tophat?
seqguy is offline   Reply With Quote
Old 03-20-2011, 12:50 PM   #12
lei
Member
 
Location: germany

Join Date: Mar 2010
Posts: 14
Default

the first script is :

#!/bin/bash

#PBS -N Tophat_lib1
#PBS -l nodes=1pn=8:lsdf
#PBS -l walltime=50:00:00
#PBS -l mem=5g
#PBS -M l.gu@gmail.com
#PBS -m ae
#PBS -j eo
#PBS -e /home/gu/solid/lib1/error

# environment variable for the indexes
BOWTIE_INDEXES=/home/gu/solid/bowtieIndex/
export BOWTIE_INDEXES
reads=/home/gu/solid/lib1/138000338_20101201_FRAG_BC_SEQ0014_bcSample1_lib1_F3.csfasta
qual=/home/gu/solid/lib1/138000338_20101201_FRAG_BC_SEQ0014_bcSample1_lib1_F3_QV.qual
outdir= /home/gu/solid/lib1
gtffile=/ibios/co01/hutter/PlassData/Ensembl57_transcripts_hg19.gtf

tophat -G $gtffile --color --quals -p 8 -o $outdir --library-type fr-secondstrand hg19 $reads $qual

the error code is :Error: you must set the mean inner distance between mates with -r
lei is offline   Reply With Quote
Old 03-20-2011, 12:53 PM   #13
lei
Member
 
Location: germany

Join Date: Mar 2010
Posts: 14
Default

the econd script is :

#!/bin/bash

#PBS -N Tophat_lib1
#PBS -l nodes=1pn=8:lsdf
#PBS -l walltime=50:00:00
#PBS -l mem=5g
#PBS -M l.gu@gmail.com
#PBS -m ae
#PBS -j eo
#PBS -e /home/gu/solid/lib1/error

# environment variable for the indexes
BOWTIE_INDEXES=/home/gu/solid/bowtieIndex/
export BOWTIE_INDEXES
#reads=/home/gu/solid/lib1/138000338_20101201_FRAG_BC_SEQ0014_bcSample1_lib1_F3.csfasta
#qual=/home/gu/solid/lib1/138000338_20101201_FRAG_BC_SEQ0014_bcSample1_lib1_F3_QV.qual
#outdir= /home/gu/solid/lib1
#gtffile=/ibios/co01/hutter/PlassData/Ensembl57_transcripts_hg19.gtf

tophat --color --quals -p 8 -o /home/gu/solid/lib1 --library-type fr-secondstrand -G /ibios/co01/hutter/PlassData/Ensembl57_transcripts_hg19.gtf /icgc/lsdf/mb/analysis/hutter/bowtieIndex/hg19 /home/gu/solid/lib1/138000338_20101201_FRAG_BC_SEQ0014_bcSample1_lib1_F3.csfasta /home/gu/solid/lib1/138000338_20101201_FRAG_BC_SEQ0014_bcSample1_lib1_F3_QV.qual

the error code is :
[Thu Mar 17 16:12:28 2011] Beginning TopHat run (v1.2.0)
-----------------------------------------------
[Thu Mar 17 16:12:28 2011] Preparing output location /home/gu/solid/lib1/
[Thu Mar 17 16:12:28 2011] Checking for Bowtie index files
[Thu Mar 17 16:12:28 2011] Checking for reference FASTA file
[Thu Mar 17 16:12:28 2011] Checking for Bowtie
Bowtie version: 0.12.7.0
[Thu Mar 17 16:12:28 2011] Checking for Samtools
Samtools Version: 0.1.12a
[Thu Mar 17 16:12:28 2011] Checking reads
min read length: 50bp, max read length: 50bp
format: fasta
[Thu Mar 17 16:14:07 2011] Reading known junctions from GTF file
[Thu Mar 17 16:18:51 2011] Mapping reads against hg19 with Bowtie
[Thu Mar 17 16:18:51 2011] Joining segment hits
Traceback (most recent call last):
File "/ibios/tbi_cluster/11.0/x86_64/bin/tophat", line 2346, in <module>
sys.exit(main())
File "/ibios/tbi_cluster/11.0/x86_64/bin/tophat", line 2304, in main
user_supplied_deletions)
File "/ibios/tbi_cluster/11.0/x86_64/bin/tophat", line 1972, in spliced_alignment
segment_len)
File "/ibios/tbi_cluster/11.0/x86_64/bin/tophat", line 1545, in split_reads
reads_file = open(reads_filename)
IOError: [Errno 2] No such file or directory: '/home/gu/solid/lib1/tmp/left_kept_reads_missing.fq'
lei is offline   Reply With Quote
Reply

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 05:57 PM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2020, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO