[GenoMax]

I did not pay close attention to your original question (i.e. you are getting the error while running tophat itself and not the actual script). My fault.

Let us start over.

Is caref_ncbiall available in the same directory that you are running tophat from? If not, can you provide the full path to that file when you execute tophat. The same should apply to cp04.fastq and caref_seq.gff. Also make certain that your gff file matches the format specification as noted here: http://cufflinks.cbcb.umd.edu/gff.html

[varshacp]

Hi GenoMAx

All the files are in the same directory. I also made sure that the names are same in gff and genome file.


Thank you

Varsha

[varshacp]

Hi

I forgot to mention that the fastq file is also in same directory. The gff file and genome sequences were downloaded from NCBI (NCBI has separate fasta file for each chromosomes and all the unplaced scafolds are in one fasta file. I concatenated these files to make the genome file and renamed it as per the gff file. The same gff file is working with other genome sequence file which does not have unplaced sequences.


Thank you

Varsha

[GenoMax]

TopHat is picky about the order of options on the command line. Can you try the following:

$ tophat -o cp04_thout5 -p 2 -G caref_seq.gff caref_ncbiall cp04.fastq

Let me also verify that the basename for your genome index files is "caref_ncbiall", that is there are several files (that comprise of the index) that have that prefix?

[GenoMax]

Hi

I forgot to mention that the fastq file is also in same directory. The gff file and genome sequences were downloaded from NCBI (NCBI has separate fasta file for each chromosomes and all the unplaced scafolds are in one fasta file. I concatenated these files to make the genome file and renamed it as per the gff file. The same gff file is working with other genome sequence file which does not have unplaced sequences.


Thank you

Varsha

Does it mean that you have not created the "index" files for this combined fasta reference file? You will need to index the reference in order to use tophat. You can build the reference index using the directions here: http://tophat.cbcb.umd.edu/tutorial.shtml#ref

[varshacp]

HI GenoMAx

The genome index was created using the same fasta file and is in the same directory

Thankx

Varsha

[GenoMax]

Can you post a listing of the files in this directory?

Also see my previous post about the order of options. If the genome index is correctly created then give that command line a try.

[varshacp]

TopHat is picky about the order of options on the command line. Can you try the following:

$ tophat -o cp04_thout5 -p 2 -G caref_seq.gff caref_ncbiall cp04.fastq

Let me also verify that the basename for your genome index files is "caref_ncbiall", that is there are several files (that comprise of the index) that have that prefix?

Hi

The basename is caref_ncbiall for the index files

[GenoMax]

Were you able to get tophat working?

[varshacp]

Hi Genomax

The index is also build using the same genome sequence file in the same directory

[GenoMax]

Are things working now? Or are you still seeing an error?

[varshacp]

HI GenoMax

I am still getting the same error.


Thank you

[GenoMax]

Varsha: Without seeing a listing of the files (related to this error, e.g. caref_ncbiall) in the directory you are running this from there is not much further help I can offer.

[varshacp]

Hi GenoMax

The following is the list of file in the directory from which I am running the tophat command

caref_ncbiall.fa (genome sequence file)
caref_ncbiall.1.bt2 (bowtie index files)
caref_ncbiall.2.bt2
caref_ncbiall.3.bt2
caref_ncbiall.4.bt2
caref_ncbiall.rev.1.bt2
caref_ncbiall.rev.2.bt2
cp04.fastq (reads files)
caref_seq.gff (genome annotation file)

Thank you

Kind regards


Varsha

[varshacp]

HI

I checked the log file and besides the run.log which I posted earlier I get the following error in the g2f.log file

terminate called after throwing an instance of 'std:ut_of_range'
what(): basic_string::substr


Help me to understand this

Thank you
Varsha