Tophat Error

[kregan1]

Hi!

I am trying to run tophat, but I keep getting this very general error and I am not sure why.


[2014-05-27 09:46:02] Beginning TopHat run (v2.0.11)
-----------------------------------------------
[2014-05-27 09:46:02] Checking for Bowtie
Bowtie version: 2.2.2.0
[2014-05-27 09:46:02] Checking for Samtools
Samtools version: 0.1.19.0
[2014-05-27 09:46:02] Checking for Bowtie index files (genome)..
[2014-05-27 09:46:02] Checking for reference FASTA file
[2014-05-27 09:46:02] Generating SAM header for /afs/crc.nd.edu/user/k/kregan1/Daphnia/genome/Daphnia_pulex.main_genome.scaffolds.fasta
Error: Opening file /afs/crc.nd.edu/user/k/kregan1/Daphnia/genome

The file that it is trying to open is actually a directory that holds my reference genome and the bowtie index files. Have any of you seen this error and been able to fix it? Thanks so much!

[blancha]

You probably made a mistake in your command line.
You're supposed to provide TopHat2 with the "basename of the genome index to be searched."
With your input, TopHat2 thinks that "/afs/crc.nd.edu/user/k/kregan1/Daphnia/genome/Daphnia_pulex.main_genome.scaffolds.fasta" is the basename of the genome index, which is clearly not the case.
If you cannot figure out the correct command, you should post the full command you used here.

The other possibility is that TopHat2 does not like that you named your folder genome, but I don't think that is the case. It's more likely that you did not specify the basename of the genome index correctly, or that you did not organize the directory correctly.

---

Here is an example of a correct TopHat2 command, for stranded RNA-Seq data.

tophat \
--no-novel-juncs \
--library-type fr-firststrand \
-G /sb/project/afb-431/genomes//Mus_musculus/Ensembl/NCBIM37/Annotation/Genes/genes.gtf \
-o ../../tophat/Hp_SHAM_C1_002 \
/sb/project/afb-431/genomes//Mus_musculus/Ensembl/NCBIM37/Sequence/Bowtie2Index/genome \
../../../FASTQ/trimmed/HI.1596.002.Index_5.VM_Hp-SHAM-C1_R1.fastq.gz \
../../../FASTQ/trimmed/HI.1596.002.Index_5.VM_Hp-SHAM-C1_R2.fastq.gz

---

Here is the start of the log file.

[2014-04-12 10:18:25] Beginning TopHat run (v2.0.10)
-----------------------------------------------
[2014-04-12 10:18:25] Checking for Bowtie
Bowtie version: 2.1.0.0
[2014-04-12 10:18:29] Checking for Samtools
Samtools version: 0.1.19.0
[2014-04-12 10:18:29] Checking for Bowtie index files (genome)..
[2014-04-12 10:18:29] Checking for reference FASTA file
[2014-04-12 10:18:29] Generating SAM header for /sb/project/afb-431/genomes//Mus_musculus/Ensembl/NCBIM37/Sequence/Bowtie2Index/genome
[2014-04-12 10:24:54] Reading known junctions from GTF file

---

Here is the content of the folder Bowtie2Index.

[blancha@lg-1r17-n04 tophat]$ ls /sb/project/afb-431/genomes//Mus_musculus/Ensembl/NCBIM37/Sequence/Bowtie2Index/
genome.1.bt2 genome.2.bt2 genome.3.bt2 genome.4.bt2 genome.fa genome.rev.1.bt2 genome.rev.2.bt2