I need to create the Tophat reference index of the human genome. I therefore downloaded the GTF file (humRef.gtf) from Ensembl and used Bowtie to create an index of the human genome with a single fasta file (dna.toplevel) with all human chromsomes, which I renamed humRef.fa, as follows:
bowtie2-build -f ./humRef.fa humRef
this created the following files: humRef.1.bt2, humRef.2.bt2, humRef.3.bt2, humRef.4.bt2, humRef.rev.1.bt2 and humRef.rev.2.bt2.
I ran tophat with the following command:
tophat2 -G humRef.gtf --transcriptome-index=humRef_tr humRef
but I got the following error:
[2016-03-30 20:05:55] Building transcriptome files with TopHat v2.1.1 ----------------------------------------------- [2016-03-30 20:05:55] Checking for Bowtie Bowtie version: 18.104.22.168 [2016-03-30 20:06:44] Checking for Bowtie index files (genome).. [2016-03-30 20:06:44] Checking for reference FASTA file [2016-03-30 20:06:44] Building transcriptome data files humRef_tr/humRef [2016-03-30 20:09:02] Building Bowtie index from humRef.fa [FAILED] Error: Couldn't build bowtie index with err = 1
Could you please tell me what is wrong? It must be a syntax issue but I don't know which one. I tried to provide paths to the files but nothing, and also changing the names of the index files. All files are in the same directory.
I also tried to remove the .fa extension of humRef.fa file, following a suggestion from internet, but the answer was:
[2016-03-30 20:30:49] Checking for reference FASTA file Warning: Could not find FASTA file humRef.fa [2016-03-30 20:30:49] Reconstituting reference FASTA file from Bowtie index
a solution that I don't think is efficient since the file is available