Question: Unable to find Bowtie2 index

Question

[mra8187]

hello i indexed genome by bowtie2 and then use them to tophat2 but still give error .....

tophat2 -p 4 -r 200 --mate-std-dev 50 -G '/media/mra/4A3B44B64CA65A93/propooooooosssssssssal SNP/DATA/Genom/hashie nevisi/Apis_mellifera.GCA_000002195.1.32.gtf' -o '/media/mra/4A3B44B64CA65A93/propooooooosssssssssal SNP/DATA/drone larvae/SRR1571716/Tophat/tophat_out_SRR1571716' ref '/media/mra/4A3B44B64CA65A93/propooooooosssssssssal SNP/DATA/Genom/refrense genome/Bowtie2/indexed-refrence-genome.fa' '/media/mra/4A3B44B64CA65A93/propooooooosssssssssal SNP/DATA/drone larvae/SRR1571716/Trimmomatic/80/SRR1571716-1.trimmed.fastq.gz' '/media/mra/4A3B44B64CA65A93/propooooooosssssssssal SNP/DATA/drone larvae/SRR1571716/Trimmomatic/80/SRR1571716-2.trimmed.fastq.gz' 

[2016-10-31 02:16:16] Beginning TopHat run (v2.0.9)
-----------------------------------------------
[2016-10-31 02:16:16] Checking for Bowtie
          Bowtie version:    2.1.0.0
[2016-10-31 02:16:16] Checking for Samtools
        Samtools version:    0.1.19.0
[2016-10-31 02:16:16] Checking for Bowtie index files (genome)..
Error: Could not find Bowtie 2 index files (ref.*.bt2)

[GenoMax]

Please don't use spaces in file/directory names. That can only lead to trouble with TopHat. I suggest that you replace them with "_" so you can remove the single quotes you have had to use everywhere.

You are also using a 2+ year old version of TopHat which you should try and upgrade to the latest.

Answer 1

[WouterDeCoster]

As specified in the manual:

The basename of the genome index to be searched. The basename is the name of any of the index files up to but not including the first period. Bowtie first looks in the current directory for the index files, then looks in the indexes subdirectory under the directory where the currently-running bowtie executable is located, then looks in the directory specified in the BOWTIE_INDEXES (or BOWTIE2_INDEXES) environment variable. Please note that it is highly recommended that a FASTA file with the sequence(s) the genome being indexed be present in the same directory with the Bowtie index files and having the name <genome_index_base>.fa. If not present, TopHat will automatically rebuild this FASTA file from the Bowtie index files.

Your command is quite hard to read because of the absurd paths you use but if I'm not mistaken you have '/media/mra/4A3B44B64CA65A93/propooooooosssssssssal SNP/DATA/Genom/refrense genome/Bowtie2/indexed-refrence-genome.fa which is a fasta file and as such not how you should provide the index

Answer 2

[mra8187]

thank you for answer... i am provide the index by using :

 bowtie2-build /media/mra/4A3B44B64CA65A93/SNP/DATA/Genom/refrense_genome/Bowtie2/refrence.fa indexed-refrence

and it give me six files with .bt2 format ... i do it for several time but tophat not recognize my indexed refrence genome i using now tophat v2.1.1 and Bowtie version 2.2.8.0... but still give me same error .........................................................................................

 tophat2 -p 4 -r 200 --mate-std-dev 50  /media/mra/4A3B44B64CA65A93/SNP/DATA/Genom/refrense_genome/Bowtie2/refrence.fa /media/mra/4A3B44B64CA65A93/SNP/DATA/drone larvae/SRR1571716/Trimmomatic/80/SRR1571716-1.trimmed.fastq.gz /media/mra/4A3B44B64CA65A93/SNP/DATA/drone larvae/SRR1571716/Trimmomatic/80/SRR1571716-2.trimmed.fastq.gz 

[2016-11-03 17:05:27] Beginning TopHat run (v2.1.1)
-----------------------------------------------
[2016-11-03 17:05:27] Checking for Bowtie
          Bowtie version:    2.2.8.0
[2016-11-03 17:05:27] Checking for Bowtie index files (genome)..
Error: Could not find Bowtie 2 index files (/media/mra/4A3B44B64CA65A93/SNP/DATA/Genom/refrense_genome/Bowtie2/refrence.fa.*.bt2l)

[GenoMax]

In this case your "basename" for the index appears to be indexed-refrence since you set it that way. So run TopHat with

tophat2 -p 4 -r 200 --mate-std-dev 50  /media/mra/4A3B44B64CA65A93/SNP/DATA/Genom/refrense_genome/Bowtie2/indexed-refrence larvae/SRR1571716/Trimmomatic/80/SRR1571716-1.trimmed.fastq.gz /media/mra/4A3B44B64CA65A93/SNP/DATA/drone larvae/SRR1571716/Trimmomatic/80/SRR1571716-2.trimmed.fastq.gz

What is this /media/mra/4A3B44B64CA65A93/SNP/DATA/drone in the original command?

[mra8187]

finally its done ... thank you ... you are great..... ......

[mra8187]

i am working on honey bee RNA-seq drone = male bee

[GenoMax]

TopHat does not like having an extraneous bee in bonnet :)

[WouterDeCoster]

Right, good catch :P

[WouterDeCoster]

Please use ADD COMMENT to reply to earlier answers, as such this thread remains logically structured and easy to follow.

As I wrote before, you don't have to use the fasta file when you specify the index for tophat2. If I'm not mistaken, in your case you should specify /media/mra/4A3B44B64CA65A93/SNP/DATA/Genom/refrense_genome/Bowtie2/refrence as index.

The basename is the name of any of the index files up to but not including the first period.