Error: Could not find Bowtie 2 index files (Reference.fas.*.bt2l)

Question

[michaela_boell]

My tophat2 seems to look for something it does not need.

I have a fasta file of my very small reference genome which is just a small plasmid (~800bp). Of this reference genome I created a bowtie index with the following command:

bowtie2-build -f pJF686.fas pJF686.fas.index

Which nicely results in the expected 6 index files. I have two fasta files (in .txt) of paired-end reads that were the returns from an ilumina sequencing of a plant genome that has fragments of the plasmid supposedly existing at some sites in it. Then I want to align my reads to my reference with the following command:

tophat2 pJF686.fas C49E6ACXX_EP_17_14s002318-1-1_Ibberson_lane814s002318_1_sequence.txt, C49E6ACXX_EP_17_14s002318-1-1_Ibberson_lane814s002318_2_sequence.txt

This returns the following:

[2016-07-28 11:10:38] Beginning TopHat run (v2.1.1)
-----------------------------------------------
[2016-07-28 11:10:38] Checking for Bowtie
          Bowtie version:    2.2.9.0
[2016-07-28 11:10:38] Checking for Bowtie index files (genome)..
Error: Could not find Bowtie 2 index files (pJF686.fas.*.bt2l)

I looked up what a .bt2l file is and found out, that it merely labels bowtie indeces that are large. Since my plasmid is small, bowtie does not make a .bt2l file but a .bt2 file. This .bt2 should be fine. So I guess the error is unspecific.

What could be the problem?

Answer 1

[GenoMax]

You can't use a comma to separate the file names that you are providing to TopHat when you are using only two files (if the above command line is correct).

If you used pJF686.fas.index (i.e. your index files are pJF686.fas.index.b*) as the basename for the index then you need to use that same name with TopHat command.

[michaela_boell]

Aha! the basename was the problem. The comma was incorrect, too. Thank you.