Question: Bowtie 2 index files exist but return Error: Could not find Bowtie 2 index files (humanGRCh38.*.bt2l)
gravatar for skurkure
15 months ago by
skurkure0 wrote:

As I have stated above, I have received an error in running tophat, with the output below. The command I supplied was tophat humanGRCh38 gerald_C2DBEACXX_3.fq,gerald_C1TD1ACXX_8_ACAGTG.fq , as I was running tophat from the directory in which the bt2l files were installed. I have supplied the prefix as well, but it did not work. I have gotten the same error when I supply the entire present working directory and the prefix, I got the same problem. Anyone else run into this, and if yes how did you fix it? I know that the version of bowtie that I am using is correct because the files are in the correct format.

$ ls

gerald_C1TD1ACXX_8_ACAGTG.bam  Homo_sapiens.GRCh38.96.gtf     humanGRCh38.3.bt2l

gerald_C1TD1ACXX_8_ACAGTG.fq   Homo_sapiens.GRCh38.dna.toplevel.fa  humanGRCh38.4.bt2l

gerald_C2DBEACXX_3.bam  humanGRCh38.1.bt2l  tophat_out gerald_C2DBEACXX_3.fq  humanGRCh38.2.bt2l

$ tophat humanGRCh38 gerald_C2DBEACXX_3.fq,gerald_C1TD1ACXX_8_ACAGTG.fq

[2019-07-02 08:34:27] Beginning TopHat run (v2.1.1)


[2019-07-02 08:34:27] Checking for Bowtie

  Bowtie version:

[2019-07-02 08:34:27] Checking for Bowtie index files (genome)..

Error: Could not find Bowtie 2 index files (humanGRCh38.*.bt2l)

index rna-seq tophat bowtie2 • 672 views
ADD COMMENTlink modified 13 months ago by Biostar ♦♦ 20 • written 15 months ago by skurkure0

Did you make this index yourself? Has it been tested before this?

What do you get if you do:

bowtie2-inspect -n humanGRCh38

You should see the names of chromosomes being listed like this (truncated, may not look exactly like this, we are testing to make sure you don't get an error).

chr1  AC:CM000663.2  gi:568336023  LN:248956422  rl:Chromosome  M5:6aef897c3d6ff0c78aff06ac189178dd  AS:GRCh38
chr2  AC:CM000664.2  gi:568336022  LN:242193529  rl:Chromosome  M5:f98db672eb0993dcfdabafe2a882905c  AS:GRCh38
chr3  AC:CM000665.2  gi:568336021  LN:198295559  rl:Chromosome  M5:76635a41ea913a405ded820447d067b0  AS:GRCh38
chr4  AC:CM000666.2  gi:568336020  LN:190214555  rl:Chromosome  M5:3210fecf1eb92d5489da4346b3fddc6e  AS:GRCh38
ADD REPLYlink modified 15 months ago • written 15 months ago by genomax91k

No, I did not build this index myself. I used a pre-built index from

When I do bowtie2-inspect -n humanGRCh38 I get an error

$ bowtie2-inspect -n humanGRCh38
bowtie2-inspect-l: word_io.h:125: T readU(FILE*, bool) [with T = long unsigned int; FILE = _IO_FILE]: Assertion `false' failed.
ADD REPLYlink modified 15 months ago by genomax91k • written 15 months ago by skurkure0

I see the following files in the tar archive you linked above. So it looks like you did not get the full set of files or uncompressing went wrong somewhere.

$ tar -tvf GCA_000001405.15_GRCh38_no_alt_analysis_set.fna.bowtie_index.tar.gz 
-rw-r--r-- kitts/gassmbly 982525150 2014-11-18 17:09 GCA_000001405.15_GRCh38_no_alt_analysis_set.fna.bowtie_index.1.bt2
-rw-r--r-- kitts/gassmbly 733719120 2014-11-18 17:09 GCA_000001405.15_GRCh38_no_alt_analysis_set.fna.bowtie_index.2.bt2
-rw-r--r-- kitts/gassmbly     10880 2014-11-18 16:01 GCA_000001405.15_GRCh38_no_alt_analysis_set.fna.bowtie_index.3.bt2
-rw-r--r-- kitts/gassmbly 733719113 2014-11-18 16:01 GCA_000001405.15_GRCh38_no_alt_analysis_set.fna.bowtie_index.4.bt2
-rw-r--r-- kitts/gassmbly 982525150 2014-11-18 18:16 GCA_000001405.15_GRCh38_no_alt_analysis_set.fna.bowtie_index.rev.1.bt2
-rw-r--r-- kitts/gassmbly 733719120 2014-11-18 18:16 GCA_000001405.15_GRCh38_no_alt_analysis_set.fna.bowtie_index.rev.2.bt2
ADD REPLYlink modified 15 months ago • written 15 months ago by genomax91k

Unless absolutely essential you may want consider using a current RNAseq splice aware aligner like STAR, bbmap instead of TopHat.

ADD REPLYlink written 15 months ago by genomax91k
Please log in to add an answer.


Use of this site constitutes acceptance of our User Agreement and Privacy Policy.
Powered by Biostar version 2.3.0
Traffic: 999 users visited in the last hour