I am utilizing Kallisto in Anaconda/miniconda for RNA sequencing. I have successfully made an index; using said index to analyze RNA sequencing data has yielded an error of:

"Error: index input file could not be opened!"

The implication is that the code used does in fact work but raises the question as to why the index file could not be opened. I have tried searching this error to see if others may have faced the same error message, but unfortunately no luck. Any recommendations and or solutions would be greatly appreciated. The code and output can be found below.

kallisto quant -i Homo_sapiens.GRCh38.cdna.all -o test -t 8 --single -l 250 -s 30 SRR8668755_1M_subsample.fastq

[quant] fragment length distribution is truncated gaussian with mean = 250, sd = 30

Error: index input file could not be opened!


1 Answer 1


Usually errors like these occur when an input file doesn't exist or is corrupt. But you might also see these errors if you don't have permissions to access the file for whatever reason. Although unlikely, these can also occur if/when your system is out of memory.

Assuming the index is corrupt, I think just retry the command. For example:

curl -O ftp://ftp.ensembl.org/pub/release-108/fasta/homo_sapiens/cdna/Homo_sapiens.GRCh38.cdna.all.fa.gz

kallisto index \
    -i Homo_sapiens.GRCh38.cdna.all.release-108_k31.idx \

And test:

kallisto quant \
    -i Homo_sapiens.GRCh38.cdna.all.release-108_k31.idx \
    -o test \
    -t 8 \
    --single \
    -l 250 \
    -s 30 \

To get this working using Windows/Anaconda Powershell, I just downloaded the cDNA sequences from https://ftp.ensembl.org/pub/release-108/fasta/homo_sapiens/cdna/ and moved them to my working directory. I also downloaded and extracted the Windows Kallisto executable v0.46.1 from https://pachterlab.github.io/kallisto/download.html. Then, with the FASTQ file in my working directory:

 .\kallisto_windows-v0.46.1\kallisto\kallisto.exe index `
    -i Homo_sapiens.GRCh38.cdna.all.release-108_k31.idx `

And tested with:

.\kallisto_windows-v0.46.1\kallisto\kallisto.exe quant `
    -i .\Homo_sapiens.GRCh38.cdna.all.release-108_k31.idx `
    -o test `
    -t 8 `
    --single `
    -l 250 `
    -s 30 `

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