Bowtie2 index file problem in RNA-Seq mapping

Question

[rekren]

Hello colleagues,

I have .fastq files obtained by RNA-Seq and I'm trying to map them into customly merged and generated miRNA reference which is taken from miRBase.

I am taking these error.

Could not locate a Bowtie index corresponding to basename "db"
Error: Encountered internal Bowtie 2 exception (#1)
Command: /usr/bin/bowtie2-align-s --wrapper basic-0 -q -x db --passthrough -U /home/graug/Desktop/mirna/2_R.mirna.cleaned.fastq 
(ERR): bowtie2-align exited with value 1

To generate index files, I've typed the following code.

bowtie-build mirna_db.fa db

To map RNA-Seq .fastq files into custom miRNA database of mine, following code was used.

bowtie2 -x db -U -q  /mirna/2_R.mirna.cleaned.fastq -S /mirna/2_R.mirna.sam --no-unal

I've checked the db index withbowtie-inspect command and index seems pretty sanity. Couldn't figure out what am I missing ? Can you help me to find it, please.

[Arup Ghosh]

Use bowtie2-build instead of bowtie-build.

[rekren]

I wasn't aware that bowtie2 had different index format. Thanks a lot.

On the other hand, I noticed that the order of command has to be like this;

bowtie2 -x db -S /mirna/2_R.mirna.sam --no-unal -U -q  /mirna/2_R.mirna.cleaned.fastq

It gave another error on the previous order of my command which says;

Warning: Output file '/home/user_name/Desktop/miRNA/2_R1.cleaned.fastq' was specified without -S.  This will not work in future Bowtie 2 versions.  Please use -S instead.
Warning: Could not open read file "-q" for reading; skipping...
Error: No input read files were valid
(ERR): bowtie2-align exited with value 1

Answer 1

[GenoMax]

Did you build the indexes with bowtie-build (which is v.1) instead of bowtie2-build?

Bowtie 2’s .bt2 index format is different from Bowtie 1’s .ebwt format, and they are not compatible with each other.

You should use bowtie (v.1.x) to align miRNA data though this post claims otherwise: A: Mapping miRNA to the Human Genome with Bowtie2.

In any case make sure you use the correct indexes with correct aligner version.