'bowtie2' is not recognized.

[Coryza]

Hi all,

I'm currently trying to use bowtie2 to map our RNAseq data to the mRNA reference / chromosomes reference. Building indexes went ok, however when trying to use bowtie2 -p 1 -x [path to 6.bt indexes] [fastq file] > [sam file] it says 'bowtie2' is not recognized as an internal or external command, operable program or batch file.

I'm using the cmd on windows. Anyone who can help me out here?

[dpryan]

You might have to type the full PATH to bowtie2. As an aside, it's going to be a lot of hassle to deal with bioinformatics programs on windows (try to borrow someone's Mac).

Also, unless your organism doesn't have much splicing, you'll probably get better results using tophat2.

[Coryza]

Ah it worked now, doing bowtie2-align instead of bowtie2. Anyhow, I was trying to map 60 homo sapiens mRNA sequences against the indexed reference mRNA of the homo sapiens, and 100.00% aligned 0 times.

Is this normal? O_O

[dpryan]

Probably not, but I'd have to see the reads and know more about how they were generated to tell you for sure. Try using the "--local" option to see if that changes things. Also, did you quality/adapter trim your reads at all?

[Coryza]

Reads were generated with Illumina MiSeq 2 paired-end reading. Adapters & Barcodes were trimmed off.

I'm using command bowtie-align -x hg19 -1 [file1] -2 [file2] -S [output] -fr -q

Using extra parameter --local generates 92.50% aligned 0 times.

[dpryan]

You might try quality trimming things to see if that solves the problem. Alternatively, try blasting a few reads and see how they align. It may be that you simply need to change the defaults for bowtie (e.g., score-min).