no reads pseudoalign with stranded option in Kallisto

Question

[divya.nandakumar]

I am trying to use Kallisto to get transcript counts. I know my RNA Seq data is stranded. However, when I use either the -fr-stranded or -rf-stranded option in Kallisto, I see that 0 reads get pseudoaligned. However, if I dont choose the strandedness, I get ~ 75% of the reads aligning. These are the commands I have been using

kallisto quant --rf-stranded -i kallistoIdx.idx -o kallisto_aligned_rfStranded R1.gz R2.gz

kallisto quant --fr-stranded -i kallistoIdx.idx -o kallisto_aligned_frStranded R1.gz R2.gz

kallisto quant -i kallistoIdx.idx -o kallisto_aligned R1.gz R2.gz

Am I doing something wrong in the stranded command? I see this is how it is indicated in the kallisto manual. I would really appreciate any help.

I tried with both kallisto/0.43 and the latest version.

[divya.nandakumar]

Thanks! I realized that as I was editing the question. Works well now!

[ATpoint]

Answer 1

[swbarnes2]

The second file should be R2. You gave it R1 twice.