Hi,

I am new to bioinformatics and am trying to perform differential expression analyses on some mouse RNA-seq data. We performed Tru-Seq Strand Specific Large Insert RNA Sequencing - High Coverage (50M pairs) on the sample. I am now trying to pseudo-align the reads to the mouse transcriptome using Kallisto. I ran bam2fq to obtain two fasts files, and also generated a mouse reference transcriptome index from both Ensemble( Mus_musculus.GRCm38.cdna.all.fa) and UCSC Genome Browser (refMrna.fa.gz).

I ran kallisto using the following command: kallisto quant -i index -o output pairA1.fastq pairA2.fastq For all the samples, the resulting run_info.json output looks similar to the example below:

"ntargets": 42184, "nbootstraps": 0, "nprocessed": 73044298, "npseudoaligned": 33281349, "nunique": 19777682, "ppseudoaligned": 45.6, "punique": 27.1, "kallistoversion": "0.45.0", "index_version": 10,

I would really appreciate any help in troubleshooting this issue. Is it an issue with the data quality, or should I be running Kallisto with additional arguments (strand specific, etc.)

Thank you very much for your help and please let me know if I can provide any additional information.