strand specificity

Question

[aimanbarki]

Hello,

I have downloaded some RNAseq data from arrayexpress. I want to use salmon and/or Hisat2. I am confused about what is the strand specificity and how to select the strand specificity parameter for hisat2 or salmon (e.g Salmon options.) . Can some one please explain it either is forward or reverse and how can i figure that out.

Preparation protocol is as follows: RNA was extracted using BioRad Aurum Total RNA Fatty and Fibrous Tissue Kit (cat no. 732-6830) following manufacturers instructions. Illumina compatible barcoded mRNA sequencing libraries were made from 100 ng of total RNA after polyA selection for mature mRNAs using the TruSeq Stranded mRNA Sample Preparation Kit’s high sample protocol (Illumina). Sequencing was performed on a single HiSeq 3000 150 bp paired end lane with the data exported as a pair of fastq formatted data files for each sample. Sample quality was assessed prior to sequencing.

[GenoMax]

Illumina TruSeq kit should be reverse stranded: https://github.com/igordot/genomics/blob/master/notes/rna-seq-strand.md

[ATpoint]

Most common stranded Illumina kits have ISR as option for salmon but as @Rob (the salmon author by the way) says, you can tell salmon to infer it.

Answer 1

[Rob]

If you pass the "automatic" library type -l A to salmon, it will attempt to infer the strand specificity itself by looking for biases in the mapping direction. Also, the resulting quantification directory will contain a file specifying how many mappings were observed in each possible orientation.