Stranded or unstranded? That is the question

Question

[juliayork]

Hey all, I'm hoping I can get someone to clarify for me if my RNASeq data is stranded or unstranded. I assembled a transcriptome de novo and mapped the reads back with bowtie2, and then did quantification using salmon. Salmon is helpful in that it give me some information about the library type in a .json file:

"expected_format": "IU",
"compatible_fragment_ratio": 1.0,
"num_compatible_fragments": 20834392,
"num_assigned_fragments": 20834392,
"num_frags_with_concordant_consistent_mappings": 33966029,
"num_frags_with_inconsistent_or_orphan_mappings": 189540,
"strand_mapping_bias": 0.47332512729115319,
"MSF": 0,
"OSF": 0,
"ISF": 17889054,
"MSR": 0,
"OSR": 0,
"ISR": 16076975,
"SF": 96618,
"SR": 92922,
"MU": 0,
"OU": 0,
"IU": 0,
"U": 0

What is confusing me is that the strand mapping bias near 0.5 should mean it is unstranded according to this google groups. However, all the reads are counted in ISF/SF or ISR/SR, which are the stranded categories. See here for the definitions of the library types..

Please help!

Answer 1

[Rob]

Answer taken from here.

It is unstranded in this case. A specific read always has a stranded orientation (it maps to one strand or the other). The strandedness of the library is a property of the collection of all reads. Here, approximately half map in each orientation, so the library is unstranded, since there is not a strong, systematic bias for one orientation over the other.

Best, Rob