Hi
I have some RNA-Seq data that I received from a colleague. I am trying to figure out if the data is unstranded, firststrand, or secondstrand. Is there an easy way to tell? I am running the data through salmon and this is what the lib_format_counts.json file is giving me. I think it is secondstranded?
read_files "Sample1"
expected_format "SR"
compatible_fragment_ratio 0.9945605200682789
num_compatible_fragments 18794420
num_assigned_fragments 18897211
num_consistent_mappings 56858584
num_inconsistent_mappings 328289
MSF 0
OSF 0
ISF 0
MSR 0
OSR 0
ISR 0
SF 328289
SR 56858584
MU 0
OU 0
IU 0
U 0
Any ideas?
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williamsbrian5064
Yes, this appears to be the standard dUTP/secondstrand/ISR library. As an aside, this is the most likely library prep type these days.
Awesome! So the sequencing data is single end. Would this then be considered SR instead ISR?
Yes, SR rather than ISR.
Thanks you so much!
If you have no clue, take a look at RSeQC ( infer_experiment.py )
This program is used to “guess” how RNA-seq sequencing were configured, particulary how reads were stranded for strand-specific RNA-seq data, through comparing the “strandness of reads” with the “standness of transcripts”.
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Maybe ask the colleague?:)
What fun is that!
haha I would be they haven't been the easiest to get in contact with. You know how it goes.
Sometimes colleagues forget what they did or remember incorrectly. The data, on the other hand, doesn't lie.
There are some excellent answers posted already, but just in case you want to learn more about strandness, check this previous post: Read pair orientation : Illumina TruSeq Stranded mRNA library
Great link! Very helpful info! I appreciate it!