Salmon lib_format_counts.json - explanation

Question

[Lada]

Hi guys,

Here's an RNAseq-related question. I have RF library, I made de novo transcriptome assembly in Trinity with basic parameters and a library flag set to RF. I ran Salmon and got a Mapping rate = 92.5671%. I wanted to check json file and what confused me was the "strand_mapping_bias": 0.0,. Is that ok? I expected it to be 1,0 since I have a stranded library and transcriptome assembly, but this is just my logic.

"expected_format": "ISR",
"compatible_fragment_ratio": 1.0,
"num_compatible_fragments": 207752880,
"num_assigned_fragments": 207752880,
"num_frags_with_concordant_consistent_mappings": 196914626,
"num_frags_with_inconsistent_or_orphan_mappings": 11528807,
"strand_mapping_bias": 0.0,
"MSF": 0,
"OSF": 0,
"ISF": 0,
"MSR": 0,
"OSR": 0,
"ISR": 196914626,
"SF": 6134642,
"SR": 5394165,
"MU": 0,
"OU": 0,
"IU": 0,
"U": 0

}

Answer 1

[Rob]

This looks totally reasonable. The strand bias parameter is measure of the fraction of reads mapping in a particular orientation. Specifically, if the first sequenced read (or the only read in single-end sequencing) always maps to the forward strand, the strand bias is 1. If the first read always maps to the reverse complement strand, the strand bias is 0. If half of the reads map to each, the strand bias is 0.5. That is, strand bias is _not_ relative to the specified library type, but rather is an absolute measure of the fraction of fragments whose first sequenced read maps in the forward orientation.

[Lada]

Hi Rob,

thank you so much for answering my question! :) Now it's so much clearer to me.

I hope you don't mind if I ask a follow-up question. Can I use Salmon to somehow test if the library itself was successfully made stranded because I have some doubts? I will use the same example as in my previous question (RF-made library and de novo assembly). I was curious to see what happens if I play with the library flag in Salmon so I put IU and I got 96% mapping rate, but strand_mappng bias was 0,2. Does it mean that my library is not completely stranded?

{
    "read_files": "[ PCV_G1DD_C1_1.fq.gz, PCV_G1DD_C1_2.fq.gz]",
    "expected_format": "IU",
    "compatible_fragment_ratio": 1.0,
    "num_compatible_fragments": 13181223,
    "num_assigned_fragments": 13181223,
    "num_frags_with_concordant_consistent_mappings": 15035520,
    "num_frags_with_inconsistent_or_orphan_mappings": 866482,
    "strand_mapping_bias": 0.19533777348571916,
    "MSF": 0,
    "OSF": 0,
    "ISF": 2937005,
    "MSR": 0,
    "OSR": 0,
    "ISR": 12098515,
    "SF": 457160,
    "SR": 409322,
    "MU": 0,
    "OU": 0,
    "IU": 0,
    "U": 0