Hi all,

I am currently performing DTU analysis following salmon quantification. I started with converting BAM files, which were aligned to the reference genome via STAR, back to fastq files (paired-end) with bam2fq since I do not have the original fastq files at hand right now. When running salmon with these files (automatic LIB detection), I get the following warning:

"Detected a potential strand bias > 1% in an unstranded protocol check the file SALMON/lib_format_counts.json for details"

According to the corresponding file, the library is stranded:

{
"read_files": "[ sample_r1.fq, sample_r2.fq]",
"expected_format": "ISR",
"compatible_fragment_ratio": 1.0,
"num_compatible_fragments": 22131862,
"num_assigned_fragments": 22131862,
"num_frags_with_concordant_consistent_mappings": 19984860,
"num_frags_with_inconsistent_or_orphan_mappings": 2245202,
"strand_mapping_bias": 0.000004803613277907642,
"MSF": 0,
"OSF": 0,
"ISF": 96,
"MSR": 0,
"OSR": 0,
"ISR": 19984860,
"SF": 1069736,
"SR": 1175370,
"MU": 0,
"OU": 0,
"IU": 0,
"U": 0}

Also, when coloring reads in IGV according to "first-in-pair read strand" it seems that the library is stranded as almost all reads have the same color. Since I double-checked the new fastq files and the strandness already appears in the BAM file, I am pretty sure that the conversion to fastq went well.

However, we did not perform stranded RNA-seq library protocol and I do not understand why I got these results. Does anyone of you have any idea why this might be the case? And do you think this fact has an influence on the further analysis (e.g. DRIMseq) or can I still use the quantification?

I really appreciate any help you can provide.