We did a stranded RNA-seq library prep on a bacterial sample (Mycobacterium tuberculosis). When using this kit for human/mouse/fly, the correct strand is always "second" or "reverse" strand (different tools have different nomenclature). This time, I am getting 90% of the reads on the other strand. It doesn't make much sense to me that the reads would somehow be on the wrong strand. At worst, they should be 50/50. Do the bacterial annotations have different assumptions? Should I be concerned?
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Hi,
a lot of strand-specific RNA-Seq kits produce "reverse" reads. See http://seqanswers.com/forums/showthread.php?t=50459 and http://seqanswers.com/forums/showthread.php?t=50711.
I'd guess it is more a library-prep issue than an annotation issue; so I don't think you should be concerned.
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Originally posted by Michael.Ante View PostHi,
a lot of strand-specific RNA-Seq kits produce "reverse" reads. See http://seqanswers.com/forums/showthread.php?t=50459 and http://seqanswers.com/forums/showthread.php?t=50711.
I'd guess it is more a library-prep issue than an annotation issue; so I don't think you should be concerned.
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Originally posted by pmiguel View PostWhat kit are you using?
Yes, if you use the same kit in the same way your strandedness should remain the same.
My first suspicion would be that the annotation for your Mycobacterium is wrong.
I am also concerned that the annotation is wrong, but I am using the references from Ensembl (http://bacteria.ensembl.org/mycobact..._2/Info/Index/). I assume their info would be consistent across all species. I never ran into any issues like this with non-bacterial references.
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It would be odd to have entire genome annotation wrong from Ensembl (but strange things do happen). Are you using the GFF file Ensembl is providing?
Can you compare the annotation from NCBI: http://www.ncbi.nlm.nih.gov/genome/?...culosis+H37Rv?
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When you write "This time, I am getting 90% of the reads on the other strand." what do you mean by "getting". I presume you use some program or browser to derive this figure? What program or browser did you use? Was it the same one you used for previous projects?
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Phillip
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Originally posted by GenoMax View PostIt would be odd to have entire genome annotation wrong from Ensembl (but strange things do happen). Are you using the GFF file Ensembl is providing?
Can you compare the annotation from NCBI: http://www.ncbi.nlm.nih.gov/genome/?...culosis+H37Rv?
I use Picard CollectRnaSeqMetrics to collect the stats. It also generates graphs for normalized coverage across transcript. Usually, the middle is much higher than the ends. With this RNA-seq, the ends are actually slightly higher.
It's really bizarre. I've never seen anything like this.
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