Hi all,
Wondering if anyone has a better understanding of how the NuGEN Ovation strand specific RNA-seq kits (specifically the Ovation® Human FFPE RNA-Seq Multiplex System) work. I'm having a bit of difficulty understanding the correct Tophat library-type and htseq-count parameters to use.
For TruSeq kits, library-type=fr-firststrand and stranded=reverse are the correct parameters. See:
But when I inspect the GAPDH locus with data from the Ovation kit, I see quite the opposite:
So it seems that read 1 of the pair is mapping to the sense (+ for GAPDH) strand, and read 2 of the pair is mapping to the antisense (- for GAPDH) strand?
Furthermore, from htseq-count, using stranded=reverse:
using stranded=yes
Based on this, I would guess that library-type=fr-secondstrand and stranded=yes would be the correct options for the Ovation RNA-seq kits, even though on the surface they appear to use the same dUTP approach. NuGEN tech support confirmed that read 1 is indeed on the same strand as the original RNA, but they seemed fairly unsure.
If anyone has used the strand specific Ovation kits and has some insight, it would be greatly appreciated!
Wondering if anyone has a better understanding of how the NuGEN Ovation strand specific RNA-seq kits (specifically the Ovation® Human FFPE RNA-Seq Multiplex System) work. I'm having a bit of difficulty understanding the correct Tophat library-type and htseq-count parameters to use.
For TruSeq kits, library-type=fr-firststrand and stranded=reverse are the correct parameters. See:
But when I inspect the GAPDH locus with data from the Ovation kit, I see quite the opposite:
Code:
Flags Count 177 3 89 3 329 4 161 8 81 8 65 10 355 13 403 13 353 15 401 15 137 16 163 39 83 39 153 875 145 6030 97 6035 73 7442 147 16934 99 16934
Furthermore, from htseq-count, using stranded=reverse:
Code:
>tail -n5 test.reverse.counts __no_feature 458267 __ambiguous 473 __too_low_aQual 0 __not_aligned 0 __alignment_not_unique 58918
Code:
>tail -n5 test.yes.counts __no_feature 195770 __ambiguous 10645 __too_low_aQual 0 __not_aligned 0 __alignment_not_unique 58918
If anyone has used the strand specific Ovation kits and has some insight, it would be greatly appreciated!
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