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We did RNA-seq on sperm cells. RNA is more on the degraded size. Because there is no good ribosomal removal kit for this species, we proceeded with KAPA hyper kit which is poly-dT based.
Now, upon mapping data is quite odd, never seen before. Reads are not aligning to the exons, instead, it seems quite random, going virtually anywhere in patches. see attached igv image for an example.
Anyone out there to understand what is going on and any way to resolve it?
Reads are mapping sporadically and we can see split reads as well, so it is not from DNA. Also, we use DNase. It is not annotation related either, as we have tried data from three major centers, ie, Ensembl, UCSC and NCBI using three different genome versions. We got a similar result, even remaking the library won't help.
Any suggestions?
Thanks,
Dibyendu
Now, upon mapping data is quite odd, never seen before. Reads are not aligning to the exons, instead, it seems quite random, going virtually anywhere in patches. see attached igv image for an example.
Anyone out there to understand what is going on and any way to resolve it?
Reads are mapping sporadically and we can see split reads as well, so it is not from DNA. Also, we use DNase. It is not annotation related either, as we have tried data from three major centers, ie, Ensembl, UCSC and NCBI using three different genome versions. We got a similar result, even remaking the library won't help.
Any suggestions?
Thanks,
Dibyendu