Hi Forum,
We have some RNA-Seq data generated using Nugen's Ovation RNA-Seq System V2 kit.I plan to do denovo transcript assembly using cufflinks with this data. But when I look at the BAM files, I see many genes which have quite a lot of reads in introns. My feeling is that most of it comes from pre-mRNA (the kit uses both random primers and oligo-dT primers) although a fraction of it may be biologically meaningful (retained introns/alternate exons etc). Since cufflinks depends on junction reads for denovo assembly, having lots of premRNA will affect these types of reads and I have no idea how this will affect the results... Does anyone have any experience with denovo assembly using libraries made with this or similar kits?
Thanks!
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My guess is that it will simply create different isoforms, with/without introns, so in some cases one of your isoforms might just be your pre-mrna and another one your mature mrna. Nothing beats trying it out.