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I was just wondering what general approach and experience groups have had sequencing mixed fragment size cDNA (transcriptome sequencing LMW 70 – 500 bp AND > 1.5 kb in one sample). Not only that, but a collaborator I’m working with at the moment is intent on sequencing normalized cDNA (Evrogen kit). One bonus is that the collaborator has plenty of RNA to start with.
The reason I bring this up is because I’ve done some mixed fragment size cDNA sequencing of normalized material in the past and it was difficult to generate a useful library even though size exclusion steps had been taken. The sequence out put for this sample was very low and the whole lot was full of short reads. Any mixed fragment size, normalized cDNA sequencers out there that can share their experiences? If so it’d be great to hear from you.
The reason I bring this up is because I’ve done some mixed fragment size cDNA sequencing of normalized material in the past and it was difficult to generate a useful library even though size exclusion steps had been taken. The sequence out put for this sample was very low and the whole lot was full of short reads. Any mixed fragment size, normalized cDNA sequencers out there that can share their experiences? If so it’d be great to hear from you.