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Old 11-03-2009, 05:09 AM   #12
pmiguel
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Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
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My impression of Roche's policy towards cDNA library construction is that they were not satisfied with any of the methods available and therefore they refused to recommend any of them. In the fullness of time Roche would release a method or kit that would produce acceptable results. I find this maddening as both Applied Biosystems and Illumina have had expression analysis solutions available for many months. Roche/454, who have been in the "next gen" game a year longer, are still dragging their feet.

But I also get the impression there were a couple of methods that at least approached the Platonic ideal of a perfect 454 cDNA library. These were (1)ribosomal depletion (eg, polyA+ isolation) of total RNA, random primed cDNA synthesis. Nebulization, etc as for genomic libraries or (2) oligo dT 1st strand synthesis using an offset restriction enzyme site (eg Mme or EcoP) adaptor. The adaptor and most of the polyA/T tail then is removed from the cDNA via restriction digest. The latter would likely involve some tedious tricks (eg methylation) to avoid cutting Mme or EcoP sites in the cDNA proper.

Anyway, we are dancing around something here and Roche is actually somewhat less mysterious than I think would normally be their wont. That is, they explicitly warn of the negative effects of polyA or polyT tracts. These tracts will blaze bright, obscuring nearby beads and possibly consuming all the dATP (or dTTP) in the flow before reaching the end of the tract. Hence the creation of the "broken oligo dT primer". This method apparently does not satisfy Roche, but their disapproval is somewhat muted.

But I think there is a second, related, issue in play. About this, Roche is more typically inscrutable, but one hears whispers. That is, one is led to believe that the image analysis software is sensitive to well-to-well bleed-over. Two beads sitting in adjacent wells of a PTP, if they emit too similar a sequence, are presumed to be "overlapping" and one or both are discarded. I'm not privy to what the threshold is for such a filter, if it does even exist, to kick in. It is of some significance, however; because SMRT cap adaptors, for instance, if they are sufficient to trigger high levels of bleed-over filtration, would be problematic. Especially if, as I believe is the case, the fragmentation broken ends are less often ligated than the extant capped ends.

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Phillip
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