Friday, February 13, 2009
cDNA with Titanium
When I went to the Titanium chemistry release party back in September 2008, I had the opportunity to chat with a bunch of other 454 faciity directors. One comment that I heard several times was that no one really ran cDNA (mostly due to the adapter issue, see previous post on cDNA preparation). I feel pretty comfortable with sequencing cDNA (mostly Evrogen and Bio S&T normalized) with the FLX though it does give lower amounts of data (usually 60-80 Mb). Now with Titanium, I'm hearing from the Roche/454 people that cDNA is not "ideal". Does anyone have any experience with cDNA on Titanium? We've done a few and the results have been disappointing (low filter pass, only ~20% of anticipated data, etc).
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Were the cDNA libraries you sequenced using Titanium prepared following the protocol you mentioned in your previous post "cDNA for pyrosequencing" with Sfi1 digestion etc?
ReplyDeleteHi Freya- Thanks for your comment. The cDNA for this particular run was constructed by Evrogen using their Trimmer Direct kit.
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ReplyDeleteAs I was trying to say before I accidentally deleted my comment - we have heard that the Evrogen Kit in particular may cause problems for 454 using the Titanium chemistry.
ReplyDeleteI have completed a library with Titanium chemistry and got close to a million reads. We got about 350 MB of sequence data total. The average contig size after assembly (lucy/cap3 worked better than newbler) was about 600 bp. The range was up to 9 kb for the largest contig.
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ReplyDelete...you can download my protocol here: http://gsl.cals.ncsu.edu/index.php?option=com_content&task=view&id=22&Itemid=40. I've used it with luck on the titanium platform even though it was originally put together when I was using FLX.
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