The reduction in ambiguities is a huge advantage of 454 or any next-gen system. As noted above, we found the multiplexing with the Roche kits to be limiting and cost prohibitive. And the throughput is only 5 samples, high-res, per Jr run. That gets expensive fast.
So yes, I'm a fan of the home-brew, where we can massively increase throughput and decrease cost. Of course you need to spend the time to get it working and order lots of primers for the multiplexing.
Email me at simon.lank[at]gmail.com if you'd like a copy of the most recent manuscript describing our higher-throughput bench and analysis methods for HLA typing on the Jr.
Simon
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We are about to purchase a GS-Jr (/Ion Torrent) to do HLA typing of patient and donors for HSCT. How did it perform with the HLA kits (if you decided to use it) or did you go for the home-brew solution? What about ambiguities, we do standard Sanger SBT at the moment and struggle with ambiguities?Leave a comment:
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I'm a little biased, since I do home-brew HLA typing with Roche/454 (not using their kits) but I don't think it's possible to add anything to their kits since they come with the primers already added to the plate. If you're interested in non-kit based methods you can drop me a line and I'd be happy to let you know how we achieve high-resolution typing by multiplexing > 48 patients per GS-Jr run (384 per FLX run).
[email protected]Leave a comment:
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Additional MIDs in the HLA kits
We're looking to start using the HLA kits on our Junior
They're supplied as a pack of 4 identical plates, each plate allowing you to run 10 separate samples each with their own MID.
However, the MIDs used are identical on each of the plates.
We were discussing whether it would be feasible to somehow add a second MID into the amplicons which would allow for more than 10 patients to be run on a single chip.
Either that or convince Roche to use a separate set of MIDs for each of the 4 plates!
Any thoughts anybody?
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