A Bit of a Surprise

At his blog, Keith Robison says that his stalled Ion Torrent sequencing project has moved forward, and given him a bit of a surprise. He writes that when he designed his primers for the project back in March, there was little information from Ion to build off of, but when he did later see the application note, that there was no cause to be concerned about his primers. "Run on the 314 chip (since none other is broadly available), the expected yield would be around 100K reads of up to 100bp. Instead, we had about 28K reads after filtering, which dropped to just under 25K mappable reads," Robison says, adding, "Once we get to just what is allegedly my library, the big hit is 51% poor signal. So why so poor?" His library was too big; from Ion, he learned that the protocol is for inserts smaller than 150 basepairs, not his 150 to 205 basepair size. "Given that this is such a crucial point in amplicon design, why isn't it in the Application Note?" Robison asks. "I can make two guesses. One is a pure failure to think through what a customer might want to find in the note, as opposed to what marketing would like them to see. The other is worse: that the information was left out because it would need to change with each advance in the Ion chemistry."