The service provider conferred with Ion and came back with the fact my library was too big; the protocols are designed for inserts smaller than 150 bp, and my amplicons were carefully designed to be 150-205 in size.
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Ion Fragment Library Preparation kit. It even has a very helpful appendix on Amplicon Sequencing, The only real hint about the size limitation is the statement that "Target regions from 75 to 150 nucleotides in length must be sequenced bidirectionally". Clearly this is insufficiently emphatic! In the fragment library preparation information, the more serious warning does appear: "Libraries with a mean sze >~220 bp yield results of reduced sequencing quality" (and that size is after adding adapters).
Kevin: I just love how small details like these screw up your experiments. Be Forewarned!
from
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Ion Fragment Library Preparation kit. It even has a very helpful appendix on Amplicon Sequencing, The only real hint about the size limitation is the statement that "Target regions from 75 to 150 nucleotides in length must be sequenced bidirectionally". Clearly this is insufficiently emphatic! In the fragment library preparation information, the more serious warning does appear: "Libraries with a mean sze >~220 bp yield results of reduced sequencing quality" (and that size is after adding adapters).
Kevin: I just love how small details like these screw up your experiments. Be Forewarned!
from
Paying a Painful 75% Secrecy Tax
In a post a while back, I mentioned that my Ion Torrent sequencing project was stalled because my service provider couldn't get some of the key kits, despite an Ion representative posting that no such shortages existed. I've been remiss in updating that; last Tuesday the kits showed up and Monday I got my data -- and a bit of a shock.
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