Important footnotes in Ion Fragment Library Preparation Kit

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.
 .....
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.

Read more »

Simple Copy Number Determination with Reference Query Pyrosequencing (RQPS)

Simple Copy Number Determination with Reference Query Pyrosequencing (RQPS)

Zhenyi Liu^1,3, Daniel L. Schneider¹, Kerry Kornfeld¹, and Raphael Kopan^{1,2,3 1} Department of Developmental Biology, School of Medicine, Washington University, St. Louis, Missouri 63110, USA
² Division of Dermatology, Department of Medicine, School of Medicine, Washington University, St. Louis, Missouri 63110, USA

The accurate measurement of the copy number (CN) for an allele is often desired. We have developed a simple pyrosequencing-based method, reference query pyrosequencing (RQPS), to determine the CN of any allele in any genome by taking advantage of the fact that pyrosequencing can accurately measure the molar ratio of DNA fragments in a mixture that differ by a single nucleotide. The method involves the preparation of an RQPS probe, which contains two linked DNA fragments that match a reference allele with a known CN and a query allele with an unknown CN.