Sequenom recently
released a novel genotyping assay termed iPLEX™ whose increase in design
efficiency and flexibility is possible through the use of novel,
mass-modified terminator nucleotides.
Introduction
Figure 1. The iPLEX™ Assay. The
scheme depicts a single assay.
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The iPLEX assay has several advantages
over previous genotyping assays for homogeneous MassEXTEND® (hME) (1)
chemistry. All reactions are terminated after a single-base extension (SBE),
in contrast to hME that uses a multiple-base extension process. The use of
SBE with hME chemistry is possible using standard ddNTP terminators.
However, mass separation of SBE products was too small (9-40 Da); the
iPLEX assay alleviates this issue by incorporating mass-modified
terminators (16-80 Da). One consequence is that the allele bias observed
in hME is greatly reduced from 60:40 to 52:48, allowing for the use of
more stringent calling thresholds and performance at twice the plexing
level. This application note presents data from feedbacks of 13
independent studies in which over 2200 iPLEX assays were designed.
Experimental Conditions
All assays were designed at Sequenom
using the Assay Design software version 3.0. In addition to considering
novel terminator masses, new options were added, including addition of
non-templated nucleotides at the 5' end of the extend primers, used to
further increase multiplexing and flexibility in design. The laboratory
work was performed following the previously recommended protocol, using
factorial methods of experimental design (2).
Results
Table I. iPLEX™ Assay Performance
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Table I shows a compilation of assay
design and genotyping performance obtained. Each institute was required to
use 24 to 96 DNA samples with previously known genotypes for accuracy
estimation. The plexing average was 23 during initial testing and
increased to 25 since product launch in June 2005. The compiled accuracy
was estimated at 99.84% with a first-pass success rate of 93%, increasing
to 97% after removal of weak assays. Near perfect accuracy can be achieved
following such quality control.
Conclusions
The iPLEX application has the
capacity to produce up to 100,000 genotypes per day on the MassARRAY
system. The assay design flexibility allows high efficiency, ranging from
50 to thousands of SNPs assays. Moderate to large SNP panels can be plexed
at plex levels up to 29, resulting in a significant cost reduction per
genotype data point.
References:
1. http://www.sequenom.com/Assets/pdfs/appnotes/Multiplexing_hME_App_Note.pdf
2. http://www.sequenom.com/Assets/pdfs/appnotes/8876-006.pdf
