Casey Molecular Diagnostic Laboratory has developed a new next-generation sequencing panel method using simplex PCR reactions run on a chip with over 5,000 individual wells, with each primer set duplicated to avoid allele dropout. This new approach offers unmatched target enrichment of over 98% and average coverage over 500x. Every novel variation is manually checked to avoid false positives or negatives. While other labs sequence more broadly, Casey believes their targeted approach focusing on relevant genes optimizes mutation detection.

1. To our valuable clients and families,
Casey Molecular Diagnostic Laboratory has exciting news to share with you. In our pursuit of
developing the most comprehensive, specific and cost effective method for our NGS panels, we
have combined the unmatched specificity of simplex PCR with the SmartChip platform from
WaferGen. Simplex PCR reaction (100 nl per reaction) is run on a chip with 5,184 individual
wells. An individual PCR primer set is printed into each well for the simplex PCR reaction. In
order to avoid allele dropout and random PCR failure, each primer set is duplicated on the chip.
This new development was made possible by the continuous effort to improve our PCR primer
library. With this new platform, we now have the fastest, most consistent and simplest target
enrichment method. Equally important, primers can now be managed easily because they are
stored at room temperature inside the individually wrapped chip.
Our new approach offers unmatched on-target enrichment of sequencing targets (>98%). Our
average coverage is >500X. Regions with coverage of less than 100X are further enriched by
adding extra primers to the newer chip versions. Therefore, we do not need to rely on
“sophisticated” variation filtering. Every novel variation, regardless of the scores, is checked
manually in order to avoid false positive or false negative reporting.
While many laboratories in the molecular diagnostics field are trying to improve detection by
sequencing more broadly, we still believe that “sequencing smart” is the best approach.
Specificity, coverage and choice of targeted genes are all important to the improvement of
mutation detection rate. By focusing only on genes relevant to the patient’s condition, our testing
approach has also been optimized to detect mutations in regions of genes frequently missed (such
as introns and highly repetitive sequences) using methods such as whole exome sequencing. We
aim to continuously improve our mutation detection rate by improving coverage and by adding
newly discovered and relevant genes. Because we sequence smart, the (hidden) cost of data
storage and the bioinformatics pipeline is significantly reduced. We believe our current approach
offers the best value to our clients.
The family of SmartPanel:
Retinal Dystrophy panel v8 ($2,500): 244 genes; 4,894 simplex PCR duplicated on two chips
(=9,788 simplex PCR).
Stargardt/Macular dystrophy panel v3 ($500): 10 genes (each coding exon is covered by two
primer designs; 511 simplex PCR duplicated on one chip (=1,022 simplex PCR; each coding
exon is covered by four separate reactions).
Pigmentation panel v3 ($1,500): 29 genes (each coding exon is covered by two primer designs;
1506 simplex PCR duplicated on one chip (=3,012 simplex PCR; each coding exon is covered by
four separate reactions).
Developmental eye disease panel v2 ($950): 45 genes; 606 simplex PCR duplicated on one
chip (=1,212 simplex PCR).

2. Neurocutaneous panel v2: 8 genes; 672 simplex PCR duplicated on one chip (=1,344 simplex
PCR).
Vitreoretinopathies panel v1: 19 genes; 1,615 simplex PCR duplicated on one chip (=3,230
simplex PCR).