Challenges of Next-Generation– Sequencing- Based Molecular Diagnostics - Next Generation Sequencing Technologies in Medical Genetics - page 85

a clinical laboratory, the initial setup for an NGS test could be very burdensome;
however, it is necessary to go through this process to understand the benefi t and
limitation of NGS tests to provide clinical interpretations with confi dence. For
WES/WGS, the focus of validation is shifted more towards developing metrics that
defi ne a high quality exome/genome such as the average coverage across the exome/
genome and the percentage of bases that meet a set minimum coverage threshold.
An evaluation of the concordance of SNPs identifi ed compared to the reference
should be made for WES. In practice, this may entail sequencing a larger number of
samples to cover suffi cient numbers of variants. Several clinical laboratories have
used 95-98 % concordance as the minimum acceptable level. In addition, homolo-
gous sequences such as pseudogenes pose a challenge for all short read sequencing
approaches. The limited length of NGS sequence reads can lead to false positive
variant calls when reads are incorrectly aligned to a homologous region, but also to
false negative results when variant-containing reads align to homologous loci.
Therefore, the clinical laboratory must develop a strategy for detecting disease-
causing variants within regions with known homology in its validation protocol.
Rescue or conformational testing may be warranted based on the indication and the
specifi city of the test defi ned by the laboratory. Test development costs, analytical
sensitivity and specifi city, and analysis complexity are important factors that must
be evaluated when considering development of NGS services. An ongoing QC
monitoring of NGS testing is also critical given the fast development of the testing
and analyzing strategies to ensure quality results. Any major change of testing pro-
tocol should be thoughtfully revalidated before implementing it for clinical service.
The clinical laboratory should inform ordering health providers with these changes
on a timely manner.

9.5 Quality Management of Next-Generation-

Sequencing Testing

Multiple commercial NGS platforms have been developed, all of which have the
capacity to sequence millions of DNA fragments in parallel. Differences in the
sequencing chemistry of each platform result in differences in total sequence capac-
ity, sequence read length, sequence run time, and fi nal quality and accuracy of the
data. These characteristics may infl uence the choice of platform to be used for a
specifi c clinical application. The clinical laboratory must develop quality control
(Q/C) measures and apply these to every run. These can vary depending on the cho-
sen methods and sequencing instrument but typically include measures to identify
sample preparation failures as well as measures to identify failed sequencing runs.
The clinical laboratory must also track sample identity throughout the testing pro-
cess, which is especially important given that NGS testing commonly entails pool-
ing of barcoded samples. Profi ciency testing protocols must also be established and
executed periodically according to CLIA and CAP regulations.