Challenges of Next-Generation– Sequencing- Based Molecular Diagnostics - Next Generation Sequencing Technologies in Medical Genetics - page 81

Chapter 9
Challenges of Next-Generation-
Sequencing-Based Molecular Diagnostics

9.1 Introduction

Although targeted sequencing by next-generation-sequencing (NGS) technology
has gained much attention in the fi eld of molecular diagnostics, several limitations
have delayed its application to medical genetics. It is diffi cult to standardize the
procedures because of the use of various sequencing platforms and target enrich-
ment methods, which are updated rapidly. The cutoff thresholds for accurate variant
identifi cation, including the minimum read depth, range of variant percentage com-
pared with the wild type, and quality score, have not been fully defi ned. Variable
coverage depth across target regions and misalignment between homologous
sequences or pseudogenes may also affect the accuracy of sequencing data. The fast
falling sequencing cost and rapid development of NGS analyzing bioinformatics
pipeline make it possible to use gene panels, whole exome sequencing (WES), and
whole genome sequencing (WGS) for clinical diagnoses. However, there are many
challenges in developing and implementing NGS technology for clinical use.
Understanding these challenges can help molecular diagnostic professionals and
ordering health professionals better gauge the utility of NGS clinical tests and the
interpretation of the NGS results. NGS is a complex sequencing technology and
many challenging steps need to be carried out to complete the test to generate clini-
cally useful interpretations.
In this chapter, we will address the challenges of NGS technologies at various
steps of the process including sample processing, data analyses, testing validation
and revalidation, quality management of NGS testing, reporting and interpretation,
informed consent and genetic counseling, training and education, and cost and
reimbursement.