Diagnosis of Inherited Neuromuscular Disorders by Next-Generation–Sequencing - Next Generation Sequencing Technologies in Medical Genetics - page 58

100 % when compared to Sanger sequencing due to the low coverage regions
which is an inherent problem of the enrichment technologies and not the sequenc-
ing platform. However, in regions with greater than 20X read depth, the genotyp-
ing data showed that both enrichment technologies produced suitable calls for use
in clinical laboratories.
Several parameters are worth comparing between the two enrichment technolo-
gies and contrasting them to Sanger sequencing; namely, clinical implementation,
cost, requirement for specialized equipment, ease of use, analytical sensitivity and
specifi city, and scalability (Valencia et al. 2012 ). RDT offers the lowest enrichment
cost per amplicon when compared to SS and Sanger sequencing. The RDT and SS
DNA requirements are similar, but the Sanger method requires a signifi cantly higher
amount of DNA. RDT is more appropriate for situations requiring multiple genes
with long exons because it can enrich a target interval up to 1 Mb. Up to eight
samples can be processed per day. Similarly, SS can be used to process eight patients
per day, but it can enrich a much larger target interval, up to the entire exome. By
contrast, only one patient sample may be processed by Sanger sequencing in a day
because it is such a large panel (383 amplicons). One important diagnostic issue is
the ability to distinguish between gene and pseudogene targets; RDT and Sanger
can readily address this issue by correctly choosing locations where the primers are
to hybridize on the genomic DNA template. Since SS is a hybridization-based
method, its limitation is not being able to distinguish between the gene and pseudo-
gene targets. Therefore, RDT is more appropriate for a clinical laboratory, due to
excellent sequence specifi city and uniformity, reproducibility, high coverage of the
target exons, and the ability to distinguish the active gene versus known pseudo-
genes. Regardless of the method, exons with highly repetitive and high GC regions
are not well enriched and require Sanger sequencing for completeness. This study
demonstrated the successful application of targeted sequencing in conjunction with
NGS to screen for mutations in hundreds of exons in a genetically heterogeneous
human disorder.
A recent study from the same group investigated the diagnostic yields from the
implementation of the RDT CMD sequencing panel as compared to the single-gene
Sanger sequencing approach (Valencia et al. 2013 ). Following a successful analyti-
cal validation, a clinical CMD NGS panel was launched at EGL and has been used
successfully by clinicians in CMD cases presenting with overlapping phenotypes,
inconclusive biochemical studies, and nondiagnostic brain or muscle MRIs. This
expedited approach to molecular diagnosis avoids the diagnostic odyssey and cost
associated with a serial gene testing approach. Valencia et al. demonstrated that the
CMD panel approach convincingly showed better mutation detection or diagnostic
yield compared to a single-gene analysis. The effi ciency and better yield of the
panel approach is better illustrated by the analysis of the 20 blinded samples
included in the study. Several samples, which underwent a series of single-gene
tests, and others which remained CMD of unknown molecular etiology due to
inconclusive biochemical or immunologic assays, all received a defi nitive diagnosis
through this NGS approach. Furthermore, the percent diagnostic yield of all EGL
CMD tests and single-gene tests was 41 % and 17 %, respectively.