Biomedical Engineering Reference
the analysis to narrow down the variants and increase the chance of fi nding caus-
ative variants. In addition, any potential causative variant has to be Sanger
sequencing- confi rmed, the gold standard in clinical laboratories.
Clinical Laboratories Offering Exome Sequencing
The major genetic centers in the United States offering WES as a diagnostic test are
Cincinnati Children's Medical Center's Molecular Genetics Laboratory, Emory
Genetics Laboratory, Baylor College of Medicine, GeneDx, and Ambry Genetics.
Generally, the turnaround time of the exome test is between 10 and 16 weeks.
Pricing varies from $5,000 to $15,000 depending on the level of analysis and
whether only the proband or a trio is analyzed. For trio analysis, all laboratories
provide a report only for the proband. To aid in interpretation, a pedigree, patient
history, medical records, and clinical form are typically requested and some labora-
tories require this information before the test can be started. An extensive consent
form needs to be fi lled out prior to test commencement. To ensure the advantages
and limitations of the test are fully understood by the family members involved,
pre- and post-counseling is recommended. Generally, the Illumina HiSeq 2000/2500
sequencer and the NimbleGen capture are the platforms of choice for performing
this test in clinical laboratories.
Clinical Exome Data and Interpretation Challenges
There are various challenges that should be noted when performing exome assays
for a diagnostic purpose. The assay only covers between 90 and 97 % of the targeted
regions at a coverage level of 10X. This coverage depends on the type of capture
technology that is used with the key players in the fi eld being NimbleGen, Illumina,
and Agilent Technologies (Su et al. 2011 ). In addition, many regions of the exome
remain unexplored and novel genes found in these locations have not yet been asso-
ciated with disease. However, these gene regions cannot be ruled out in the analysis
and may require reanalysis of the data a year from the initial analysis to see if other
new disease associations are obtained (Klee et al. 2011 ). Another challenge of
exome analysis is that regions that play a role in regulating gene expression will not
be captured; expanded strategies such as genome will play a role in complementing
WES in this area. Alternatively, other exome capture products that include 5
UTR regions are starting to make their way into the marketplace. Due to the limited
size of the sequence reads of 100 bp, short sequence deletion and insertions (indels)
detection will be limited to about 1/3 of the read length. Larger indels will not be
detected for comprehensive mutation inclusion. This will improve by complement-
ing assembly-like methods before alignment in a stepwise manner (Wong 2013b ).
Furthermore, there are a high number of false positive fi ndings for various reasons.