A Review of DNA Enrichment Technologies - Next Generation Sequencing Technologies in Medical Genetics - page 26

to amplify a high percentage of regions is the limitation of highly multiplexed PCR.
Moreover, multiplexes are diffi cult to decipher as to which PCR products are ampli-
fying and which one are not unless sequencing verifi cation is performed. The paral-
lel use of many primer pairs can generate a high level of nonspecifi c amplifi cation,
caused by interaction between the primers.
To overcome these shortcomings, PCR amplifi cation, by RDT, in a microfl uidic
environment has been successfully performed (Tewhey et al. 2009 ). In essence, oil
microdroplets segregate thousands of individual PCRs in the same reaction tube
(Fig. 3.1 ). Specifi cally, each droplet supports an independent PCR by containing a
single primer pair with genomic DNA and other reagents. The droplet population is
hundreds to thousands of distinct primer pairs and is subjected to thermal cycling,
after which this emulsion is broken and products are recovered. The mixture of
DNA amplicons are subjected to shotgun library construction and NGS sequencing.
This technology prevents different primer pairs from interacting with each other and
removes the key constraint of multiplex PCR. However, the initial cost of purchas-
ing a large amount of PCR primers and the cost of equipment are very substantial in
commercial offerings. This technology has been recently applied in clinical labora-
tories to aid in the diagnosis of congenital disorders of glycosylation, congenital
muscular dystrophy, and hearing loss (Jones et al. 2011 ; Sivakumaran et al. 2013 ;
Valencia et al. 2012 , 2013 ). The application of this technology to congenital muscu-
lar dystrophy and hearing loss is discussed in detail in Chaps. 6 and 7 .

3.3 Hybridization-Based Enrichment Methods

Hybridization-based methods are the most popular targeted enrichment strategies
and have been extended to capture the whole human exome reliably (Clark et al.
2011 ; Majewski et al. 2011 ). Such an approach can be classifi ed as liquid-based
hybridization (Bainbridge et al. 2010 ) or solid phase-based (e.g., microarray-based)
hybridization (Choi et al. 2009 ; Hodges et al. 2007 ), depending on how the reac-
tions are experimentally implemented.

3.3.1 Solid Phase Capture

The principle of enrichment through direct selection is well known and it includes
several procedural steps, namely, shotgun fragment library hybridization to immo-
bilized probe, nonspecifi c hybrids are washed away, and captured DNA is eluted
(Lovett et al. 1991 ). Roche NimbleGen made this technology compatible with NGS
(Fig. 3.1 ; Albert et al. 2007 ; Hodges et al. 2009 ; Okou et al. 2007 ). Such a method
can usually enrich targets by about 1,000- to 2,000-fold in one round of hybridiza-
tion reaction (Albert et al. 2007 ). The enrichment effi ciency has been increased by
multiple enrichment cycles (Summerer et al. 2009 ). Originally, a single microarray