A Review of DNA Enrichment Technologies - Next Generation Sequencing Technologies in Medical Genetics - page 27

with 385,000 probes was hybridized to a library DNA (HD1 NimbleGen array) with
a region of interest around 4-5 Mb. More recent microarrays contain 2.1 million
probes and can capture greater than 60 Mb, the exome. Following the capture, NGS
can be performed using any of the sequencing platforms including Illumina HiSeq
2500. In contrast, Agilent's Capture Arrays, with only 244,000 probes, are the most
direct competitor to NimbleGen's HD1 arrays. One key factor for a successful target
enrichment outcome is the probe design, which is dependent on gene annotation
databases such as the RefSeq database. Therefore, unknown disease causing genes,
noncoding genomic regions, and regulatory sequences are not included in the
designs. Also high GC percentage regions are poorly captured (Okou et al. 2007 ).
Microarray target enrichment of large genomic regions is faster and less labor-
intensive than comparable PCR-based approaches. However, the drawbacks of this
technology are that the hardware is expensive and the DNA requirement is large,
around 10-15

g, though this is irrespective of whether the capture experiment is
for 100 kb or an entire exome (Mamanova et al. 2010 ). In addition, the number of
microarrays that can be processed by a single person per day is a limiting factor to
scale up the operation in a clinical laboratory. Despite its disadvantages, solid phase
hybridization methods have successfully been used for enrichment of genes involved
in deafness, retinitis pigmentosa, cardiomyopathies, and immunodefi ciencies
(Daiger et al. 2010 ; Ghosh et al. 2012 ; Meder et al. 2011 ; Shearer et al. 2010 ;
Simpson et al. 2011 ).

μ

3.3.2 Solution-Based Capture

Both Agilent and NimbleGen have developed and commercialized solution cap-
tures. Experimentally, the capture consists of a number of general steps. Biotinylated
DNA or RNA probes are hybridized, for several days, with fragmented target DNA.
The DNA/probe hybrids in the solution are captured by magnetic streptavidin beads
immobilized by a strong magnet. DNA fragments not targeted in the liquid phase
are removed by repeated washes. Targeted DNA fragments are eluted from the
beads by increasing the pH with NaOH in order to break the biotin streptavidin bond
and degrade the RNA probes. Only the enriched DNA fragments are sequenced by
NGS protocols.
Solution captures were introduced to overcome the aforementioned disadvan-
tages of microarray captures (Fig. 3.1 ). Agilent (150-mer RNA probes) and
NimbleGen (60- to 90-mer DNA probes) solution captures differ in regard to the
nature of the probes. Solution target enrichment can be performed in 96-well plates,
using a thermal cycler, so it is more readily scalable than microarray enrichment and
does not require expensive equipment (Mamanova et al. 2010 ). Microarray and
solution captures use specifi c probes designed to target regions of interest. The dis-
tinguishing feature is the probe to library ratio during the hybridization. Microarray
target enrichment uses a vast excess of DNA library over probes, whereas, solution
capture has an excess of probes over template, driving the hybridization reaction