Biomedical Engineering Reference
In-Depth Information
emulsion droplets and then pooled to create a primer library (Tewhey et al. 2009 ).
Separately, emulsion droplets containing genomic DNA and PCR reagents are
prepared. Following the merging of the droplets, DNA is amplifi ed by the PCR and
subsequently processed for NGS.
The intent of this chapter is to provide a cursory view of the enrichment tech-
nologies that are commercially available, to lay the principles of each technology,
and provide examples of which methods have been used in clinical laboratories.
Non-hybridization-Based Enrichment Methods
Molecular Inversion Probes
PCR-based non-hybridization gene enrichment schemes include MIPs (Fig. 3.1 ;
Deng et al. 2009 ; Porreca et al. 2007 ; Turner et al. 2009 ). Briefl y, single-stranded
oligonucleotides with a common linker fl anked by target-specifi c sequences are
annealed to their target sequence and a ligase circularizes them (Landegren et al.
2004 ; Nilsson et al. 1994 ). Any uncircularized molecule is digested by exonuclease
treatment to reduce background, and circularized molecules are PCR-amplifi ed via
primers directed at the common linker. To perform exon capture in combination
with NGS, a DNA polymerase can be used to gap-fi ll between target-specifi c MIP
sequences designed to fl ank a full or partial exon, before ligase-driven circulariza-
tion, thus capturing a copy of the intervening sequence (Porreca et al. 2007 ). The
assay is affl icted by low uniformity due to ineffi ciencies of the capture reaction.
However, an optimized, simplifi ed protocol for MIP-based exon capture has been
reported (Turner et al. 2009 ). The main disadvantages of using MIPs for target
enrichment are capture uniformity compared to hybridization-based methods.
Additionally, MIP oligonucleotides are costly and not readily available in large
numbers to cover large target sets. In contrast, the current view of MIP-based cap-
ture followed by direct sequencing may be most relevant for projects involving rela-
tively small numbers of targets but large numbers of samples. Unfortunately, this
method was never developed into a commercially available product.
Highly Multiplex Droplet PCR
For over 20 years, PCR has been the method of choice for amplifi cation before
sequencing a sample (Mamanova et al. 2010 ). This approach is compatible with
Sanger sequencing because a single amplicon is generated that can be easily subse-
quently sequenced and the length of an amplicon is comparable to the length of
sequence produced. PCR may be compatible with NGS platforms as long as a large
number of PCR products for a region of interest can be generated. However, failure
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