Biomedical Engineering Reference
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Fig. 2.1 Schematic of the next-generation-sequencing workfl ow. Following DNA isolation, target
sequences are enriched by amplifi cation (RainDance) or capture-based methods, sequenced by a
next-generation platform (HiSeq 2500), and analyzed by open source or commercial software
package, such as NextGENe from Softgenetics, to obtain the variants that will then be fi lter priori-
tized to identify the potentially causative gene(s)
Fundamentals of NGS Platforms
Generally, NGS is composed of four steps; DNA isolation, target sequences enrich-
ment, sequencing by an NGS platform, and bioinformatic analysis (Fig. 2.1 ). During
the analysis, fragment sequences are aligned and variant calls are obtained and pri-
oritized by applying various fi lters to identify the potentially causative gene(s). An
optional report generation step exists in clinical laboratories after potential caus-
ative variants are Sanger-confi rmed. A detailed description of the principles and
platforms of NGS is mentioned in the next sections.
Massively parallel sequencing is one common feature shared by almost all cur-
rent NGS platforms, following clonally amplifi ed single DNA molecules, sepa-
rated in a defi ned microchamber (called fl ow cells, fl owchips, or picotiter plate;
Voelkerding et al. 2009 ). One exception to this is Pacifi c Biosciences which uses
single-molecule sequencing technology without clonal amplifi cation (Eid et al.
2009 ). In contrast, Sanger sequencing has orders of magnitude lower throughput
by sequencing products produced in individual sequencing reactions. NGS is fi rst
carried out by fragmenting the genomic DNA into small pieces, usually in the
range of 300-500 bps (Borgström et al. 2011 ). Then, platform-specifi c adapters
are ligated to the ends of the DNA segments, permitting their attachment and
sequencing. In the NGS execution, sequencing results are obtained by reading
optical signals during repeated cycles from either polymerase-mediated fl uores-
cent nucleotide extensions of four different colors (e.g., Illumina's HiSeq system),
or from iterative cycles of fl uorescently labeled oligonucleotide ligation (e.g., ABI
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