A Survey of Next-Generation–Sequencing Technologies - Next Generation Sequencing Technologies in Medical Genetics - page 13

SOLiD system), or by the principle of pyrosequencing (e.g., Roche 454 system;
Fig. 1.1 ; Margulies et al. 2005 ; Ruparel et al. 2005 ). Nonoptical DNA sequencing
by detecting the hydrogen protons generated by template-directed DNA poly-
merase synthesis on semiconductor-sensing ion chips has recently been developed
as well (Fig. 1.1 ; Rothberg et al. 2011 ). In such a massively parallel sequencing
process, NGS platforms produce up to 600 Gb of nucleotide sequence from a sin-
gle instrument run (e.g., Illumina's HiSeq 2000; Clark et al. 2011 ). The sequenced
fragments are called “reads,” which could be 25-100 bps from one or both ends.
The massive capacity of NGS allows the sequencing of many randomly overlap-
ping DNA fragments; therefore, each nucleotide in targeted regions may be
included in many reads, allowing repeated analysis which provides depth of cover-
age. Increased depth of coverage usually improves sequencing accuracy, because a
consensus voting algorithm is used in determining the fi nal nucleotide calls (Lin
et al. 2012 ).

2.3 Roche/454 Life Sciences

Roche 454 utilizes pyrosequencing technology and was the fi rst commercial NGS
platform (Fig. 1.1 ) . In contrast to Sanger that uses dideoxynucleotides to terminate
the chain amplifi cation, pyrosequencing technology detects the pyrophosphate
released during nucleotide incorporation. The library DNAs with 454-specifi c
adapters are denatured into single strands and captured by amplifi cation beads fol-
lowed by emulsion PCR (Liu et al. 2012 ). Then on a picotiter plate, one of dNTPs
(dATP, dGTP, dCTP, dTTP) will complement the bases of the template strand with
the help of ATP sulfurylase, luciferase, luciferin, DNA polymerase, and adenosine
5 phosphosulfate (APS) and release pyrophosphate (PPi) which equals the amount
of incorporated nucleotide. The ATP transformed from PPi drives the luciferin into
oxyluciferin and generates visible light (Liu et al. 2012 ). At the same time, the
unmatched bases are degraded by apyrase. Then another dNTP is added into the
reaction system and the pyrosequencing reaction is repeated.
The read length of Roche 454 was initially 100-150 bp in 2005, 200 K+ reads,
and could output 20 Mb per run (Mardis 2008 ). In 2008, after the launching of the
new 454 GS FLX Titanium system, its read length could reach 700 bp with accuracy
of 99.9 % and output of 0.7 Gb data per run within 24 h. In 2009, Roche combined
the GS Junior a benchtop system into the 454 sequencing system which simplifi ed
the library preparation and data processing, and output was also upgraded to 14 Gb
per run (Huse et al. 2007 ). The most notable feature of this system is its run time of
approximately 10 h from sequencing to completion. The longer read length is also
a distinguishing advantage compared with other NGS systems. In addition, the
manpower is reduced by the automation of the library construction and semiauto-
mation of the emulsion PCR. However, the high cost of reagents and the relatively
high error rate in terms of polybases longer than 6 bp remain a challenge.