Biomedical Engineering Reference
In-Depth Information
In 2006, the Genome Analyzer (GA) became commercially available and was
initially released by Solexa and acquired by Illumina in 2007 (Fig. 1.1 ) . In principle,
this system obtains sequences by sequencing by synthesis (SBS). The GA uses a
fl ow cell that has an optically transparent slide with eight lanes with surfaces that
bind oligonucleotide anchors (Voelkerding et al. 2009 ). Fragmented template DNA
is end-repaired to generate 5
-phosphorylated blunt ends. The adapter oligonucle-
otides are complementary to the fl ow-cell anchors. The adapter-modifi ed, single-
stranded template DNA is added to the fl ow cell and immobilized by hybridization
to the anchors. DNA templates are amplifi ed in the fl ow cell by “bridge” amplifi ca-
tion resulting in clusters each containing approximately 1,000 clonal molecules. For
sequencing, clusters are denatured, chemically cleaved, and washed to leave only
forward strands for single-end sequencing. Sequencing of the forward strands is
accomplished by primer hybridization complementary to the adapter sequences,
addition of polymerase, and by mixing four terminator nucleotides (ddATP, ddGTP,
ddCTP, ddTTP) containing a different cleavable fl uorescent dye. The terminators
are incorporated according to sequence complementarity. After incorporation,
excess reagents are washed away, the clusters are optically interrogated, and the
fl uorescence signal is captured by a charge-coupled device (CCD) (Mardis 2008 ).
With successive chemical steps, the reversible dye terminators are unblocked, the
fl uorescent labels are cleaved and washed away, and the next sequencing cycle is
Initially, the Solexa GA's output was 1 Gb per run. In 2009, the output of GA
increased in three upgrades to 20 Gb per run (75PE), 30 Gb per run (100PE), and
50 Gb per run (150PE), respectively, by improvements in polymerase, buffer, fl ow
cell, and software. The latest GAIIx can reach 85 Gb of sequence per run (Liu et al.
2012 ). In early 2010, using the same sequencing strategy with GA, Illumina
launched the HiSeq 2000 system. The original output of this system was 200 Gb per
run and improved to 600 Gb per run which may be completed in 8 days. The error
rate of 100PE could be below 2 % on average after fi ltering. Compared with 454 and
Sequencing by Oligo Ligation Detection (SOLiD), HiSeq 2000 provides the lowest
cost per million bases ($0.02/Mb). With multiplexing incorporated in P5/P7 primers
and adapters, it could handle thousands of samples simultaneously. HiSeq 2000
needs (HiSeq control software) HCS for program control, (real-time analyzer soft-
ware) RTA to do on-instrument base-calling, and CASAVA for secondary analysis.
With the introduction of Truseq v3 reagents and associated software, the HiSeq
2000 instrument can better sequence high GC regions. In 2012, an upgrade to the
HiSeq 2000 instrument to generate the HiSeq 2500 system was introduced. The
HiSeq 2500 system features two run modes, rapid run and high output run, and the
ability to process one or two fl ow cells simultaneously. As with the HiSeq 2000, the
HiSeq 2500 in the high output mode can generate 600 Gb per run. However, the
HiSeq 2500 has a rapid mode that can generate up to 180 Gb per run in about 40 h.
This provides a fl exible and scalable platform that supports the broadest range of
applications and study sizes.
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