Comparative Genomics-Basic and Applied Research.pdf

16 Comparative Genomics
A. B.
E.
(iii)
(ii)
C. D.
(i)
FIGURE 2.1 (See color figure in the insert following page 48.) 454 Life Sciences sequencing.
(A) DNA preparation: Isolated genomic DNA is fragmented, ligated to adaptors, and separated
into single strands. (B) Emulsion PCR: Single-stranded DNAs are bound to beads under
conditions that favor one DNA molecule per bead. An oil-PCR reaction mixture is added to
encapsulate bead–DNA complexes into single oil droplets, onto which PCR amplification is
performed to create beads containing several million copies of the same template sequence.
(C) Deposition of the PCR-amplified beads into individual wells in the PTP is followed by
the addition of smaller beads immobilized with ATP surfurylase and luciferase (D), which
convert inorganic pyrophosphate into a light signal. (E) Schematic of the GS20 instrument,
which consists of the following subsystems: (i) fluidic assembly for delivery of dATP, dCTP,
dGTP, and dTTP reagents; (ii) PTP; and (iii) CCD camera. Figure reprinted from Margulies
et al., Nature 437, 376–380, 2005, by permission from Macmillan Publishers Ltd., copyright
(2005).
high-resolution charged coupled device (CCD) camera, permitting detection of the light
generated from each PTP well undergoing the pyrosequencing reaction (Figure 2.1E).
With a pass rate of ~50% and a read length of 100 bases, one run will produce about
30–40 million bases of sequence data in 4–5 hours.
The Genome Sequencer 20 (GS20) instrument was launched by 454 Life Sciences
in 2005. More than 40 articles have since been published on the GS20 platform,
describing sequencing of bacterial genomes, 28,30–34 surveying microbial environments
(i.e., metagenomics), 35–40 profiling expressed sequence tags (ESTs), 41–44 and wholegenome
surveys of ancient DNA. 45–47 Many of these studies highlight the advantages
and disadvantages of the GS20, depending on the intended goals of the research effort.
For example, Hofreuter et al. reported the sequencing and characterization of the
highly pathogenic Campylobacter jejuni strain 81-176. 34 Two 454 Life Sciences runs
were performed, generating 60,905,794 high-quality bases from 558,331 successful
reads (i.e., the average read length was 109 bases). A de novo assembly produced a
genome with 34x coverage (i.e., on average, each nucleotide in the assembly was called
by 34 different reads) in 43 contigs (contiguous sequence represented by two or more