Biology Reference
In-Depth Information
Table 7.2: Features of Several Next-Generation Sequencing Platforms.
Platform
PCR type
Sequencing by synthesis
Read length (bp)
a
Roche 454
Emulsion
Pyrosequencing
500-1000
Illumina (Solexa)
Bridge
Reversible terminators
20-40
SOLiD
Emulsion
Ligase
35
Polonator
Emulsion
Ligase
13
HeliScope
Single molecule
Polymerase
30
a
Read length and cost change as the companies involved improve their platforms.
many copies of a single DNA fragment. All polonies are attached to the array,
and reagents are then applied to amplify all polonies in the array in parallel.
Image-based detection of fluorescent labels incorporated with each PCR exten-
sion is used to obtain sequence data in parallel. Successive iterations of PCR
cycles and imaging are used to develop a contiguous sequencing read for each
component of the array. 454 sequencing (Roche Applied Science), Illumina
(Solexa technology), the SOLiD platform (Applied Biosystems), the Polonator
(Dover/Harvard), and the HeliScope Single Molecule Sequencer (Helicos) all use
this general scheme. The biochemistry of each platform is different; however,
the workflows are similar. For details about each platform, go to the companies'
websites for diagrams and additional references. The following descriptions pro-
vide a general overview, as does
Table 7.2
, of these sequencing systems.
7.11.1.1 Roche (454) GS FLX Sequencer
This was the first NextGen platform available commercially and involves pro-
duction of adaptor-flanked libraries of DNA fragments that are 400-600 bp
(
Dressman et al. 2003, Margulies et al. 2005
). The double-stranded DNA frag-
ments are separated into single strands, each with a special adaptor. These frag-
ments are loaded onto micrometer (
μ
m)-sized beads using a proprietary process,
each bead containing one fragment. Emulsion PCR (PCR that occurs within small
water droplets contained in a synthetic oil) generates millions of copies on each
capture bead. After breaking the emulsion, the beads are cleaned and beads
lacking DNA are eliminated, whereas beads holding more than one type of
DNA fragment are filtered out during the signal processing. Sequencing occurs
by synthesis: a single-stranded DNA fragment is copied, making the fragment
double-stranded. Starting at one end, the enzyme sequentially adds a single
nucleotide that is the match of the nucleotide on the single strand. After the
amplification of DNA strands, the DNA-capture beads are placed into tiny wells
on a plate for sequencing. The wells are 44
μ
m in diameter, and each well can
hold only one bead. Each plate contains 1.6 million wells. After the wells are
