Biomedical Engineering Reference
In-Depth Information
template that differ in length with the last base fluorescently labeled with a charac-
teristic fluorescent moiety. Separating these fragments by size through electropho-
resis, the sequence can be determined from the color of fluorescence produced at a
given fragment length. Though serviceable, this procedure is problematic for several
reasons. The template read length using this method is limited to ~800 bp with base-
calling quality dropping as read length increases. This introduces significant
challenges, especially for de novo sequencing, requiring that either chromosome
walking or shotgun sequencing be used, which are logistically challenging
and require computationally intensive assembly of the completed sequence. The
chain termination reaction itself is also time consuming, as is electrophoretic
separation [
3
]. However, the overarching problems with Sanger sequencing are
the relatively large amounts (~100
g) of DNA required - amplification leads to
errors - and the expense due to reagents for labeling and separation.
Lately, Sanger sequencing has been superseded by next generation sequencing
(NGS) technologies such as Illumina's Genome Analyzer, Roche's 454 sequencer
and Applied Biosystems' SOLiD System, which can resequence a human genome
for less than $100,000. NGS produces an enormous volume of DNA sequencing
data - in excess of one billion short reads per instrument per day [
4
]. NGS methods
generally involve randomly shearing genomic DNA into smaller fragments from
which sequencing libraries are created. Many NGS technologies, including cyclic
reversible termination, single-nucleotide addition and single molecule real time
(sic), can be categorized as sequencing-by-synthesis that involves DNA polymer-
ase. These short templates are immobilized in a flow cell allowing billions of
sequencing reactions to be performed simultaneously. The immobilized libraries
are subjected to either sequencing-by-synthesis techniques (cyclic reversible termi-
nation, single nucleotide addition) or sequencing-by-ligation. Finally, NGS uses
either fluorescent or luminescent detection of nucleotide incorporation in combina-
tion with imaging to readout the sequence one reaction at a time.
Libraries are prepared either through clonally amplified templates or single DNA
molecule templates. In both cases the nucleic acid is first fragmented, then adaptor
primers are annealed to the end to make templates. In the case of clonal amplifica-
tion, individual fragments are then amplified using emulsion PCR, isothermal bridge
amplification, or another technique to cluster a group of clonal amplicons. This
amplification produces greater signal as each of the amplicons will contribute signal
to the sequencing process. For example, Illumina relies on the amplification of
clonal clusters and a sequencing-by-synthesis reversible terminator method that
uses reversible versions of dye-terminators, adding one nucleotide at a time and
then detecting fluorescence at each position by repeated removal of the blocking
group to allow polymerization of another nucleotide. Pyrosequencing as used by 454
also uses clonal amplification along with DNA polymerization, adding one nucleo-
tide at a time and then detecting the nucleotides added to a location through the light
emitted through release of the attached pyrophosphates. Since nucleotides are added
to the templates in a given cycle, each cycle demands a high fidelity addition process.
Incomplete extension or misincorporation of
m
the template results in signal
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