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Sequencing Review
Sequencing Review
454 instruments are pyrosequencers that carry out many reactions at a time (parallel sequencing) in wells of
a PicoTiter Plate. Beads coated with thousands of homogeneous DNA fragments are added to individual
wells on the plate. The DNA fragments are amplified in an oil emulsion mixture with DNA polymerase and
primers[1]. dNTPs are sequentially added to the wells one at a time and washed. The process of continuous
washing and the sequencial addition of dNTPs, DNA polymerase, luciferase, and ATP-sulfurylase explains
the high reagent costs of sequencing. ATP-sulfurylase converts the PPi released from each dNTP addition
to the complementary strand of the original ssDNA to ATP. ATP fuels luciferase in each well [2]. The light
produced is detected with a flourescence microscope [3]. The current (2009) 454 FLX system has the
ability to sequence 100 Mb DNA in 8 hours with an average read of 250 bp and raw accuracy of 99.5% [4].
The amplification of DNA using universal primers covalently bonded to the surface of the flow cell
produces 500-1000 clonal copies of the DNA fragments [8]. Fluorescently labeled nucleotides are
cyclically washed over the flow cell. These nucleotides are conjugated with reversible terminators so that
the four nucleotide bases can be simultaneously incorporated base by base across the flow cell. Laser
induced excitation of the cell allows imaging of the excited flourophores [9]. Before the next cycle, tris(2-
carboxyethyl)pho-sphine (TCEP) is added to knock off the flourescent dye and side chain (reversible
terminator) and bring back the 3' hydroxyl group, allowing for the next nucleotide addition [10]. The use of
a flow cell and reversible terminator allows the Illumina Genome Analyzer to produce 600 Mb of DNA per
day with only 36 bp reads. The trade-off between pyrosequencing methods and the flow cell method is
increased throughput for shorter reads. The raw accuracy of the Illumina genome analyzer is over 98.5%.
Increased coverage is necessary when using sequencers with high raw error rates [11].