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Molecular Pathology

DNA sequencing

Roche 454 FLX Pyrosequencer


Author: Rodney E. Shackelford, D.O., Ph.D., Tulane University (see Reviewers page)
Revised: 13 February 2011, last major update February 2011
Copyright: (c) 2008-2011, PathologyOutlines.com, Inc.

General
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● The Roche 454 Pyrosequencer was introduced in 2004, and for many applications, has replaced Sanger sequencing based instruments due to its high accuracy, low cost and relatively long reads
● It uses pyrosequencing technology, initially fragmenting a genome by nebulization into 300-800 bp fragments which are then “polished” to produce blunt ends; the ends are ligated to two specific adaptor sequences, one with a 5’-biotin tag for immobilization of the DNA onto 28 mm streptavidin-coated beads
● The DNA is separated into single strands and bound to beads under conditions which favor the binding of one DNA fragment per bead
● The beads are compartmentalized in droplets of PCR reaction mixture in oil emersion, and the PCR reactions occurs within each droplet, resulting in up to ten million DNA fragment copies bound to the bead
● Next, the emulsion is broken, the DNA strands are denatured, and each bead carrying many copies of the now single-stranded DNA fragments are deposited into an individual well in a fiber-optic slide
● Each well has a diameter of about 44 mm and each slide has about 1.6 million wells
● To reduce well to well "cross talk", usually not all the wells are filled; this reduces interference of light signals from adjacent wells, which would lower sequencing accuracy and efficiency
● In general, enough wells are filled to allow about 400,000 parallel reads
● Smaller beads carrying immobilized enzymes required for pyrosequencing are then deposited in each well
● The DNA sequence is read as light pulses when the luciferin-produced photon emissions are compared to the dNTP used in the reaction
● The sequencing reaction is less efficient with homopolymer runs of six of more nucleotides
● The 454 FLX Sequencer combines initial sequencing data, while screening out poor-quality sequences, mixed sequences (i.e. more than one initial DNA fragment/bead), and sequences lacking the initial priming sequence used to create the expanded DNA sequence
● The 454 FLX Pyrosequencer can give 100 Mb of quality data, sufficient to sequence the smaller genomes of bacteria and viruses
● The first large sequencing done with this platform was the 580,069 bp Mycoplasma genitalium genome in 2004 (Nature 2005;437:376)

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