Molecular markers
DNA sequencing
Illumina genome analyzer

Author: Rodney E. Shackelford, D.O., Ph.D. (see Authors page)

Revised: 30 May 2018, last major update February 2011

Copyright: (c) 2008-2018, PathologyOutlines.com, Inc.

PubMed Search: Illumina genome analyzer[TIAB]

Table of Contents
Definition / general
Cite this page: Shackelford, R.E. Illumina genome analyzer. PathologyOutlines.com website. http://www.pathologyoutlines.com/topic/molecularpathdnaseqillumina.html. Accessed July 17th, 2018.
Definition / general
  • The Illumina Genome Analyzer, similar to the 454 FLX sequencer, begins by constructing a sequence mixed library of DNA fragments of up to several hundred bp
  • Adaptor sequences with forward and reverse PCR primers are attached to each end of these DNA fragments
  • The adaptor linked sequences are then amplified by bridge PCR, where both the forward and reverse PCR primers are tethered to a solid substrate by a flexible linker
  • During PCR, the amplified sequences remain immobilized and clustered in one location on the array; eventually there are 1,000 to 1 million clustered amplicons, sufficient for reporting incorporated bases as the required signal intensity
  • Several million different clusters can be amplified and distinguished at different locations within eight independent lanes that are on a single flow cell; thus, eight different libraries can be sequenced simultaneously
  • Once the amplification step is completed, the amplicons are made single stranded and a universal sequencing primer is hybridized to a universal sequence flanking the DNA to be sequenced
  • Sequencing occurs with each single base addition, using a modified DNA polymerase and a mix of four modified dNTPs, each with two different modifications:
    1. The dNTPs have a chemically cleavable moiety (a "reversible terminator") at their 3' hydroxyl position that allows only one nucleotide incorporation
    2. Each dNTP has a distinct fluorescent label that can also be chemically cleaved
  • Thus, after one round of dNTP incorporation has occurred, it can be photometically measured
  • The reversible terminator and fluorescent label are then chemically cleaved, washed away and the DNA sequencing can proceed by adding another labeled dNTP series
  • The read length with this technique is about 36 bp; longer reads are possible but have higher error rates
  • The main limiting factors are incomplete cleavage of the fluorescent labels and terminating moieties, all of which result in signal decay and dephasing
  • Homopolyer stretches are far less of a problem with this sequencing platform that with the 454 FLX sequencer
  • Combining different sequences allows this platform to sequence several hundred nucleotides per run