Single Molecule DNA Sequencing: from Demonstration to Application

Abstract

In recent years, single molecule DNA sequencing by cyclic synthesis has progressed from the demonstration stage [1] to a working system with high throughput DNA [2,3], cDNA [4], and direct RNA [5] unbiased sequencing capabilities. In this system, fluorescence microscopy is used to individually monitor tens of millions of immobilized DNA or RNA molecules for incorporation of labeled nucleotides. This process yields read lengths with sufficient sequence information to allow reliable and unique alignment of most tested fragments to a reference sequence, supporting a sequencing method that is amplification-free, fast and cheap. In this presentation, various aspects of single molecule sequencing by cyclic synthesis will be discussed. Low cost and high throughput DNA and RNA sequencing methods will usher in a new era of personal medicine. 1. Braslavsky I, Hebert B, Kartalov E, Quake SR: Sequence information can be obtained from single DNA molecules. Proceedings Of The National Academy Of Sciences Of The United States Of America 2003, 100:3960–3964. 2. Harris TD, Buzby PR, Babcock H, Beer E, Bowers J, Braslavsky I, Causey M, Colonell J, Dimeo J, Efcavitch JW, et al.: Single-molecule DNA sequencing of a viral genome. Science 2008, 320:106–109. 3. Pushkarev D, Neff NF, Quake SR: Single-molecule sequencing of an individual human genome. Nature Biotechnology 2009, 27:847–850. 4. Lipson D, Raz T, Kieu A, Jones DR, Giladi E, Thayer E, Thompson JF, Letovsky S, Milos P, Causey M: Quantification of the yeast transcriptome by single-molecule sequencing. Nature Biotechnology 2009, 27:652–658. 5. Ozsolak F, Platt AR, Jones DR, Reifenberger JG, Sass LE, McInerney P, Thompson JF, Bowers J, Jarosz M, Milos PM: Direct RNA sequencing. Nature 2009, advance online publication.