The properties and applications of single-molecule DNA sequencing

Abstract

Single-molecule sequencing enables DNA or RNA to be sequenced directly from biological samples, making it well-suited for diagnostic and clinical applications. Here we review the properties and applications of this rapidly evolving and promising technology.

Figure 1

Sequence database submissions from 1982 to 2010. Nucleotides submitted to the classical version of GenBank (diamonds, thin line) and to the Sequence Read Archive (circles, thick line) are shown as a function of time. Data for GenBank up to 2008 were obtained from the NCBI website [68] and subsequent years were obtained from GenBank publications [69,70]. Data for SRA was obtained from publications for 2008 to 2010 [71-73] and estimated for 2007 on the basis of 44 projects being in the database at the end of the year [74] and using February 2008 data from NCBI [75] to estimate the approximate number of bases likely to have been submitted from that spectrum of projects. Key advances in sequencing technology are shown with arrows. The development of second generation sequencing technologies and single-molecule sequencing has had a dramatic increase in the number of sequences deposited in public databases. Less than a year after its initiation, the SRA had already surpassed classical GenBank and it now accounts for over 95% of all new sequence deposits.

Figure 2

Overview of single-molecule sequencers. The three most advanced single-molecule sequencing systems all carry out sequencing-by-synthesis using laser excitation to generate a fluorescent signal from labeled nucleotides, which is then detected using a camera. (a) In the Helicos BioSciences system [18], single nucleotides, each with a fluorescent dye attached to the base, are sequentially added. (b,c) In the Pacific Biosciences [35] and Life Technologies [41] systems, four different nucleotides, each with a different color dye attached to the phosphates, are continuously added. Background fluorescence is minimized differently in the three systems. (a) Helicos uses total internal reflectance fluorescence (TIRF) to create a narrow evanescent field of light in which the intensity of the light decays exponentially away from the glass surface. Only dyes that are in the TIRF evanescent field can fluoresce. (b) Pacific Biosciences uses a zero mode waveguide (ZMW), which limits illumination to a narrow region near the bottom of the well containing the polymerase. Only dyes near the opening of the ZMW can fluoresce. (c) Life Technologies uses fluorescence resonance energy transfer (FRET) between the initially absorbing quantum dot on the polymerase and the emitting dye on the nucleotide. Only dyes close to the polymerase-attached quantum dot can be excited by FRET and then fluoresce. For the three systems, DNA is immobilized for viewing over time by a surface-attached sequencing primer (Helicos (a)), by interaction with a surface-bound polymerase (Pacific Biosciences (b)), or by ligating to a surface-attached oligonucleotide (Life Technologies (c)). For Helicos (a), the polymerase is replaced after every cycle of nucleotide addition. For Life Technologies (c), the polymerase can be replaced on a given DNA molecule after each read is completed. For Pacific Biosciences (b), the polymerase cannot be replaced.

Figure 3

The attributes of single-molecule sequencing technology. The current read counts and read lengths for single-molecule sequencing technologies are shown by the dots. Each technology is striving for improvements in their key attributes with the research aimed in the directions shown by the arrow.