High-Speed DNA Sequencing: An Approach Based Upon Fluorescence Detection of Single Molecules

Automation for genomics has enabled a 43-fold increase in the total finished human genomic sequence in the world in the past four years. This is the second half of a two-part, noncomprehensive review that presents an overview of different types of automation equipment used in genome sequencing. The first part of the review, published in the previous issue, focused on automated procedures used to prepare DNA for sequencing or analysis. This second part of the review presents a look at available DNA sequencers and array technology and concludes with a look at future technologies. Alternate sequencing technologies including mass spectrometry, biochips, and single molecule analysis are included in this review.Sequencing data, essential for many of the medical breakthroughs of today and tomorrow, is currently accumulating at an exponential rate, largely because of advances in sequencing methods and laboratory automation. Instruments are available that can automate nearly every step in the large-scale sequencing process.The first article in this two-part review (Meldrum 2000) presented a description of automation designed for isolating DNA, cloning or amplifying DNA, preparing enzymatic sequencing reactions, and purifying DNA. Part one of the review also showed that the majority of genome centers use both in-house automation and commercial automation (Collins et al. 1998;Wendl et al. 1998;Mullikin and McMurragy 1999;Spurr et al. 1999; see http://www.genome.org for a supplementary table providing a snapshot view of automation used at a number of different genome centers).In this, the second part of the review, the focus is on automation after DNA preparation and sequencing reactions-on obtaining the sequence and its analysis. A discussion of future technologies for DNA sequencing projects is also included. (See the web sites referenced for photos or further details on the instruments presented.)

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“…Again, this approach may yield a very rapid, low cost sequencing method. See also Jett et al (1989) and Davis et al (1991).…”

Section: Single Molecule Analysismentioning

confidence: 99%

Automation for Genomics, Part Two: Sequencers, Microarrays, and Future Trends

Meldrum¹

2000

Genome Res.

116

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“…While the origins of SMS date back to 1989 [3], it is only now becoming practical. The Heliscope sequencer, sold by Helicos, is the first commercial product that allows for the sequencing of DNA with SMS.…”

Section: Introductionmentioning

confidence: 99%

Read Mapping Algorithms for Single Molecule Sequencing Data

Yanovsky¹,

Rumble²,

Brudno

2008

Lecture Notes in Computer Science

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Abstract. Single Molecule Sequencing technologies such as the Heliscope simplify the preparation of DNA for sequencing, while sampling millions of reads in a day. Simultaneously, the technology suffers from a significantly higher error rate, ameliorated by the ability to sample multiple reads from the same location. In this paper we develop novel rapid alignment algorithms for two-pass Single Molecule Sequencing methods. We combine the Weighted Sequence Graph (WSG) representation of all optimal and near optimal alignments between the two reads sampled from a piece of DNA with k-mer filtering methods and spaced seeds to quickly generate candidate locations for the reads on the reference genome. We also propose a fast implementation of the Smith-Waterman algorithm using vectorized instructions that significantly speeds up the matching process. Our method combines these approaches in order to build an algorithm that is both fast and accurate, since it is able to take complete advantage of both of the reads sampled during two pass sequencing.

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“…2 Using a 0.6-pL sample volume with conventional optics, Nguyen et al" demonstrated a detection limit of 800 molecules of Rhodamine 6G. Dovichi and co-workers' 4 ' 6 applied ultrasensitive detection techniques to the problem of amino acid analysis by capillary zone electrophoresis. Using BPhycoerythrin, a large molecule containing the equivalent of 25 Rhodamine 6G (R6G) chromophores, Peck et al 7 observed nonrandom features in the autocorrelation of the signal collected with the passage of many molecules and indicated that they had achieved a detection efficiency of 15% with a false detection rate of 3 errors/s.…”

Section: Introductionmentioning

confidence: 99%

“…A new urgency came to the field when it was suggested that DNA may be sequenced at rates many times those of current methods by sequential enzymatic cleavage of an appropriately labeled single DNA strand followed by detection and identification of the individual labeled nucleotides. 4 ' 5 Hirschfeld 6 ' 7 first reported the detection of a single biological molecule (MW 20,000) that was tagged with 80-100 chromophores. Dovichi et al 8 ' 9 later suggested laserinduced fluorescence as an approach for single-molecule detection in liquids.…”

Section: Introductionmentioning

confidence: 99%

Detection and identification of single molecules in solution

1992

Self Cite

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We have extended our recent experiments in the detection of single fluorescent molecules in solution to the exploration of spectroscopy at the single-molecule level. As a first step we have developed a technique that can efficiently distinguish between two species of dye molecules on the basis of differences in their emission spectra. We have also demonstrated that another spectroscopic property, fluorescence lifetime, can be accurately determined at the single-molecule level. Spectroscopic properties can be used to identify fluorescent molecules and to reveal static or slowly varying aspects of the microenvironment of each molecule, thereby yielding information unavailable from bulk studies.

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High-Speed DNA Sequencing: An Approach Based Upon Fluorescence Detection of Single Molecules

Cited by 147 publications

References 20 publications

Automation for Genomics, Part Two: Sequencers, Microarrays, and Future Trends

Automation for Genomics, Part Two: Sequencers, Microarrays, and Future Trends

Read Mapping Algorithms for Single Molecule Sequencing Data

Detection and identification of single molecules in solution

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