Single-Molecule DNA Sequencing of a Viral Genome

Harris, T. D.; Buzby, Phillip R.; Babcock, Hazen P.; Beer, E. George; Bowers, Jayson; Braslavsky, Ido; Causey, Marie; Colonell, Jennifer; DiMeo, James; Efcavitch, J. William; Giladi, Eldar; Gill, J.; Healy, John M.; Jarosz, Mirna; Lapen, Dan; Moulton, Keith; Quake, Stephen R.; Steinmann, Kathleen E.; Thayer, Edward C.; Tyurina, Anastasia; Ward, R. Matthew; Weiss, Howard; Xie, Zhoujie

doi:10.1126/science.1150427

Cited by 597 publications

(417 citation statements)

References 14 publications

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“…Next-generation sequencing (NGS) platforms have transformed genetic variation studies by a massive reduction of cost and sequencing effort (Shendure et al 2004(Shendure et al , 2005Margulies et al 2005;Bentley 2006;Johnson et al 2007;Harris et al 2008). However, this technology advance has not yet been matched by an equal improvement at the front end: the isolation of target DNA sequences for analysis (Garber 2008).…”

mentioning

confidence: 99%

Microarray-based multicycle-enrichment of genomic subsets for targeted next-generation sequencing

Summerer¹,

Wu²,

Haase³

et al. 2009

Genome Res.

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The lack of efficient high-throughput methods for enrichment of specific sequences from genomic DNA represents a key bottleneck in exploiting the enormous potential of next-generation sequencers. Such methods would allow for a systematic and targeted analysis of relevant genomic regions. Recent studies reported sequence enrichment using a hybridization step to specific DNA capture probes as a possible solution to the problem. However, so far no method has provided sufficient depths of coverage for reliable base calling over the entire target regions. We report a strategy to multiply the enrichment performance and consequently improve depth and breadth of coverage for desired target sequences by applying two iterative cycles of hybridization with microfluidic Geniom biochips. Using this strategy, we enriched and then sequenced the cancer-related genes BRCA1 and TP53 and a set of 1000 individual dbSNP regions of 500 bp using Illumina technology. We achieved overall enrichment factors of up to 1062-fold and average coverage depths of 470-fold. Combined with high coverage uniformity, this resulted in nearly complete consensus coverages with >86% of target region covered at 20-fold or higher. Analysis of SNP calling accuracies after enrichment revealed excellent concordance, with the reference sequence closely mirroring the previously reported performance of Illumina sequencing conducted without sequence enrichment.

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mentioning

confidence: 99%

Microarray-based multicycle-enrichment of genomic subsets for targeted next-generation sequencing

Summerer¹,

Wu²,

Haase³

et al. 2009

Genome Res.

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“…These approaches also have the potential to identify unknown cofactors of a protein complex. When combined with single-molecule ELISA (enzyme-linked immunosorbent assay) [57], single-molecule sequencing technologies [58][59][60], single-molecule multi-color FRET (fluorescence resonance energy transfer) [61][62][63], and screening tools, the new techniques discussed in this review will acquire versatility and be used for practical analysis in the near future, complementing mass spectroscopy techniques.…”

Section: Discussionmentioning

confidence: 99%

Bringing single-molecule spectroscopy to macromolecular protein complexes

Joo

Fareh

Kim

2013

Trends in Biochemical Sciences

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Single-molecule fluorescence spectroscopy offers real-time, nanometer-resolution information. Over the past two decades, this emerging single-molecule technique has been rapidly adopted to investigate the structural dynamics and biological functions of proteins. Despite this remarkable achievement, single-molecule fluorescence techniques must be extended to macromolecular protein complexes that are physiologically more relevant for functional studies. In this review, we present recent major breakthroughs for investigating protein complexes within cell extracts using single-molecule fluorescence. We outline the challenges, future prospects and potential applications of these new single-molecule fluorescence techniques in biological and clinical research. Single-molecule protein studiesSingle-molecule techniques have become potent tools for the discovery of novel protein mechanisms by allowing high spatiotemporal resolution. Sub-nanometer resolution, the ultimate scale of biological systems, has been reached with single-molecule fluorescence microscopy[1] and spectroscopy [2]; single-molecule force and torque spectroscopy [3,4]; atomic force microscopy [5] and spectroscopy [6]; and nanopores [7]. Measurements can be carried out on the biologically relevant time scales of microseconds to minutes.Among these techniques, single-molecule fluorescence spectroscopy has enabled researchers to unveil the action mechanism of a protein by imaging protein activity in real time. Benefitting from advances in general microscopy[1], detection devices [8], and fluorophore physics [9,10], single-molecule fluorescence spectroscopy has reached sub-nanometer spatial resolution [11] and microsecond temporal resolution [12,13]. Single-molecule fluorescence imaging is carried out primarily with total internal reflection, confocal, and zero-mode waveguide microscopy [2]. Among these, total internal reflection fluorescence microscopy has been employed by several research teams in the development of novel techniques described in this review [14][15][16][17] Single-molecule observations using cell extractsUnlike purified recombinant proteins, crude cell extracts provide more biologically relevant environment in which molecules of interest can interact not only with their partners but also with other cellular components. It was anticipated to combine this approach with single molecule techniques and observe the assembly and function of macromolecular complexes in real time. However, technical hurdles related to the specificity and efficiency of labeling molecules of interest within crude cell extracts had been reported [22][23][24]. In order to overcome this limitation, several groups recently developed original approaches. Hoskins et al. used protein complexes specifically tagged with fluorophores [14,19], and Yardimci et al. immunostained reaction products using specific fluorescent antibodies [15,20]. Real-time observation of spliceosome assemblyDespite the relatively small number of genes in the human genome, we have extraordin...

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“…The third generation technologies can be divided into categories based on the method they use to detect the DNA sequence: Fluorescence Most of the third generation sequencing platforms under development using fluorescence detection are based on the standard sequencing-by-synthesis method. The first single molecule sequencing platform to market was the Heliscope from Helicos Bioscience (Harris et al 2008), launched in 2009, which is based on a similar methodology to that described for the Solexa/Illumina second generation platform. However this is a single molecule method and all the nucleotides have the same fluorescent label which acts as the terminating moiety.…”

Section: Third Generation Sequencingmentioning

confidence: 99%

Review of massively parallel DNA sequencing technologies

Moorthie

Mattocks

Wright

2011

HUGO J

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Since the development of technologies that can determine the base-pair sequence of DNA, the ability to sequence genes has contributed much to science and medicine. However, it has remained a relatively costly and laborious process, hindering its use as a routine biomedical tool. Recent times are seeing rapid developments in this field, both in the availability of novel sequencing platforms, as well as supporting technologies involved in processes such as targeting and data analysis. This is leading to significant reductions in the cost of sequencing a human genome and the potential for its use as a routine biomedical tool. This review is a snapshot of this rapidly moving field examining the current state of the art, forthcoming developments and some of the issues still to be resolved prior to the use of new sequencing technologies in routine clinical diagnosis.

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Single-Molecule DNA Sequencing of a Viral Genome

Cited by 597 publications

References 14 publications

Microarray-based multicycle-enrichment of genomic subsets for targeted next-generation sequencing

Microarray-based multicycle-enrichment of genomic subsets for targeted next-generation sequencing

Bringing single-molecule spectroscopy to macromolecular protein complexes

Review of massively parallel DNA sequencing technologies

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