Direct RNA sequencing.

Our understanding of human biology and disease is ultimately dependent on a complete understanding of the genome and its functions. The recent application of microarray and sequencing technologies to transcriptomics has changed the simplistic view of transcriptomes to a more complicated view of genome-wide transcription where a large fraction of transcripts emanates from unannotated parts of genomes, and underlined our limited knowledge of the dynamic state of transcription. Most of this broad body of knowledge was obtained indirectly because current transcriptome analysis methods typically require RNA to be converted to complementary DNA (cDNA) before measurements, even though the cDNA synthesis step introduces multiple biases and artefacts that interfere with both the proper characterization and quantification of transcripts. Furthermore, cDNA synthesis is not particularly suitable for the analysis of short, degraded and/or small quantity RNA samples. Here we report direct single molecule RNA sequencing without prior conversion of RNA to cDNA. We applied this technology to sequence femtomole quantities of poly(A)(+) Saccharomyces cerevisiae RNA using a surface coated with poly(dT) oligonucleotides to capture the RNAs at their natural poly(A) tails and initiate sequencing by synthesis. We observed transcript 3' end heterogeneity and polyadenylated small nucleolar RNAs. This study provides a path to high-throughput and low-cost direct RNA sequencing and achieving the ultimate goal of a comprehensive and bias-free understanding of transcriptomes.

Virtual terminator nucleotides for next-generation DNA sequencing.

We synthesized reversible terminators with tethered inhibitors for next-generation sequencing. These were efficiently incorporated with high fidelity while preventing incorporation of additional nucleotides, and we used them to sequence canine bacterial artificial chromosomes in a single-molecule system that provided even coverage for over 99% of the region sequenced. This single-molecule approach generated high-quality sequence data without the need for target amplification and thus avoided concomitant biases.

A Rapid, Accurate, Single Molecule Counting Method Detects Clostridium difficile Toxin B in Stool Samples.

We describe a new rapid and accurate immunoassay-based technology capable of counting single target molecules using digital imaging without magnification. Using the technology, we developed a rapid test for Clostridium difficile toxin B, which is responsible for the pathology underlying potentially fatal C. difficile infections (CDI). There are currently no tests for CDI that are rapid, sensitive, and specific. The MultiPath C. difficile toxin B test images and counts complexes of target-specific magnetic and fluorescent particles that have been tethered together by toxin B molecules in minimally processed stool samples. The performance characteristics of the 30 minute test include a limit of detection of 45 pg/mL, dynamic range covering 4-5 orders of magnitude, and coefficient of variation of less than 10%. The MultiPath test detected all toxinotypes and ribotypes tested, including the one most commonly occurring in the US and EU; shows no cross reactivity with relevant bacterial species; and is robust to potential interferants commonly present in stool samples. On a training set of 320 clinical stool samples, the MultiPath C. difficile toxin B test showed 97.0% sensitivity (95% CI, 91.4-99.4%); 98.3% specificity (95% CI, 96.8-99.2%); and 98.2% accuracy (95% CI, 96.7-99.0%) compared to the cellular cytotoxicity neutralization assay (CCNA) reference method. Based on these compelling performance characteristics, we believe the MultiPath technology can address the lack of rapid, sensitive, specific, and easy-to-use diagnostic tests for C. difficile.

Single-molecule DNA sequencing of a viral genome.

The full promise of human genomics will be realized only when the genomes of thousands of individuals can be sequenced for comparative analysis. A reference sequence enables the use of short read length. We report an amplification-free method for determining the nucleotide sequence of more than 280,000 individual DNA molecules simultaneously. A DNA polymerase adds labeled nucleotides to surface-immobilized primer-template duplexes in stepwise fashion, and the asynchronous growth of individual DNA molecules was monitored by fluorescence imaging. Read lengths of >25 bases and equivalent phred software program quality scores approaching 30 were achieved. We used this method to sequence the M13 virus to an average depth of >150x and with 100% coverage; thus, we resequenced the M13 genome with high-sensitivity mutation detection. This demonstrates a strategy for high-throughput low-cost resequencing.

Sequential ATP hydrolysis by Cdc6 and ORC directs loading of the Mcm2-7 helicase.

Loading of the Mcm2-7 DNA replicative helicase onto origin-proximal DNA is a critical and tightly regulated event during the initiation of eukaryotic DNA replication. The resulting protein-DNA assembly is called the prereplicative complex (pre-RC), and its formation requires the origin recognition complex (ORC), Cdc6, Cdt1, and ATP. ATP hydrolysis by ORC is required for multiple rounds of Mcm2-7 loading. Here, we investigate the role of ATP hydrolysis by Cdc6 during pre-RC assembly. We find that Cdc6 is an ORC- and origin DNA-dependent ATPase that functions at a step preceding ATP hydrolysis by ORC. Inhibiting Cdc6 ATP hydrolysis stabilizes Cdt1 on origin DNA and prevents Mcm2-7 loading. In contrast, the initial association of Mcm2-7 with the other pre-RC components does not require ATP hydrolysis by Cdc6. Importantly, these coordinated yet distinct functions of ORC and Cdc6 ensure the correct temporal and spatial regulation of pre-RC formation.

ATP hydrolysis by ORC catalyzes reiterative Mcm2-7 assembly at a defined origin of replication.

The origin recognition complex (ORC) is a six-subunit, ATP-regulated, DNA binding protein that is required for the formation of the prereplicative complex (pre-RC), an essential replication intermediate formed at each origin of DNA replication. In this study, we investigate the mechanism of ORC function during pre-RC formation and how ATP influences this event. We demonstrate that ATP hydrolysis by ORC requires the coordinate function of the Orc1 and Orc4 subunits. Mutations that eliminate ORC ATP hydrolysis do not support cell viability and show defects in pre-RC formation. Pre-RC formation involves reiterative loading of the putative replicative helicase, Mcm2-7, at the origin. Importantly, preventing ORC ATP hydrolysis inhibits this repeated Mcm2-7 loading. Our findings indicate that ORC is part of a helicase-loading molecular machine that repeatedly assembles Mcm2-7 complexes onto origin DNA and suggest that the assembly of multiple Mcm2-7 complexes plays a critical role in origin function.

Mapping subunit location on the Saccharomyces cerevisiae origin recognition complex free and bound to DNA using a novel nanoscale biopointer.

The Saccharomyces cerevisiae origin recognition complex (ORC) is composed of six subunits and is an essential component in the assembly of the replication apparatus. To probe the organization of this multiprotein complex by electron microscopy, each subunit was tagged on either its C or N terminus with biotin and assembled into a complex with the five other unmodified subunits. A nanoscale biopointer consisting of a short DNA duplex with streptavidin at one end was used to map the location of the N and C termini of each subunit. These observations were made using ORC free in solution and bound to the ARS1 origin of replication. This mapping confirms and extends previous studies mapping the sites of subunit interaction with origin DNA. In particular, we provide new information concerning the stoichiometry of the ORC-ARS1 complex and the changes in conformation that are associated with DNA binding by ORC. This versatile, new approach to mapping protein structure has potential for many applications.