Dramatic improvements in DNA sequencing technology have revolutionized our ability to characterize most genomic diversity. However, accurate resolution of large structural events has remained challenging due to the comparatively shorter read lengths of second-generation technologies. Emerging third-generation sequencing technologies, which yield markedly increased read length on rapid time scales and for low cost, have the potential to address assembly limitations. Here we combine sequencing data from second- and third-generation DNA sequencing technologies to assemble the two-chromosome genome of a recent Haitian cholera outbreak strain into two nearly finished contigs at > 99.9% accuracy. Complex regions with clinically significant structure were completely resolved. In separate control assemblies on experimental and simulated data for the canonical N16961 reference we obtain 14 and 8 scaffolds greater than 1kb, respectively, correcting several errors in the underlying source data. This work provides a blueprint for the next generation of rapid microbial identification and full-genome assembly.

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The global structure of the hammerhead ribozyme was determined in the absence of Mg(2+) by solution NMR experiments. The hammerhead ribozyme motif forms a branched structure consisting of three helical stems connected to a catalytic core. The (1)H-(15)N and (1)H-(13)C residual dipolar couplings were measured in a set of differentially (15)N/(13)C-labeled ribozymes complexed with an unlabeled noncleavable substrate. The residual dipolar couplings provide orientation information on both the local and the global structure of the molecule. Analysis of the residual dipolar couplings demonstrated that the local structure of the three helical stems in solution is well modeled by an A-form conformation. However, the global structure of the hammerhead in solution in the absence of Mg(2+) is not consistent with the Y-shaped conformation observed in crystal structures of the hammerhead. The residual dipolar couplings for the helical stems were combined with standard NOE and J coupling constant NMR data from the catalytic core. The NOE data show formation of sheared G-A base pairs in domain 2. These NMR data were used to determine the global orientation of the three helical stems in the hammerhead. The hammerhead forms a rather extended structure under these conditions with a large angle between stems I and II ( approximately 153 degrees ), a smaller angle between stems II and III ( approximately 100 degrees ), and the smallest angle between stems I and III ( approximately 77 degrees ). The residual dipolar coupling data also contain information on the dynamics of the molecule and were used here to provide qualitative information on the flexibility of the helical domains in the hammerhead ribozyme-substrate complex.

An improved method is presented for the preparation of milligram quantities of homogenous-length RNAs suitable for nuclear magnetic resonance or X-ray crystallographic structural studies. Heterogeneous-length RNA transcripts are processed with a hammerhead ribozyme to yield homogenous-length products that are then readily purified by anion exchange high-performance liquid chromatography. This procedure eliminates the need for denaturing polyacrylamide gel electrophoresis, which is the most laborious step in the standard procedure for large-scale production of RNA by in vitro transcription. The hammerhead processing of the heterogeneous-length RNA transcripts also substantially improves the overall yield and purity of the desired RNA product.