Comparison of next generation sequencing technologies for transcriptome characterization

<p>Abstract</p> <p>Background</p> <p>We have developed a simulation approach to help determine the optimal mixture of sequencing methods for most complete and cost effective transcriptome sequencing. We compared simulation results for traditional capillary sequencing with "Next Generation" (NG) ultra high-throughput technologies. The simulation model was parameterized using mappings of 130,000 cDNA sequence reads to the <it>Arabidopsis </it>genome (NCBI Accession SRA008180.19). We also generated 454-GS20 sequences and <it>de novo </it>assemblies for the basal eudicot California poppy (<it>Eschscholzia californica</it>) and the magnoliid avocado (<it>Persea americana</it>) using a variety of methods for cDNA synthesis.</p> <p>Results</p> <p>The <it>Arabidopsis </it>reads tagged more than 15,000 genes, including new splice variants and extended UTR regions. Of the total 134,791 reads (13.8 MB), 119,518 (88.7%) mapped exactly to known exons, while 1,117 (0.8%) mapped to introns, 11,524 (8.6%) spanned annotated intron/exon boundaries, and 3,066 (2.3%) extended beyond the end of annotated UTRs. Sequence-based inference of relative gene expression levels correlated significantly with microarray data. As expected, NG sequencing of normalized libraries tagged more genes than non-normalized libraries, although non-normalized libraries yielded more full-length cDNA sequences. The <it>Arabidopsis </it>data were used to simulate additional rounds of NG and traditional EST sequencing, and various combinations of each. Our simulations suggest a combination of FLX and Solexa sequencing for optimal transcriptome coverage at modest cost. We have also developed ESTcalc <url>http://fgp.huck.psu.edu/NG_Sims/ngsim.pl</url>, an online webtool, which allows users to explore the results of this study by specifying individualized costs and sequencing characteristics.</p> <p>Conclusion</p> <p>NG sequencing technologies are a highly flexible set of platforms that can be scaled to suit different project goals. In terms of sequence coverage alone, the NG sequencing is a dramatic advance over capillary-based sequencing, but NG sequencing also presents significant challenges in assembly and sequence accuracy due to short read lengths, method-specific sequencing errors, and the absence of physical clones. These problems may be overcome by hybrid sequencing strategies using a mixture of sequencing methodologies, by new assemblers, and by sequencing more deeply. Sequencing and microarray outcomes from multiple experiments suggest that our simulator will be useful for guiding NG transcriptome sequencing projects in a wide range of organisms.</p

Nodeomics: Pathogen Detection in Vertebrate Lymph Nodes Using Meta-Transcriptomics

The ongoing emergence of human infections originating from wildlife highlights the need for better knowledge of the microbial community in wildlife species where traditional diagnostic approaches are limited. Here we evaluate the microbial biota in healthy mule deer (Odocoileus hemionus) by analyses of lymph node meta-transcriptomes. cDNA libraries from five individuals and two pools of samples were prepared from retropharyngeal lymph node RNA enriched for polyadenylated RNA and sequenced using Roche-454 Life Sciences technology. Protein-coding and 16S ribosomal RNA (rRNA) sequences were taxonomically profiled using protein and rRNA specific databases. Representatives of all bacterial phyla were detected in the seven libraries based on protein-coding transcripts indicating that viable microbiota were present in lymph nodes. Residents of skin and rumen, and those ubiquitous in mule deer habitat dominated classifiable bacterial species. Based on detection of both rRNA and protein-coding transcripts, we identified two new proteobacterial species; a Helicobacter closely related to Helicobacter cetorum in the Helicobacter pylori/Helicobacter acinonychis complex and an Acinetobacter related to Acinetobacter schindleri. Among viruses, a novel gamma retrovirus and other members of the Poxviridae and Retroviridae were identified. We additionally evaluated bacterial diversity by amplicon sequencing the hypervariable V6 region of 16S rRNA and demonstrate that overall taxonomic diversity is higher with the meta-transcriptomic approach. These data provide the most complete picture to date of the microbial diversity within a wildlife host. Our research advances the use of meta-transcriptomics to study microbiota in wildlife tissues, which will facilitate detection of novel organisms with pathogenic potential to human and animals

Folylpoly-gamma-glutamate synthetase: Kinetics of multiple glutamate ligations.

Folylpoly-gamma-glutamate synthetase (FPGS) is an ATP-dependent ligase that catalyzes the formation of the poly-gamma-glutamate chain of folates and anti-folates such as methotrexate and lometrexol(TM). While the chemical mechanism of the reaction catalyzed by FPGS is known, it is unknown whether a single glutamate residue or multiple glutamate residues are added for each folate binding event. These are the two mechanistic possibilities, distributive or processive respectively, for successive glutamate additions to the folate substrate. In a distributive mechanism, the enzyme and products dissociate between each addition and the enzyme then randomly distributes itself among all available termini. A processive mechanism is one in which the enzyme processes along the same substrate chain catalyzing multiple additions without dissociating. This research is focused on determining which of these mechanisms is utilized by FPGS and developing methods for the study of multiple ligation reactions. Specifically, recombinant human FPGS (hFPGS) with the antifolate drug lometrexol(TM) were chosen as the enzyme-substrate pair. Two assays have been developed for studying hFPGS. The first is a continuous spectrophotometric assay utilizing the pyruvate kinase/lactate dehydrogenase enzymatic coupling system, used in the study of an ATPase reaction catalyzed by hFPGS. The second assay is a non-continuous, radiation-based assay that relies on ion-pair HPLC separation of the folate products of the hFPGS reaction followed by liquid scintillation counting. A new synthesis of lometrexol(TM) ((6R )-5,10-dideazatetrahydrofolic acid), radio-labeled lometrexol(TM), and the poly-gamma-glutamates of lometrexol(TM) for use in the study of the hFPGS reaction is reported. These tools allowed a careful analysis of the time course of formation of the various chain-length products to be accomplished. These time course data were used to determine the relative rates of extension of the different polyglutamate substrates. Another set of experiments that directly addressed the processivity of hFPGS were substrate trapping and pulse-chase experiments. These experiments utilized a pulse of [14C]-lometrexol(TM) and a chase or trap of unlabeled lometrexol(TM) and provided a direct measure of folate release during multiple glutamate additions. Together, the results of these experiments indicate that hFPGS catalyzes the processive addition of approximately four glutamate residues onto the antifolate lometrexol(TM).Ph.D.BiochemistryPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/124958/2/3163951.pd

Examination of the Reactivity of Benzoxaboroles and Related Compounds with a <i>cis</i>-Diol

Benzoxaboroles have been emerging as an interesting and useful scaffold in drug discovery due to their apparently unique reactivity toward diols under physiological conditions. In this work, the reaction of benzoxaborole with the diol-containing, fluorescent dye Alizarin Red S is probed. Steady-state and presteady-state experiments have been conducted for the characterization of the reactions over a wide range of pH. Results indicate that Alizarin Red S reacts with both the boronic (neutral, trigonal) form as well as the boronate (anionic, tetrahedral) form of benzoxaborole in a reaction largely analogous to that previously determined for the simple phenylboronic acid. However, in certain key aspects, the reactivity of the benzoxaborole was found to differ from that of simple phenylboronic acid. The structural origin of these differences has been explored by examination of compounds related to both benzoxaborole and phenylboronic acid. These results may be applied to rational drug discovery efforts aimed at expanding the use of benzoxaboroles in medicine

Elucidation of the Mechanism of the Reaction between Phenylboronic Acid and a Model Diol, Alizarin Red S

In this work, the reaction between phenylboronic acid and the diol-containing, fluorescent dye Alizarin Red S (<b>ARS</b>) was probed. Fluorescence titrations, ¹¹B NMR measurements, and both pre- and steady-state kinetic experiments were used for the characterization of this reaction over a large pH range (4–10.5). It was shown that <b>ARS</b> preferentially reacted with the boronic (neutral, trigonal) form of phenylboronic acid; however, the boronate (anionic, tetrahedral) form was also reactive. All in all, four reactant species were implicated in the formation of four different adduct species. The rate of a given adduct formation depended on the combination of the solution pH and the p<i>K</i>_a’s of both <b>ARS</b> and the arylboronic acid. The reaction was found to proceed in two distinct kinetic steps with the products and starting materials in facile exchange. In addition, the elucidation of the mechanism indicated the presence of two fluorescent products with the structure of the major contributor differing from what had been cited in the literature

Ring Structure and Aromatic Substituent Effects on the p<i>K</i>_a of the Benzoxaborole Pharmacophore

In this work, we present an investigation into the physical properties of a unique class of aromatic boronic acids, the benzoxaboroles. Using spectrophotometric methods, the ionization constants of a family of substituted benzoxaboroles are determined. Heterocyclic ring modifications are examined to determine their effects on the ionization of the boronic acid moiety. It is also shown that the substituent effects about the aromatic ring follow a Hammett relationship with the compounds' measured p<i>K</i>_a values. Finally, these substituent effects are also shown to extend to the sugar binding properties of these compounds under physiologically relevant conditions. Combined, these data will inform medicinal chemists wishing to tailor the ionization and/or ability of this class of compound to bind diol-containing biomolecules

Protection of the Benzoxaborole Moiety: Synthesis and Functionalization of Zwitterionic Benzoxaborole Complexes

The synthesis and utility of three benzoxaborole protecting groups are reported. These protecting groups improve organic solubility and allow otherwise incompatible reactions (oxidations, substitutions, and mild reductions) to be achieved in the presence of the benzoxaborole moiety. 3-(<i>N</i>,<i>N</i>-Dimethylamino)-1-propanol was determined to be useful in one-step sequences and is readily cleaved upon workup. Two other groups, <i>N</i>-methylsalicylidenimine and 2-[1-(methylimino)­ethyl]­phenol, are suitable for multistep syntheses. Deprotection with mild aqueous acid allows for chromatography-free isolation of the benzoxaborole in high yields