Despite the high abundance of Aquificae in many geothermal systems, these bacteria are difficult to culture and no viruses infecting members of this phylum have been isolated. Here, we describe the complete, circular dsDNA Uncultivated Virus Genome (UViG) of Thermocrinis Octopus Spring virus (TOSV), derived from metagenomic data, along with eight related UViGs representing three additional viral species. Despite low overall similarity among viruses from different hot springs, the genomes shared a high degree of synteny, and encoded numerous genes for nucleotide metabolism, including a PolA-type DNA polymerase polyprotein with likely accessory functions, a DNA Pol III sliding clamp, a thymidylate kinase, a DNA gyrase, a helicase, and a DNA methylase. Also present were conserved genes predicted to code for phage capsid, large and small subunits of terminase, portal protein, holin, and lytic transglycosylase, all consistent with a distant relatedness to cultivated Caudovirales. These viruses are predicted to infect Aquificae, as multiple CRISPR spacers matching the viral genomes were identified within the genomes and metagenomic contigs from these bacteria. Based on the predicted atypical bi-directional replication strategy, low sequence similarity to known viral genomes, and unique position in gene-sharing networks, we propose a new putative genus, "Pyrovirus", in the order Caudovirales.

Staphylococcus aureus (S. aureus) infections range in severity due to expression of certain virulence factors encoded on mobile genetic elements (MGE). As such, characterization of these MGE, as well as single nucleotide polymorphisms, is of high clinical and microbiological importance. To understand the evolution of these dangerous pathogens, it is paramount to define reference strains that may predate MGE acquisition. One such candidate is S. aureus Tager 104, a previously uncharacterized strain isolated from a patient with impetigo in 1947.

Thermus aquaticus Y51MC23 was isolated from a boiling spring in the Lower Geyser Basin of Yellowstone National Park. Remarkably, this T. aquaticus strain is able to grow anaerobically and produces multiple morphological forms. Y51MC23 is a Gram-negative, rod-shaped organism that grows well between 50°C and 80°C with maximum growth rate at 65°C to 70°C. Growth studies suggest that Y51MC23 primarily scavenges protein from the environment, supported by the high number of secreted and intracellular proteases and peptidases as well as transporter systems for amino acids and peptides. The genome was assembled de novo using a 350 bp fragment library (paired end sequencing) and an 8 kb long span mate pair library. A closed and finished genome was obtained consisting of a single chromosome of 2.15 Mb and four plasmids of 11, 14, 70, and 79 kb. Unlike other Thermus species, functions usually found on megaplasmids were identified on the chromosome. The Y51MC23 genome contains two full and two partial prophage as well as numerous CRISPR loci. The high identity and synteny between Y51MC23 prophage 2 and that of Thermus sp. 2.9 is interesting, given the 8,800 km separation of the two hot springs from which they were isolated. The anaerobic lifestyle of Y51MC23 is complex, with multiple morphologies present in cultures. The use of fluorescence microscopy reveals new details about these unusual morphological features, including the presence of multiple types of large and small spheres, often forming a confluent layer of spheres. Many of the spheres appear to be formed not from cell envelope or outer membrane components as previously believed, but from a remodeled peptidoglycan cell wall. These complex morphological forms may serve multiple functions in the survival of the organism, including food and nucleic acid storage as well as colony attachment and organization.

Bioinformatics and functional screens identified a group of Family A-type DNA Polymerase (polA) genes encoded by viruses inhabiting circumneutral and alkaline hot springs in Yellowstone National Park and the US Great Basin. The proteins encoded by these viral polA genes (PolAs) shared no significant sequence similarity with any known viral proteins but were remarkably similar to PolAs encoded by two of three families of the bacterial phylum Aquificae and by several apicoplast-targeted PolA-like proteins found in the eukaryotic phylum Apicomplexa, which includes the obligate parasites Plasmodium, Babesia, and Toxoplasma. The viral gene products share signature elements previously associated only with Aquificae and Apicomplexa PolA-like proteins and were similar to proteins encoded by prophage elements of a variety of otherwise unrelated Bacteria, each of which additionally encoded a prototypical bacterial PolA. Unique among known viral DNA polymerases, the viral PolA proteins of this study share with the Apicomplexa proteins large amino-terminal domains with putative helicase/primase elements but low primary sequence similarity. The genomic context and distribution, phylogeny, and biochemistry of these PolA proteins suggest that thermophilic viruses transferred polA genes to the Apicomplexa, likely through secondary endosymbiosis of a virus-infected proto-apicoplast, and to the common ancestor of two of three Aquificae families, where they displaced the orthologous cellular polA gene. On the basis of biochemical activity, gene structure, and sequence similarity, we speculate that the xenologous viral-type polA genes may have functions associated with diversity-generating recombination in both Bacteria and Apicomplexa.

With the advent of more affordable “personal” next generation sequencing instruments from major platform providers, NGS applications are moving outside of core sequencing labs to enable a variety of projects. As the number of samples for sequencing increases, there is a need for library prep automation that matches throughput of the instruments while not requiring an investment equal to the sequencer itself. We are developing a combination of new NGS sample prep chemistry and a small automated platform that reduces time to final prepared library and increases efficiency and consistency. The DNA sample prep chemistry combines typical end-repair and A-tailing steps into one master mix step with buffers directly compatible with downstream ligation steps, eliminating the need for multiple cleanup steps throughout the process. The chemistry has also been optimized to drive higher A-tailing efficiencies, which reduces chimera formation from blunt fragments and loss of fragments tailed with nucleotides other than the necessary “A”. These chemistries were designed to be easily automatable, and subsequent development led to the design of a compact liquid handling platform to perform these tasks. The instrument described in this presentation allows numerous DNA libraries to be prepared simultaneously, including incorporation of barcoded adapters for multiplex PCR and sequencing. In contrast to other small, dedicated systems, the instrument is also open and programmable, meaning users can choose to utilize the platform for other applications. In this poster, we will discuss the feasibility of automating the chemistry on the instrument and the utility of an inexpensive NGS sample prep system for laboratories with a range of sample throughput needs. www.lucigen.com Democratizing Automated Sample Prep – Low Cost Automation of Next Gen Sequencing Library Prep for Moderate Throughput Laboratories Curtis Knox*, Svetlana Jasinovica*, Michael Lodes*, Sally Floyd*, David Mead*, Kevin Barrett**, Tristan Berto**, Seth Hanson** *Lucigen Corporation , Middleton, WI, USA ** Gilson, Inc., Middleton, WI, USA NxSeq® Sample Prep Technology The NxSeq DNA Sample Prep kit was developed to: • Reduce time to complete NGS sample prep. • Improve A-tailing and downstream ligation of fragments to adapters. An optimized master mix of enzymes and buffers enables end-repair and A-tailing of DNA fragments in one tube without the need for buffer exchange or sample cleanup. The result is a manual system for NGS sample prep from sheared DNA that cuts traditional workflow by up to 50% (2 hours) from the most commonly used method. In addition, hands-on time is reduced by up to 75% (see figure 1). Fig. 1 – Comparison of total time and hands-on time required for sample prep between NxSeq and common commercially available kits. NxSeq® DNA Prep Kit Validation Previously, we validated the performance of the NxSeq DNA Sample Prep kits by shearing DH10B E. coli genomic DNA and preparing identical samples with the TrueSeq® Sample prep kit v2, The Roche 454 Sample prep kit, and the NEBNext ® DNA Sample Prep Master Mix Set 1. All samples were verified for target size range on an Agilent Bioanalyzer and then run on an Illumina Genome Analyzer IIx using barcoded adapters (see figures 2 and 3). Figure 2. Bioanalizer trace comparison of DH10B Genomic DNA libraries made with NxSeq, NEB, and Roche prep kits. Equivalent performance was obtained for all kits. Figure 3. Coverage comparison of DH10B Genomic DNA libraries. Plots indicate depth of coverage achieved in a moving 100 base read window. Equivalent performance was obtained for all kits.

Genetic Engineering & Biotechnology NewsVol. 32, No. 2 Assay TutorialChimera-Free Library Prep for NGS PlatformsUsing Systems Efficiently and Economically to Obtain Desired Data at Reasonable CostMichael LodesMichael LodesMichael Lodes, Ph.D. (E-mail Address: mlodes@lucigen.com), is senior scientist at Lucigen. Web: www.lucigen.com.Search for more papers by this authorPublished Online:17 Jan 2012https://doi.org/10.1089/gen.32.2.11AboutSectionsView articleView Full TextPDF/EPUB ToolsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View articleFiguresReferencesRelatedDetailsCited ByTargeted multiplex next‐generation sequencing: advances in techniques of mitochondrial and nuclear DNA sequencing for population genomics25 January 2013 | Molecular Ecology Resources, Vol. 13, No. 2 Volume 32Issue 2Jan 2012 Information© 2012 by GEN PublishingTo cite this article:Michael Lodes.Chimera-Free Library Prep for NGS Platforms.Genetic Engineering & Biotechnology News.Jan 2012.20-21.http://doi.org/10.1089/gen.32.2.11Published in Volume: 32 Issue 2: January 17, 2012PDF download

The NxSeq ™ 40 kb Mate-Pair Cloning Kit facilitates the creation of scaffolds for de novo genome assembly. Supporting either Illumina or 454 sequencing, the kit produces long-span, mate-pair sequences with greater efficiency than existing protocols. In addition, NxSeq DNA Sample Prep Kits can be used to streamline workflow and speed up DNA library preparation for next-generation sequencing.

Please cite this paper as: Straight et al. (2010) A novel electrochemical device to differentiate pandemic (H1N1) 2009 from seasonal influenza. Influenza and Other Respiratory Viruses 4(2), 73–79. Background One of the challenges of the recent pandemic (H1N1) 2009 influenza outbreak was to differentiate the virus from seasonal influenza when confronting clinical cases. The determination of the virus has implications on treatment choice, and obvious epidemiologic significance. Objectives We set out to apply a novel electrochemical device to samples derived from clinical cases of pandemic (H1N1) 2009 influenza to examine the ability of the device to differentiate these samples from cases of seasonal influenza. Patients/Methods An IRB approved protocol allowed for the use of original nasal wash samples from 24 confirmed human cases pandemic (H1N1) 2009 influenza. Clinical samples from cases of seasonal influenza (Influenza A/H1N1, A/H3N2, and B) were included as controls. Nucleic acids were extracted and samples examined by the ElectraSense ® Influenza A assay (CombiMatrix, Inc). Samples were also examined by RT‐PCR or Luminex assays as a comparator. Results and Conclusions The ElectraSense ® Influenza A assay correctly identified 23 of 24 samples of laboratory‐confirmed pandemic (H1N1) 2009 Influenza. The assay correctly identified all samples of influenza A/H1N1 and A/H3N2, and differentiated these from pandemic (H1N1) 2009 Influenza in all cases. The ElectraSense ® Influenza A assay proved to be a useful assay to quickly and accurately differentiate pandemic (H1N1) 2009 influenza from seasonal influenza.

Micro RNAs (miRNAs) are a class of small, non-coding RNA species that play critical roles throughout cellular development and regulation. miRNA expression patterns taken from various tissue types often point to the cellular lineage of an individual tissue type, thereby being a more invariant hallmark of tissue type. Recent work has shown that these miRNA expression patterns can be used to classify tumor cells, and that this classification can be more accurate than the classification achieved by using messenger RNA gene expression patterns. One aspect of miRNA biogenesis that makes them particularly attractive as a biomarker is the fact that they are maintained in a protected state in serum and plasma, thus allowing the detection of miRNA expression patterns directly from serum. This study is focused on the evaluation of miRNA expression patterns in human serum for five types of human cancer, prostate, colon, ovarian, breast and lung, using a pan-human microRNA, high density microarray. This microarray platform enables the simultaneous analysis of all human microRNAs by either fluorescent or electrochemical signals, and can be easily redesigned to include newly identified miRNAs. We show that sufficient miRNAs are present in one milliliter of serum to detect miRNA expression patterns, without the need for amplification techniques. In addition, we are able to use these expression patterns to correctly discriminate between normal and cancer patient samples.

Although the most common use of DNA microarrays is gene expression profiling, microarrays are also used for many other applications, including genotyping, resequencing, SNP analysis, and DNA methylation assays. Here we describe genotyping arrays for Influenza A subtype identification and for upper respiratory pathogen diagnostics using standard hybridization techniques and we also describe resequencing, SNP, and methylation assays using an enzyme-based strategy [25, 26].

Bacterial and viral upper respiratory infections (URI) produce highly variable clinical symptoms that cannot be used to identify the etiologic agent. Proper treatment, however, depends on correct identification of the pathogen involved as antibiotics provide little or no benefit with viral infections. Here we describe a rapid and sensitive genotyping assay and microarray for URI identification using standard amplification and hybridization techniques, with electrochemical detection (ECD) on a semiconductor-based oligonucleotide microarray. The assay was developed to detect four bacterial pathogens (Bordetella pertussis, Streptococcus pyogenes, Chlamydia pneumoniae and Mycoplasma pneumoniae) and 9 viral pathogens (adenovirus 4, coronavirus OC43, 229E and HK, influenza A and B, parainfluinza types 1, 2, and 3 and respiratory syncytial virus. This new platform forms the basis for a fully automated diagnostics system that is very flexible and can be customized to suit different or additional pathogens. Multiple probes on a flexible platform allow one to test probes empirically and then select highly reactive probes for further iterative evaluation. Because ECD uses an enzymatic reaction to create electrical signals that can be read directly from the array, there is no need for image analysis or for expensive and delicate optical scanning equipment. We show assay sensitivity and specificity that are excellent for a multiplexed format.

Electrochemical detection has been developed and assay performances studied for the CombiMatrix oligonucleotide microarray platform that contains 12,544 individually addressable microelectrodes (features) in a semiconductor matrix. The approach is based on the detection of redox active chemistries (such as horseradish peroxidase (HRP) and the associated substrate TMB) proximal to specific microarray electrodes. First, microarray probes are hybridized to biotin-labeled targets, second, the HRP-streptavidin conjugate binds to biotin, and enzymatic oxidation of the electron donor substrate then occurs. The detection current is generated due to electro-reduction of the HRP reaction product, and it is measured with the CombiMatrix ElectraSense™ Reader. Performance of the ElectraSense™ platform has been characterized using gene expression and genotyping assays to analyze: (i) signal to concentration dependence, (ii) assay resolution, (iii) coefficients of variation, (CV) and (iv) array-to-array reproducibility and data correlation. The ElectraSense™ platform was also compared to the standard fluorescent detection, and good consistency was observed between these two different detection techniques. A lower detection limit of 0.75 pM was obtained for ElectraSense™ as compared to the detection limit of 1.5 pM obtained for fluorescent detection. Thus, the ElectraSense™ platform has been used to develop nucleic acid assays for highly accurate genotyping of a variety of pathogens including bio-threat agents (such as Bacillus anthracis, Yersinia pestis, and other microorganisms including Escherichia coli, Bacillus subtilis, etc.) and common pathogens of the respiratory tract (e.g. influenza A virus).

ABSTRACT In the face of concerns over an influenza pandemic, identification of virulent influenza A virus isolates must be obtained quickly for effective responses. Rapid subtype identification, however, is difficult even in well-equipped virology laboratories or is unobtainable in the field under more austere conditions. Here we describe a genome assay and microarray design that can be used to rapidly identify influenza A virus hemagglutinin subtypes 1 through 15 and neuraminidase subtypes 1 through 9. Also described is an array-based enzymatic assay that can be used to sequence portions of both genes or any other sequence of interest.