Complete Genome Sequences of 44 Arthrobacter Phages.

We report here the complete genome sequences of 44 phages infecting Arthrobacter sp. strain ATCC 21022. These phages have double-stranded DNA genomes with sizes ranging from 15,680 to 70,707 bp and G+C contents from 45.1% to 68.5%. All three tail types (belonging to the families Siphoviridae, Myoviridae, and Podoviridae) are represented.

Tales of diversity: Genomic and morphological characteristics of forty-six Arthrobacter phages.

The vast bacteriophage population harbors an immense reservoir of genetic information. Almost 2000 phage genomes have been sequenced from phages infecting hosts in the phylum Actinobacteria, and analysis of these genomes reveals substantial diversity, pervasive mosaicism, and novel mechanisms for phage replication and lysogeny. Here, we describe the isolation and genomic characterization of 46 phages from environmental samples at various geographic locations in the U.S. infecting a single Arthrobacter sp. strain. These phages include representatives of all three virion morphologies, and Jasmine is the first sequenced podovirus of an actinobacterial host. The phages also span considerable sequence diversity, and can be grouped into 10 clusters according to their nucleotide diversity, and two singletons each with no close relatives. However, the clusters/singletons appear to be genomically well separated from each other, and relatively few genes are shared between clusters. Genome size varies from among the smallest of siphoviral phages (15,319 bp) to over 70 kbp, and G+C contents range from 45-68%, compared to 63.4% for the host genome. Although temperate phages are common among other actinobacterial hosts, these Arthrobacter phages are primarily lytic, and only the singleton Galaxy is likely temperate.

Development of a normalized extraction to further aid in fast, high-throughput processing of forensic DNA reference samples.

The goal of this project was to develop a "normalized" extraction procedure to be used in conjunction with previously validated 3µL fast PCR reactions (42-51min utilizing KAPA2G™ Fast Multiplex PCR Kit) and alternative capillary electrophoresis (24-28min injection using POP-6™ Polymer and a 22cm array). This was the final phase of a workflow overhaul for the database unit at Cellmark Forensics to achieve a substantial reduction in processing time for forensic DNA database samples without incurring significant added costs and/or the need for new instrumentation, while still generating high quality STR profiles. Extraction normalization aimed to consistently yield a small range of DNA concentrations, thereby eliminating the need for sample quantification and dilution. This was specifically achieved using the ChargeSwitch® Forensic DNA Purification Kit and a reduction in extraction bead quantity, thereby forcing an increase in bead binding efficiency. Following development of this extraction procedure, an evaluation ensued to assess the combination of normalized extraction, 3µL fast PCR (with PowerPlex 16 HS, Identifiler Plus and Identifiler primer sets), and alternative CE detection - further referred to as new "first pass" procedures. These modifications resulted in a 37% reduction in processing time and were evaluated via an in depth validation, from which nearly 2000 STR profiles were generated, of which 554 profiles from 77 swab donors and 210 profiles from 35 buccal collector donors specifically arose from the new first pass procedures. This validation demonstrates the robustness of these processes for buccal swabs and Buccal DNA Collectors™ using the three primer sets evaluated and their ability to generate high quality STR profiles with 95-99% and 88-91% pass rates, respectively.

Genome Sequences of Streptomyces Phages Amela and Verse.

Amela and Verse are two Streptomyces phages isolated by enrichment on Streptomyces venezuelae (ATCC 10712) from two different soil samples. Amela has a genome length of 49,452, with 75 genes. Verse has a genome length of 49,483, with 75 genes. Both belong to the BD3 subcluster of Actinobacteriophage.

Validation of alternative capillary electrophoresis detection of STRs using POP-6 polymer and a 22cm array on a 3130xl genetic analyzer.

The goal of this project was to reduce capillary electrophoresis detection time on a 3130xl Genetic Analyzer for amplification product obtained from 4-dye and 5-dye STR amplification kits while still generating high quality STR profiles. This was accomplished by utilizing a more viscous polymer (POP-6™) and a shorter array (22 cm) than that which are typically used (POP-4(®) polymer and a 36 cm array) for human identification purposes. Spatial calibration and detection run modules were modified in response to the use of this polymer/array combination and to reduce detection time. Alternative detection resulted in 24-28 min run times, as compared to ∼45 min using traditional POP-4(®)/36 cm detection methods. POP-6™/22 cm detection run modules were validated for use with 4-dye Promega STR kits (e.g., PowerPlex(®) 16 and PowerPlex(®) 16HS) and 5-dye Life Technologies kits (e.g., Identifiler(®) and Identifiler(®) Plus). Three hundred ninety-five samples, controls and allelic ladders were used for the validation studies, which consisted of a comparison of alternative POP-6™/22 cm detection to traditional POP-4(®)/36 cm (including reproducibility/concordance of allele calls, resolution, ILS sizing quality, peak height and pass rates), a sizing study (precision and accuracy) and a sensitivity study to obtain a usable range of injection times. Compared to traditional POP-4(®)/36 cm detection, alternative detection resulted in 100% reproducible and concordant alleles, the ability to achieve one base resolution, slightly reduced ILS sizing quality, slightly reduced peak height and statistically similar pass rates (α=0.05). It should be noted that alternative detection offered improved resolution over that of traditional for amplicons less than ∼200 b, but had reduced resolution for products greater than ∼200 b. Additionally, alternative detection yielded acceptable precision and accuracy of sizing using Life Technologies criteria (<0.15 standard deviation of allele sizing and ±0.5b sizing differences for the same allele) and usable injection parameters of 2 kV 4-15s (compared to 3 kV 10s for traditional). The run modules developed and validated for 4-dye and 5-dye STR kits using POP-6™ polymer on a 22 cm array offer a tremendous reduction in detection time (∼40%) while still generating high quality STR profiles.

Cluster K mycobacteriophages: insights into the evolutionary origins of mycobacteriophage TM4.

Five newly isolated mycobacteriophages--Angelica, CrimD, Adephagia, Anaya, and Pixie--have similar genomic architectures to mycobacteriophage TM4, a previously characterized phage that is widely used in mycobacterial genetics. The nucleotide sequence similarities warrant grouping these into Cluster K, with subdivision into three subclusters: K1, K2, and K3. Although the overall genome architectures of these phages are similar, TM4 appears to have lost at least two segments of its genome, a central region containing the integration apparatus, and a segment at the right end. This suggests that TM4 is a recent derivative of a temperate parent, resolving a long-standing conundrum about its biology, in that it was reportedly recovered from a lysogenic strain of Mycobacterium avium, but it is not capable of forming lysogens in any mycobacterial host. Like TM4, all of the Cluster K phages infect both fast- and slow-growing mycobacteria, and all of them--with the exception of TM4--form stable lysogens in both Mycobacterium smegmatis and Mycobacterium tuberculosis; immunity assays show that all five of these phages share the same immune specificity. TM4 infects these lysogens suggesting that it was either derived from a heteroimmune temperate parent or that it has acquired a virulent phenotype. We have also characterized a widely-used conditionally replicating derivative of TM4 and identified mutations conferring the temperature-sensitive phenotype. All of the Cluster K phages contain a series of well conserved 13 bp repeats associated with the translation initiation sites of a subset of the genes; approximately one half of these contain an additional sequence feature composed of imperfectly conserved 17 bp inverted repeats separated by a variable spacer. The K1 phages integrate into the host tmRNA and the Cluster K phages represent potential new tools for the genetics of M. tuberculosis and related species.