ThermoPhyl : a software tool for selecting phylogenetically optimized conventional and quantitative-PCR taxon-targeted assays for use with complex samples

The ability to specifically and sensitively target genotypes of interest is critical for the success of many PCR-based analyses of environmental or clinical samples that contain multiple templates.Next-generation sequence data clearly show that such samples can harbour hundreds to thousands of operational taxonomic units; a richness which precludes the manual evaluation of candidate assay specificity and sensitivity using multiple sequence alignments. To solve this problem we have developed and validated a free software tool which automates the identification of PCR assays targeting specific genotypes in complex samples. ThermoPhyl uses user-defined target and non-target sequence databases to assess the phylogenetic sensitivity and specificity of thermodynamically optimised candidate assays derived from primer design software packages. ThermoPhyl takes its name from its central premise of testing Thermodynamically optimal assays for Phylogenetic specificity and sensitivity and can be used for two primer (traditional PCR) or two primers with an internal probe (e.g. TaqMan® qPCR) applications and potentially for oligonucleotide probes.Here we describe the use of ThermoPhyl for traditional PCR and qPCR assays. PCR assays selected using ThermoPhyl were validated using 454 pyrosequencing of a traditional specific PCR assay and with a set of four genotype-specific qPCR assays applied to estuarine sediment samples

Windows .NET Network Distributed Basic Local Alignment Search Toolkit (W.ND-BLAST)

BACKGROUND: BLAST is one of the most common and useful tools for Genetic Research. This paper describes a software application we have termed Windows .NET Distributed Basic Local Alignment Search Toolkit (W.ND-BLAST), which enhances the BLAST utility by improving usability, fault recovery, and scalability in a Windows desktop environment. Our goal was to develop an easy to use, fault tolerant, high-throughput BLAST solution that incorporates a comprehensive BLAST result viewer with curation and annotation functionality. RESULTS: W.ND-BLAST is a comprehensive Windows-based software toolkit that targets researchers, including those with minimal computer skills, and provides the ability increase the performance of BLAST by distributing BLAST queries to any number of Windows based machines across local area networks (LAN). W.ND-BLAST provides intuitive Graphic User Interfaces (GUI) for BLAST database creation, BLAST execution, BLAST output evaluation and BLAST result exportation. This software also provides several layers of fault tolerance and fault recovery to prevent loss of data if nodes or master machines fail. This paper lays out the functionality of W.ND-BLAST. W.ND-BLAST displays close to 100% performance efficiency when distributing tasks to 12 remote computers of the same performance class. A high throughput BLAST job which took 662.68 minutes (11 hours) on one average machine was completed in 44.97 minutes when distributed to 17 nodes, which included lower performance class machines. Finally, there is a comprehensive high-throughput BLAST Output Viewer (BOV) and Annotation Engine components, which provides comprehensive exportation of BLAST hits to text files, annotated fasta files, tables, or association files. CONCLUSION: W.ND-BLAST provides an interactive tool that allows scientists to easily utilizing their available computing resources for high throughput and comprehensive sequence analyses. The install package for W.ND-BLAST is freely downloadable from . With registration the software is free, installation, networking, and usage instructions are provided as well as a support forum

Evaluation of the bacterial diversity among and within individual venous leg ulcers using bacterial tag-encoded FLX and Titanium amplicon pyrosequencing and metagenomic approaches

<p>Abstract</p> <p>Background</p> <p>Approximately 1 out of every 100 individuals has some form of venous insufficiency, which can lead to chronic venous disease and Venous Leg Ulcer (VLU). There are known underlying pathologies which contribute to the chronic nature of VLU including biofilm phenotype infections.</p> <p>Results</p> <p>Using pyrosequencing based approaches we evaluated VLU to characterize their microbial ecology. Results show that VLU infections are polymicrobial with no single bacterium colonizing the wounds. The most ubiquitous and predominant organisms include a previously uncharacterized bacteroidales, various anaerobes, <it>Staphylococcus</it>, <it>Corynebacterium</it>, and <it>Serratia</it>. Topological analysis of VLU show some notable differences in bacterial populations across the surface of the wounds highlighting the importance of sampling techniques during diagnostics. Metagenomics provide a preliminary indication that there may be protozoa, fungi and possibly an undescribed virus associated with these wounds.</p> <p>Conclusion</p> <p>The polymicrobial nature of VLU and previous research on diabetic foot ulcers and surgical site infections suggest that the future of therapy for such wounds lies in the core of the logical and proven multiple concurrent strategy approach, which has been termed "biofilm-based wound care" and the use of individualized therapeutics rather than in a single treatment modality.</p

Inherent bacterial DNA contamination of extraction and sequencing reagents may affect interpretation of microbiota in low bacterial biomass samples

Additional file 1: Table S1. Taxonomic listing of all bacterial genera and species detected as contaminants of DNA extraction and processing kits in the present study and previously reported

Differential allergen expression in three Tyrophagus putrescentiae strains inhabited by distinct microbiome

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/152656/1/all13921.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/152656/2/all13921_am.pd

The rehydration transcriptome of the desiccation-tolerant bryophyte Tortula ruralis: transcript classification and analysis

BACKGROUND: The cellular response of plants to water-deficits has both economic and evolutionary importance directly affecting plant productivity in agriculture and plant survival in the natural environment. Genes induced by water-deficit stress have been successfully enumerated in plants that are relatively sensitive to cellular dehydration, however we have little knowledge as to the adaptive role of these genes in establishing tolerance to water loss at the cellular level. Our approach to address this problem has been to investigate the genetic responses of plants that are capable of tolerating extremes of dehydration, in particular the desiccation-tolerant bryophyte, Tortula ruralis. To establish a sound basis for characterizing the Tortula genome in regards to desiccation tolerance, we analyzed 10,368 expressed sequence tags (ESTs) from rehydrated rapid-dried Tortula gametophytes, a stage previously determined to exhibit the maximum stress induced change in gene expression. RESULTS: The 10, 368 ESTs formed 5,563 EST clusters (contig groups representing individual genes) of which 3,321 (59.7%) exhibited similarity to genes present in the public databases and 2,242 were categorized as unknowns based on protein homology scores. The 3,321 clusters were classified by function using the Gene Ontology (GO) hierarchy and the KEGG database. The results indicate that the transcriptome contains a diverse population of transcripts that reflects, as expected, a period of metabolic upheaval in the gametophyte cells. Much of the emphasis within the transcriptome is centered on the protein synthetic machinery, ion and metabolite transport, and membrane biosynthesis and repair. Rehydrating gametophytes also have an abundance of transcripts that code for enzymes involved in oxidative stress metabolism and phosphorylating activities. The functional classifications reflect a remarkable consistency with what we have previously established with regards to the metabolic activities that are important in the recovery of the gametophytes from desiccation. A comparison of the GO distribution of Tortula clusters with an identical analysis of 9,981 clusters from the desiccation sensitive bryophyte species Physcomitrella patens, revealed, and accentuated, the differences between stressed and unstressed transcriptomes. Cross species sequence comparisons indicated that on the whole the Tortula clusters were more closely related to those from Physcomitrella than Arabidopsis (complete genome BLASTx comparison) although because of the differences in the databases there were more high scoring matches to the Arabidopsis sequences. The most abundant transcripts contained within the Tortula ESTs encode Late Embryogenesis Abundant (LEA) proteins that are normally associated with drying plant tissues. This suggests that LEAs may also play a role in recovery from desiccation when water is reintroduced into a dried tissue. CONCLUSION: The establishment of a rehydration EST collection for Tortula ruralis, an important plant model for plant stress responses and vegetative desiccation tolerance, is an important step in understanding the genome level response to cellular dehydration. The type of transcript analysis performed here has laid the foundation for more detailed functional and genome level analyses of the genes involved in desiccation tolerance in plants

Influence of wet distillers grains diets on beef cattle fecal bacterial community structure

<p>Abstract</p> <p>Background</p> <p>The high demand for ethanol in the U.S. has generated large stocks of wet distillers grains (DG), a byproduct from the manufacture of ethanol from corn and sorghum grains. Little is known, however, about the potential influence of dietary DG on fecal microbial community structure. A better understanding of the microbial population in beef cattle feces could be an important monitoring tool to facilitate goals of improving nutrient management, increasing animal growth performance and decreasing odors and/or shedding of pathogens. Five diets consisting of a traditional diet fed to finishing beef cattle in the Southern High Plains of Texas-CON (steam-flaked corn control with 0% DG), and four concentrations of DG in the dietary dry matter; 10 C (10% corn-based DG), 5S (5% sorghum-based DG), 10S (10% sorghum DG), and 15S (15% sorghum DG) were fed to steers at the Texas Tech University Burnett Animal Center. Diets were essentially isonitrogenous with a formulated crude protein value of 13.5%.</p> <p>Results</p> <p>Fecal grab samples were obtained from 20 steers (n = 4 per diet) and the barcoded DNA pyrosequencing method was used to generate 127,530 16S operational taxonomic units (OTUs). A total of 24 phyla were observed, distributed amongst all beef cattle on all diets, revealing considerable animal to animal variation, however only six phyla (core set) were observed in all animals regardless of dietary treatment. The average abundance and range of abundance, respectively of the core phyla were as follows: Firmicutes (61%, 19 to 83%), Bacteroidetes (28%, 11 to 63%), Proteobacteria (3%, 0.34 to 17.5%), Tenericutes (0.15%, 0.0 to 0.35%), Nitrospirae (0.11%, 0.03 to 0.22%), and Fusobacteria (0.086%, 0.017 to 0.38%). Feeding DG-based diets resulted in significant shifts in the fecal microbial community structure compared with the traditional CON. Four low abundance phyla significantly responded to dietary treatments: Synergistetes (<it>p </it>= 0.01), WS3 (<it>p </it>= 0.054), Actinobacteria (<it>p </it>= 0.06), and Spirochaetes (<it>p </it>= 0.06).</p> <p>Conclusions</p> <p>This is, to our knowledge, the first study using this method to survey the fecal microbiome of beef cattle fed various concentrations of wet DG. Comparison of our results with other cattle DNA sequencing studies of beef and dairy cattle feces from a variety of geographical locations and different management practices identifies a core set of three phyla shared across all cattle. These three phyla, in order of relative abundance are; Firmicutes, Bacteroidetes, and Proteobacteria. The presence of large animal-to-animal variation in cattle microbiome was noted in our study as well as by others.</p