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PLoS One. 2011;6(6):e20956. Epub 2011 Jun 29.

Target region selection is a critical determinant of community fingerprints generated by 16S pyrosequencing.

Source

Division of Periodontology, College of Dentistry, The Ohio State University, Columbus, Ohio, United States of America. kumar.83@osu.edu

Abstract

Pyrosequencing of 16S rRNA genes allows for in-depth characterization of complex microbial communities. Although it is known that primer selection can influence the profile of a community generated by sequencing, the extent and severity of this bias on deep-sequencing methodologies is not well elucidated. We tested the hypothesis that the hypervariable region targeted for sequencing and primer degeneracy play important roles in influencing the composition of 16S pyrotag communities. Subgingival plaque from deep sites of current smokers with chronic periodontitis was analyzed using Sanger sequencing and pyrosequencing using 4 primer pairs. Greater numbers of species were detected by pyrosequencing than by Sanger sequencing. Rare taxa constituted nearly 6% of each pyrotag community and less than 1% of the Sanger sequencing community. However, the different target regions selected for pyrosequencing did not demonstrate a significant difference in the number of rare and abundant taxa detected. The genera Prevotella, Fusobacterium, Streptococcus, Granulicatella, Bacteroides, Porphyromonas and Treponema were abundant when the V1-V3 region was targeted, while Streptococcus, Treponema, Prevotella, Eubacterium, Porphyromonas, Campylobacer and Enterococcus predominated in the community generated by V4-V6 primers, and the most numerous genera in the V7-V9 community were Veillonella, Streptococcus, Eubacterium, Enterococcus, Treponema, Catonella and Selenomonas. Targeting the V4-V6 region failed to detect the genus Fusobacterium, while the taxa Selenomonas, TM7 and Mycoplasma were not detected by the V7-V9 primer pairs. The communities generated by degenerate and non-degenerate primers did not demonstrate significant differences. Averaging the community fingerprints generated by V1-V3 and V7-V9 primers providesd results similar to Sanger sequencing, while allowing a significantly greater depth of coverage than is possible with Sanger sequencing. It is therefore important to use primers targeted to these two regions of the 16S rRNA gene in all deep-sequencing efforts to obtain representational characterization of complex microbial communities.

PMID:
21738596
[PubMed - in process] 
PMCID: PMC3126800
Free PMC Article
Click here to readClick here to read
13.
J Invest Dermatol. 2011 Oct;131(10):2026-32. doi: 10.1038/jid.2011.168. Epub 2011 Jun 23.

Diversity of the human skin microbiome early in life.

Source

Department of Microbiology, Johnson & Johnson Consumer Companies, Skillman, New Jersey, USA.

Abstract

Within days after birth, rapid surface colonization of infant skin coincides with significant functional changes. Gradual maturation of skin function, structure, and composition continues throughout the first years of life. Recent reports have revealed topographical and temporal variations in the adult skin microbiome. Here we address the question of how the human skin microbiome develops early in life. We show that the composition of cutaneous microbial communities evolves over the first year of life, showing increasing diversity with age. Although early colonization is dominated by Staphylococci, their significant decline contributes to increased population evenness by the end of the first year. Similar to what has been shown in adults, the composition of infant skin microflora appears to be site specific. In contrast to adults, we find that Firmicutes predominate on infant skin. Timely and proper establishment of healthy skin microbiome during this early period might have a pivotal role in denying access to potentially infectious microbes and could affect microbiome composition and stability extending into adulthood. Bacterial communities contribute to the establishment of cutaneous homeostasis and modulate inflammatory responses. Early microbial colonization is therefore expected to critically affect the development of the skin immune function.

PMID:
21697884
[PubMed - in process] 
PMCID: PMC3182836
Free PMC Article
Click here to readClick here to read
14.
J Wound Care. 2011 May;20(5):232, 234-9.

Molecular diagnostics and personalised medicine in wound care: assessment of outcomes.

Source

Research and Testing Laboratory and Pathogenius Diagnostics, Lubbock, TX, USA. sdowd@pathogenresearch.org

Abstract

OBJECTIVE:

This large, level A, retrospective cohort study set out to compare healing outcomes in three large cohorts of wound patients managed universally for bioburden: standard of care group, who were prescribed systemic antibiotics on the basis of empiric and traditional culture-based methodologies; treatment group 1, who were prescribed an improved selection of systemic antibiotics based on the results of molecular diagnostics; treatment group 2 who received personalised topical therapeutics (including antibiotics) based on the results of molecular diagnostics.

METHOD:

Apart from the differences in diagnostic methods and antibiotic treatments described above, all three cohorts were subjected to the same biofilm-based wound care protocol, which included evaluation of the host and bioburden, frequent sharp debridement, use of wound dressings and comprehensive standard care (reperfusion therapy, nutritional support, offloading, compression and management of comorbidities).

RESULTS:

In all, 1378 patients were recruited into the study. In the standard of care group 48.5% of patients (244/503) healed completely during the 7-month study period. This increased to 62.4% (298/479) in treatment group 1 and 90.4% (358/396) in treatment group 2. Cox proportional hazards analysis revealed the time to complete closure decreased by 26% in treatment group 1 (p<0.001) and 45.9% in treatment group 2 (p<0.001) compared with the standard of care group. Patients in treatment group 2 had >200% better odds of healing at any given time point compared with the other cohorts.

CONCLUSION:

Implementation of personalised topical therapeutics guided by molecular diagnosis resulted in statistically and clinically significant improvements in outcome. The integration of molecular diagnostics and personalised medicine provides a directed and targeted approach to wound care. Conflict of interest: SED and RDW are owners of PathoGenius Laboratories, a clinical diagnostic laboratory. SED and RDW are owners of Research and Testing Laboratory, which develops molecular diagnostics. CJ and JK are clinical advisors for PathoGenius. CJ and JK are owners of Southeastern Medical Compounding, Savannah, GA and Southeastern Medical Technologies, Savannah, GA.

PMID:
21647068
[PubMed - indexed for MEDLINE]
Click here to read
15.
Methods Mol Biol. 2011;733:129-41.

Tag-encoded FLX amplicon pyrosequencing for the elucidation of microbial and functional gene diversity in any environment.

Source

Research and Testing Laboratory, Lubbock, TX, USA.

Abstract

Comprehensive evaluation of microbial diversity in almost any environment is now possible. Questions such as "Does the addition of fiber to the diet of humans change the gastrointestinal microbiota?" can now be answered easily and inexpensively. Tag-encoded FLX-amplicon pyrosequencing (TEFAP) has been utilized to evaluate bacterial, archaeal, fungal, algal, as well as functional genes. Using the new tag-encoded FLX amplicon pyrosequencing (bTEFAP) approach, we have evaluated the microbial diversity using a more cost-effective and largely reproducible method that would allow us to sequence the ribosomal RNA genes of microorganisms (hereafter focused on bacteria), without the need for the inherent bias of culture methods. These developments have ushered in a new age of microbial ecology studies, and we have utilized this technology to evaluate the microbiome in a wide range of systems in almost any conceivable environment.

PMID:
21431767
[PubMed - indexed for MEDLINE]
Click here to read
16.
ISME J. 2011 Sep;5(9):1426-37. doi: 10.1038/ismej.2011.25. Epub 2011 Mar 24.

Ovine pedomics: the first study of the ovine foot 16S rRNA-based microbiome.

Source

Department of Biological Sciences, School of Life Sciences, University of Warwick, Coventry, UK. l.a.calvo-bado@warwick.ac.uk

Abstract

We report the first study of the bacterial microbiome of ovine interdigital skin based on 16S rRNA by pyrosequencing and conventional cloning with Sanger-sequencing. Three flocks were selected, one a flock with no signs of footrot or interdigital dermatitis, a second flock with interdigital dermatitis alone and a third flock with both interdigital dermatitis and footrot. The sheep were classified as having either healthy interdigital skin (H) and interdigital dermatitis (ID) or virulent footrot (VFR). The ovine interdigital skin bacterial community varied significantly by flock and clinical condition. The diversity and richness of operational taxonomic units was greater in tissue from sheep with ID than H or VFR-affected sheep. Actinobacteria, Bacteriodetes, Firmicutes and Proteobacteria were the most abundant phyla comprising 25 genera. Peptostreptococcus, Corynebacterium and Staphylococcus were associated with H, ID and VFR, respectively. Sequences of Dichelobacter nodosus, the causal agent of ovine footrot, were not amplified because of mismatches in the 16S rRNA universal forward primer (27F). A specific real-time PCR assay was used to demonstrate the presence of D. nodosus, which was detected in all samples including the flock with no signs of ID or VFR. Sheep with ID had significantly higher numbers of D. nodosus (10(4)-10(9) cells per g tissue) than those with H or VFR feet.

PMID:
21430786
[PubMed - in process] 
PMCID: PMC3160683
 [Available on 2012/9/1]
Click here to read
17.
FEMS Microbiol Ecol. 2011 Jul;77(1):17-27. doi: 10.1111/j.1574-6941.2011.01079.x. Epub 2011 Mar 21.

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

Source

University of Warwick, School of Life Sciences, Coventry, UK. brian.oakley@ars.usda.gov

Abstract

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 that 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 that automates the identification of PCR assays targeting specific genotypes in complex samples. ThermoPhyl uses user-defined target and nontarget sequence databases to assess the phylogenetic sensitivity and specificity of thermodynamically optimized candidate assays derived from primer design software packages. ThermoPhyl derives 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) application 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.

© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

PMID:
21366628
[PubMed - indexed for MEDLINE]
Click here to read
18.
Open Microbiol J. 2010 Aug 11;4:47-52.

Black Box Chimera Check (B2C2): a Windows-Based Software for Batch Depletion of Chimeras from Bacterial 16S rRNA Gene Datasets.

Source

Research and Testing Laboratory of the South Plains, Lubbock, TX, USA 79407.

Abstract

The existing chimera detection programs are not specifically designed for "next generation" sequence data. Technologies like Roche 454 FLX and Titanium have been adapted over the past years especially with the introduction of bacterial tag-encoded FLX/Titanium amplicon pyrosequencing methodologies to produce over one million 250-600 bp 16S rRNA gene reads that need to be depleted of chimeras prior to downstream analysis. Meeting the needs of basic scientists who are venturing into high-throughput microbial diversity studies such as those based upon pyrosequencing and specifically providing a solution for Windows users, the B2C2 software is designed to be able to accept files containing large multi-FASTA formatted sequences and screen for possible chimeras in a high throughput fashion. The graphical user interface (GUI) is also able to batch process multiple files. When compared to popular chimera screening software the B2C2 performed as well or better while dramatically decreasing the amount of time required generating and screening results. Even average computer users are able to interact with the Windows .Net GUI-based application and define the stringency to which the analysis should be done. B2C2 may be downloaded from http://www.researchandtesting.com/B2C2.

PMID:
21339894
[PubMed] 
PMCID: PMC3040993
Free PMC Article
Click here to read
19.
Hum Mol Genet. 2011 May 1;20(9):1687-96. Epub 2011 Feb 4.

Maternal methyl-donor supplementation induces prolonged murine offspring colitis susceptibility in association with mucosal epigenetic and microbiomic changes.

Source

Department of Pediatrics, Baylor College of Medicine, Texas Children's Hospital, USDA/ARS Children's Nutrition Research Center, Houston, TX, USA.

Abstract

Developmental epigenetic changes, such as DNA methylation, have been recognized as potential pathogenic factors in inflammatory bowel diseases, the hallmark of which is an exaggerated immune response against luminal microbes. A methyl-donor (MD) diet can modify DNA methylation at select murine genomic loci during early development. The components of the MDs are routinely incorporated into prenatal human supplements. Therefore, we studied the effects of maternal MD supplementation on offspring colitis susceptibility and colonic mucosal DNA methylation and gene expression changes in mice as a model. Additionally, we investigated the offspring mucosal microbiomic response to the maternal dietary supplementation. Colitis was induced by dextran sulfate sodium. Colonic mucosa from offspring of MD-supplemented mothers following reversal to control diet at weaning was interrogated by methylation-specific microarrays and pyrosequencing at postnatal days 30 (P30) and P90. Transcriptomic changes were analyzed by microarray profiling and real-time reverse transcription polymerase chain reaction. The mucosal microbiome was studied by high throughput pyrosequencing of 16S rRNA. Maternal MD supplementation induced a striking susceptibility to colitis in offspring. This phenotype was associated with colonic mucosal DNA methylation and expression changes. Metagenomic analyses did not reveal consistent bacteriomic differences between P30 and P90, but showed a prolonged effect of the diet on the offspring mucosal microbiome. In conclusion, maternal MD supplementation increases offspring colitis susceptibility that associates with persistent epigenetic and prolonged microbiomic changes. These findings underscore that epigenomic reprogramming relevant to mammalian colitis can occur during early development in response to maternal dietary modifications.

PMID:
21296867
[PubMed - indexed for MEDLINE] 
PMCID: PMC3115577
 [Available on 2012/5/1]
Click here to read
20.
PLoS One. 2011 Jan 19;6(1):e15811.

Genome-wide polymorphism and comparative analyses in the white-tailed deer (Odocoileus virginianus): a model for conservation genomics.

Source

Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, Texas, United States of America. cseabury@cvm.tamu.edu

Erratum in

  • PLoS One. 2011;6(2). doi: 10.1371/annotation/80c6965b-ffa0-4100-8a20-3f41a08b2894.

Abstract

The white-tailed deer (Odocoileus virginianus) represents one of the most successful and widely distributed large mammal species within North America, yet very little nucleotide sequence information is available. We utilized massively parallel pyrosequencing of a reduced representation library (RRL) and a random shotgun library (RSL) to generate a complete mitochondrial genome sequence and identify a large number of putative single nucleotide polymorphisms (SNPs) distributed throughout the white-tailed deer nuclear and mitochondrial genomes. A SNP validation study designed to test specific classes of putative SNPs provides evidence for as many as 10,476 genome-wide SNPs in the current dataset. Based on cytogenetic evidence for homology between cow (Bos taurus) and white-tailed deer chromosomes, we demonstrate that a divergent genome may be used for estimating the relative distribution and density of de novo sequence contigs as well as putative SNPs for species without draft genome assemblies. Our approach demonstrates that bioinformatic tools developed for model or agriculturally important species may be leveraged to support next-generation research programs for species of biological, ecological and evolutionary importance. We also provide a functional annotation analysis for the de novo sequence contigs assembled from white-tailed deer pyrosequencing reads, a mitochondrial phylogeny involving 13,722 nucleotide positions for 10 unique species of Cervidae, and a median joining haplotype network as a putative representation of mitochondrial evolution in O. virginianus. The results of this study are expected to provide a detailed template enabling genome-wide sequence-based studies of threatened, endangered or conservationally important non-model organisms.

PMID:
21283515
[PubMed - indexed for MEDLINE] 
PMCID: PMC3023705
Free PMC Article

 

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