BARCRAWL and BARTAB: software tools for the design and implementation of barcoded primers for highly multiplexed DNA sequencing

<p>Abstract</p> <p>Background</p> <p>Advances in automated DNA sequencing technology have greatly increased the scale of genomic and metagenomic studies. An increasingly popular means of increasing project throughput is by multiplexing samples during the sequencing phase. This can be achieved by covalently linking short, unique "barcode" DNA segments to genomic DNA samples, for instance through incorporation of barcode sequences in PCR primers. Although several strategies have been described to insure that barcode sequences are unique and robust to sequencing errors, these have not been integrated into the overall primer design process, thus potentially introducing bias into PCR amplification and/or sequencing steps.</p> <p>Results</p> <p><it>Barcrawl </it>is a software program that facilitates the design of barcoded primers, for multiplexed high-throughput sequencing. The program <it>bartab </it>can be used to deconvolute DNA sequence datasets produced by the use of multiple barcoded primers. This paper describes the functions implemented by <it>barcrawl </it>and <it>bartab </it>and presents a proof-of-concept case study of both programs in which barcoded rRNA primers were designed and validated by high-throughput sequencing.</p> <p>Conclusion</p> <p><it>Barcrawl </it>and <it>bartab </it>can benefit researchers who are engaged in metagenomic projects that employ multiplexed specimen processing. The source code is released under the GNU general public license and can be accessed at <url>http://www.phyloware.com</url>.</p

Culture-Independent Microbiological Analysis of Foley Urinary Catheter Biofilms

Background: Prevention of catheter-associated urinary tract infection (CAUTI), a leading cause of nosocomial disease, is complicated by the propensity of bacteria to form biofilms on indwelling medical devices [1,2,3,4,5]. Methodology/Principal Findings: To better understand the microbial diversity of these communities, we report the results of a culture-independent bacterial survey of Foley urinary catheters obtained from patients following total prostatectomy. Two patient subsets were analyzed, based on treatment or no treatment with systemic fluoroquinolone antibiotics during convalescence. Results indicate the presence of diverse polymicrobial assemblages that were most commonly observed in patients who did not receive systemic antibiotics. The communities typically contained both Gram-positive and Gramnegative microorganisms that included multiple potential pathogens. Conclusion/Significance: Prevention and treatment of CAUTI must take into consideration the possible polymicrobial nature of any particular infection

The Human Nasal Microbiota and Staphylococcus aureus Carriage

BACKGROUND: Colonization of humans with Staphylococcus aureus is a critical prerequisite of subsequent clinical infection of the skin, blood, lung, heart and other deep tissues. S. aureus persistently or intermittently colonizes the nares of approximately 50% of healthy adults, whereas approximately 50% of the general population is rarely or never colonized by this pathogen. Because microbial consortia within the nasal cavity may be an important determinant of S. aureus colonization we determined the composition and dynamics of the nasal microbiota and correlated specific microorganisms with S. aureus colonization. METHODOLOGY/PRINCIPAL FINDINGS: Nasal specimens were collected longitudinally from five healthy adults and a cross-section of hospitalized patients (26 S. aureus carriers and 16 non-carriers). Culture-independent analysis of 16S rRNA sequences revealed that the nasal microbiota of healthy subjects consists primarily of members of the phylum Actinobacteria (e.g., Propionibacterium spp. and Corynebacterium spp.), with proportionally less representation of other phyla, including Firmicutes (e.g., Staphylococcus spp.) and Proteobacteria (e.g. Enterobacter spp). In contrast, inpatient nasal microbiotas were enriched in S. aureus or Staphylococcus epidermidis and diminished in several actinobacterial groups, most notably Propionibacterium acnes. Moreover, within the inpatient population S. aureus colonization was negatively correlated with the abundances of several microbial groups, including S. epidermidis (p = 0.004). CONCLUSIONS/SIGNIFICANCE: The nares environment is colonized by a temporally stable microbiota that is distinct from other regions of the integument. Negative association between S. aureus, S. epidermidis, and other groups suggests microbial competition during colonization of the nares, a finding that could be exploited to limit S. aureus colonization

XplorSeq: A software environment for integrated management and phylogenetic analysis of metagenomic sequence data

<p>Abstract</p> <p>Background</p> <p>Advances in automated DNA sequencing technology have accelerated the generation of metagenomic DNA sequences, especially environmental ribosomal RNA gene (rDNA) sequences. As the scale of rDNA-based studies of microbial ecology has expanded, need has arisen for software that is capable of managing, annotating, and analyzing the plethora of diverse data accumulated in these projects.</p> <p>Results</p> <p>XplorSeq is a software package that facilitates the compilation, management and phylogenetic analysis of DNA sequences. XplorSeq was developed for, but is not limited to, high-throughput analysis of environmental rRNA gene sequences. XplorSeq integrates and extends several commonly used UNIX-based analysis tools by use of a Macintosh OS-X-based graphical user interface (GUI). Through this GUI, users may perform basic sequence import and assembly steps (base-calling, vector/primer trimming, contig assembly), perform BLAST (Basic Local Alignment and Search Tool; <abbrgrp><abbr bid="B1">1</abbr><abbr bid="B2">2</abbr><abbr bid="B3">3</abbr></abbrgrp>) searches of NCBI and local databases, create multiple sequence alignments, build phylogenetic trees, assemble Operational Taxonomic Units, estimate biodiversity indices, and summarize data in a variety of formats. Furthermore, sequences may be annotated with user-specified meta-data, which then can be used to sort data and organize analyses and reports. A document-based architecture permits parallel analysis of sequence data from multiple clones or amplicons, with sequences and other data stored in a single file.</p> <p>Conclusion</p> <p>XplorSeq should benefit researchers who are engaged in analyses of environmental sequence data, especially those with little experience using bioinformatics software. Although XplorSeq was developed for management of rDNA sequence data, it can be applied to most any sequencing project. The application is available free of charge for non-commercial use at <url>http://vent.colorado.edu/phyloware</url>.</p

Microbiome profiling by Illumina sequencing of combinatorial sequence-tagged PCR products

We developed a low-cost, high-throughput microbiome profiling method that uses combinatorial sequence tags attached to PCR primers that amplify the rRNA V6 region. Amplified PCR products are sequenced using an Illumina paired-end protocol to generate millions of overlapping reads. Combinatorial sequence tagging can be used to examine hundreds of samples with far fewer primers than is required when sequence tags are incorporated at only a single end. The number of reads generated permitted saturating or near-saturating analysis of samples of the vaginal microbiome. The large number of reads al- lowed an in-depth analysis of errors, and we found that PCR-induced errors composed the vast majority of non-organism derived species variants, an ob- servation that has significant implications for sequence clustering of similar high-throughput data. We show that the short reads are sufficient to assign organisms to the genus or species level in most cases. We suggest that this method will be useful for the deep sequencing of any short nucleotide region that is taxonomically informative; these include the V3, V5 regions of the bac- terial 16S rRNA genes and the eukaryotic V9 region that is gaining popularity for sampling protist diversity.Comment: 28 pages, 13 figure

Antibiotic and Antiinflammatory Therapy Transiently Reduces Inflammation and Hypercoagulation in Acutely SIV-Infected Pigtailed Macaques

Increased chronic immune activation and inflammation are hallmarks of HIV/SIV infection and are highly correlated with progression to AIDS and development of non-AIDS comorbidities, such as hypercoagulability and cardiovascular disease. Intestinal dysfunction resulting in microbial translocation has been proposed as a lead cause of systemic immune activation and hypercoagulability in HIV/SIV infection. Our goal was to assess the biological and clinical impact of a therapeutic strategy designed to reduce microbial translocation through reduction of the microbial content of the intestine (Rifaximin-RFX) and of gut inflammation (Sulfasalazine-SFZ). RFX is an intraluminal antibiotic that was successfully used in patients with hepatic encephalopathy. SFZ is an antiinflammatory drug successfully used in patients with mild to moderate inflammatory bowel disease. Both these clinical conditions are associated with increased microbial translocation, similar to HIV-infected patients. Treatment was administered for 90 days to five acutely SIV-infected pigtailed macaques (PTMs) starting at the time of infection; seven untreated SIVsab-infected PTMs were used as controls. RFX+SFZ were also administered for 90 days to three chronically SIVsab-infected PTMs. RFX+SFZ administration during acute SIVsab infection of PTMs resulted in: significantly lower microbial translocation, lower systemic immune activation, lower viral replication, better preservation of mucosal CD4+ T cells and significantly lower levels of hypercoagulation biomarkers. This effect was clear during the first 40 days of treatment and was lost during the last stages of treatment. Administration of RFX+SFZ to chronically SIVsab–infected PTMs had no discernible effect on infection. Our data thus indicate that early RFX+SFZ administration transiently improves the natural history of acute and postacute SIV infection, but has no effect during chronic infection

Changing gender roles and attitudes and their implications for well-being around the new millennium

&lt;b&gt;Purpose&lt;/b&gt;&lt;p&gt;&lt;/p&gt; Given evidence that gender role attitudes (GRAs) and actual gender roles impact on well-being, we examine associations between GRAs, three roles (marital status, household chore division, couple employment) and psychological distress in working-age men and women. We investigate time-trends reflecting broader social and economic changes, by focusing on three age groups at two dates.&lt;p&gt;&lt;/p&gt; &lt;b&gt;Methods&lt;/b&gt;&lt;p&gt;&lt;/p&gt; We used British Household Panel Survey data from 20- to 64-year-olds in heterosexual couple households in 1991 (N = 5,302) and 2007 (N = 6,621). We examined: levels of traditional GRAs according to gender, age, date, household and employment roles; associations which GRAs and roles had with psychological distress (measured via the GHQ-12); whether psychological distress increased when GRAs conflicted with actual roles; and whether any of these associations differed according to gender, age or date.&lt;p&gt;&lt;/p&gt; &lt;b&gt;Results&lt;/b&gt;&lt;p&gt;&lt;/p&gt; Gender traditionalism was lower among women, younger people, those participating in 2007 and in ‘less traditional’ relationships and households. Psychological distress was higher among those with more traditional GRAs and, particularly among men, for those not employed, and there was some evidence of different patterns of association according to age-group. There was limited evidence, among women only, of increased psychological distress when GRAs and actual roles conflicted and/or reductions when GRAs and roles agreed, particularly in respect of household chores and paid employment.&lt;p&gt;&lt;/p&gt; &lt;b&gt;Conclusions&lt;/b&gt;&lt;p&gt;&lt;/p&gt; Although some aspects of gender roles and attitudes (traditionalism and paid employment) are associated with well-being, others (marital status and household chores), and attitude-role consistency, may have little impact on the well-being of contemporary UK adults.&lt;p&gt;&lt;/p&gt

Using fractional exhaled nitric oxide (FeNO) to diagnose steroid-responsive disease and guide asthma management in routine care

Acknowledgements We thank Robin Taylor for his informative thinking and publications on FeNO, which have helped to influence and direct the thinking of the authors. Funding Extraction of the real-life dataset was funded by Research in Real Life Limited, the analysis of the dataset and the writing of this manuscript were co-funded (50:50) by Research in Real Life Limited and Aerocrine.Peer reviewedPublisher PD

Bacterial Communities of the Coronal Sulcus and Distal Urethra of Adolescent Males

Lactobacillus-dominated vaginal microbiotas are associated with reproductive health and STI resistance in women, whereas altered microbiotas are associated with bacterial vaginosis (BV), STI risk and poor reproductive outcomes. Putative vaginal taxa have been observed in male first-catch urine, urethral swab and coronal sulcus (CS) specimens but the significance of these observations is unclear. We used 16 S rRNA sequencing to characterize the microbiota of the CS and urine collected from 18 adolescent men over three consecutive months. CS microbiotas of most participants were more stable than their urine microbiotas and the composition of CS microbiotas were strongly influenced by circumcision. BV-associated taxa, including Atopobium, Megasphaera, Mobiluncus, Prevotella and Gemella, were detected in CS specimens from sexually experienced and inexperienced participants. In contrast, urine primarily contained taxa that were not abundant in CS specimens. Lactobacilllus and Streptococcus were major urine taxa but their abundance was inversely correlated. In contrast, Sneathia, Mycoplasma and Ureaplasma were only found in urine from sexually active participants. Thus, the CS and urine support stable and distinct bacterial communities. Finally, our results suggest that the penis and the urethra can be colonized by a variety of BV-associated taxa and that some of these colonizations result from partnered sexual activity