Groundtruthing next-gen sequencing for microbial ecology-biases and errors in community structure estimates from PCR amplicon pyrosequencing

Analysis of microbial communities by high-throughput pyrosequencing of SSU rRNA gene PCR amplicons has transformed microbial ecology research and led to the observation that many communities contain a diverse assortment of rare taxa-a phenomenon termed the Rare Biosphere. Multiple studies have investigated the effect of pyrosequencing read quality on operational taxonomic unit (OTU) richness for contrived communities, yet there is limited information on the fidelity of community structure estimates obtained through this approach. Given that PCR biases are widely recognized, and further unknown biases may arise from the sequencing process itself, a priori assumptions about the neutrality of the data generation process are at best unvalidated. Furthermore, post-sequencing quality control algorithms have not been explicitly evaluated for the accuracy of recovered representative sequences and its impact on downstream analyses, reducing useful discussion on pyrosequencing reads to their diversity and abundances. Here we report on community structures and sequences recovered for in vitro-simulated communities consisting of twenty 16S rRNA gene clones tiered at known proportions. PCR amplicon libraries of the V3-V4 and V6 hypervariable regions from the in vitro-simulated communities were sequenced using the Roche 454 GS FLX Titanium platform. Commonly used quality control protocols resulted in the formation of OTUs with >1% abundance composed entirely of erroneous sequences, while over-aggressive clustering approaches obfuscated real, expected OTUs. The pyrosequencing process itself did not appear to impose significant biases on overall community structure estimates, although the detection limit for rare taxa may be affected by PCR amplicon size and quality control approach employed. Meanwhile, PCR biases associated with the initial amplicon generation may impose greater distortions in the observed community structure

Magnetoresistance Scaling Reveals Symmetries of the Strongly Correlated Dynamics in BaFe_{2}(As_{1-x}P_{x})_{2}.

The phenomenon of T-linear resistivity commonly observed in a number of strange metals has been widely seen as evidence for the breakdown of the quasiparticle picture of metals. This study shows that a recently discovered H/T scaling relationship in the magnetoresistance of the strange metal BaFe_{2}(As_{1-x}P_{x})_{2} is independent of the relative orientations of current and magnetic field. Rather, its magnitude and form depend only on the orientation of the magnetic field with respect to a single crystallographic axis: the direction perpendicular to the magnetic iron layers. This finding suggests that the magnetotransport scaling does not originate from the conventional averaging or orbital velocity of quasiparticles as they traverse a Fermi surface, but rather from dissipation arising from two-dimensional correlations

Chemostat culture systems support diverse bacteriophage communities from human feces

BACKGROUND: Most human microbiota studies focus on bacteria inhabiting body surfaces, but these surfaces also are home to large populations of viruses. Many are bacteriophages, and their role in driving bacterial diversity is difficult to decipher without the use of in vitro ecosystems that can reproduce human microbial communities. RESULTS: We used chemostat culture systems known to harbor diverse fecal bacteria to decipher whether these cultures also are home to phage communities. We found that there are vast viral communities inhabiting these ecosystems, with estimated concentrations similar to those found in human feces. The viral communities are composed entirely of bacteriophages and likely contain both temperate and lytic phages based on their similarities to other known phages. We examined the cultured phage communities at five separate time points over 24 days and found that they were highly individual-specific, suggesting that much of the subject-specificity found in human viromes also is captured by this culture-based system. A high proportion of the community membership is conserved over time, but the cultured communities maintain more similarity with other intra-subject cultures than they do to human feces. In four of the five subjects, estimated viral diversity between fecal and cultured communities was highly similar. CONCLUSIONS: Because the diversity of phages in these cultured fecal communities have similarities to those found in humans, we believe these communities can serve as valuable ecosystems to help uncover the role of phages in human microbial communities

Marketing and clinical trials: a case study.

BACKGROUND: Publicly funded clinical trials require a substantial commitment of time and money. To ensure that sufficient numbers of patients are recruited it is essential that they address important questions in a rigorous manner and are managed well, adopting effective marketing strategies. METHODS: Using methods of analysis drawn from management studies, this paper presents a structured assessment framework or reference model, derived from a case analysis of the MRC's CRASH trial, of 12 factors that may affect the success of the marketing and sales activities associated with clinical trials. RESULTS: The case study demonstrates that trials need various categories of people to buy in - hence, to be successful, trialists must embrace marketing strategies to some extent. CONCLUSION: The performance of future clinical trials could be enhanced if trialists routinely considered these factors

Whole-Retina Reduced Electrophysiological Activity in Mice Bearing Retina-Specific Deletion of Vesicular Acetylcholine Transporter

Background: Despite rigorous characterization of the role of acetylcholine in retinal development, long-term effects of its absence as a neurotransmitter are unknown. One of the unanswered questions is how acetylcholine contributes to the functional capacity of mature retinal circuits. The current study investigates the effects of disrupting cholinergic signalling in mice, through deletion of vesicular acetylcholine transporter (VAChT) in the developing retina, pigmented epithelium, optic nerve and optic stalk, on electrophysiology and structure of the mature retina. Methods & Results A combination of electroretinography, optical coherence tomography imaging and histological evaluation assessed retinal integrity in mice bearing retina-targeted (embryonic day 12.5) deletion of VAChT (VAChT(Six3-Cre-flox/flox)) and littermate controls at 5 and 12 months of age. VAChT(Six3-Cre-flox/flox) mice did not show any gross changes in nuclear layer cellularity or synaptic layer thickness. However, VAChT(Six3-Cre-flox/flox) mice showed reduced electrophysiological response of the retina to light stimulus under scotopic conditions at 5 and 12 months of age, including reduced a-wave, b-wave, and oscillatory potential (OP) amplitudes and decreased OP peak power and total energy. Reduced a-wave amplitude was proportional to the reduction in b-wave amplitude and not associated with altered a-wave 10%-90% rise time or inner and outer segment thicknesses. Significance This study used a novel genetic model in the first examination of function and structure of the mature mouse retina with disruption of cholinergic signalling. Reduced amplitude across the electroretinogram wave form does not suggest dysfunction in specific retinal cell types and could reflect underlying changes in the retinal and/or extraretinal microenvironment. Our findings suggest that release of acetylcholine by VAChT is essential for the normal electrophysiological response of the mature mouse retina

What influences recruitment to randomised controlled trials? A review of trials funded by two UK funding agencies.

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.BACKGROUND: A commonly reported problem with the conduct of multicentre randomised controlled trials (RCTs) is that recruitment is often slower or more difficult than expected, with many trials failing to reach their planned sample size within the timescale and funding originally envisaged. The aim of this study was to explore factors that may have been associated with good and poor recruitment in a cohort of multicentre trials funded by two public bodies: the UK Medical Research Council (MRC) and the Health Technology Assessment (HTA) Programme. METHODS: The cohort of trials was identified from the administrative databases held by the two funding bodies. 114 trials that recruited participants between 1994 and 2002 met the inclusion criteria. The full scientific applications and subsequent trial reports submitted by the trial teams to the funders provided the principal data sources. Associations between trial characteristics and recruitment success were tested using the Chi-squared test, or Fisher's exact test where appropriate. RESULTS: Less than a third (31%) of the trials achieved their original recruitment target and half (53%) were awarded an extension. The proportion achieving targets did not appear to improve over time. The overall start to recruitment was delayed in 47 (41%) trials and early recruitment problems were identified in 77 (63%) trials. The inter-relationship between trial features and recruitment success was complex. A variety of strategies were employed to try to increase recruitment, but their success could not be assessed. CONCLUSION: Recruitment problems are complex and challenging. Many of the trials in the cohort experienced recruitment difficulties. Trials often required extended recruitment periods (sometimes supported by additional funds). While this is of continuing concern, success in addressing the trial question may be more important than recruitment alone.Published versio

Sperm Quality of Hatchery-Reared Lake Trout Throughout the Spawning Season

The objective of this study was to investigate variation in sperm quality metrics (motility, velocity, linearity, longevity, and density) of hatchery-reared Lake Trout Salvelinus namaycush throughout the spawning season. Seasonal variation in sperm quality was investigated using both a regression and repeated-measures approach. Sperm was collected from the same 16 individuals over four sampling periods, separated by 3-week intervals. Regression analyses showed that 727% of the variation in sperm traits could be explained by seasonal variation, indicating that seasonality can have a significant impact on the quality of sperm. Significant positive linear relationships were found for percent motility and linearity at 5s postactivation. Significant negative quadratic relationships were found for velocity at 5s postactivation, longevity, and density, whereas a positive quadratic relationship was found for linearity at 10s postactivation. Repeated measures ANOVAs showed a significant effect of season for percent motility and linearity at 5 and 10s postactivation, velocity at 10s postactivation, and longevity. Our findings are important for optimizing fertilization protocols for hatchery production and can also be used to understand reproductive biology and ecology of wild Lake Trout stocks. Received March 15, 2012; accepted June 30, 201

Recruitment of young women to a trial of chlamydia screening – as easy as it sounds?

BACKGROUND: Recruiting to trials is complex and difficult. The Prevention of Pelvic Infection (POPI) trial aims to see if screening women for chlamydia and treating those found to be infected reduces the incidence of pelvic inflammatory disease in the following twelve months. It focuses on young, sexually active, multiethnic, mainly inner city, female students. The main aim of this paper is to describe our recruitment methods. Secondary aims in two small subgroups, are to compare characteristics of women recruited with those not recruited, and to explore participants' understanding of when their samples would be tested for chlamydia. METHODS: Women students attending lectures or in common rooms at 22 universities and further education colleges were recruited by female research assistants working in pairs. Participants were asked to complete a questionnaire on sexual health and to provide self-taken vaginal swabs. In addition, during 3 recruitment sessions, a female medical student asked non-participants to complete a brief anonymous questionnaire on reasons for not taking part. Finally another female medical student contacted 40 consecutive participants within a month of recruitment and asked if they understood that their samples might not be tested for a year. RESULTS: With enormous effort over 2 years we recruited 2526 women. A survey of 61 non-responders showed only 18 (30%) were eligible to take part (age <28, been sexually active and not been tested for chlamydia in the past 3 months). Eligible non-responders were of similar age to the 35 responders in the same recruitment sessions, but more likely to be from ethnic minority groups (67% 12/18 versus 29% 10/35 p < 0.01). Email and telephone contact with 35/40 (88%) of consecutive participants showed only two (6%) did not understand that their specimen might not be tested for chlamydia for a year. Thirty participants (85%) could name one or more possible consequences of untreated chlamydia infection. CONCLUSION: As in other studies, a key to attaining recruitment targets was the enthusiasm of the research team. Minority ethnic groups were probably under-represented, but understanding of participants was good