Taxonomic Classification of Bacterial 16S rRNA Genes Using Short Sequencing Reads: Evaluation of Effective Study Designs

Massively parallel high throughput sequencing technologies allow us to interrogate the microbial composition of biological samples at unprecedented resolution. The typical approach is to perform high-throughout sequencing of 16S rRNA genes, which are then taxonomically classified based on similarity to known sequences in existing databases. Current technologies cause a predicament though, because although they enable deep coverage of samples, they are limited in the length of sequence they can produce. As a result, high-throughout studies of microbial communities often do not sequence the entire 16S rRNA gene. The challenge is to obtain reliable representation of bacterial communities through taxonomic classification of short 16S rRNA gene sequences. In this study we explored properties of different study designs and developed specific recommendations for effective use of short-read sequencing technologies for the purpose of interrogating bacterial communities, with a focus on classification using naïve Bayesian classifiers. To assess precision and coverage of each design, we used a collection of ∼8,500 manually curated 16S rRNA gene sequences from cultured bacteria and a set of over one million bacterial 16S rRNA gene sequences retrieved from environmental samples, respectively. We also tested different configurations of taxonomic classification approaches using short read sequencing data, and provide recommendations for optimal choice of the relevant parameters. We conclude that with a judicious selection of the sequenced region and the corresponding choice of a suitable training set for taxonomic classification, it is possible to explore bacterial communities at great depth using current technologies, with only a minimal loss of taxonomic resolution.</p

Host genetic variation in mucosal immunity pathways influences the upper airway microbiome

Nasal microbiome alpha diversity (XLSX 43 kb

ABO antigen and secretor statuses are not associated with gut microbiota composition in 1,500 twins

Background: Host genetics is one of several factors known to shape human gut microbiome composition, however, the physiological processes underlying the heritability are largely unknown. Inter-individual differences in host factors secreted into the gut lumen may lead to variation in microbiome composition. One such factor is the ABO antigen. This molecule is not only expressed on the surface of red blood cells, but is also secreted from mucosal surfaces in individuals containing an intact FUT2 gene (secretors). Previous studies report differences in microbiome composition across ABO and secretor genotypes. However, due to methodological limitations, the specific bacterial taxa involved remain unknown.Results: Here, we sought to determine the relationship of the microbiota to ABO blood group and secretor status in a large panel of 1503 individuals from a cohort of twins from the United Kingdom. Contrary to previous reports, robust associations between either ABO or secretor phenotypes and gut microbiome composition were not detected. Overall community structure, diversity, and the relative abundances of individual taxa were not significantly associated with ABO or secretor status. Additionally, joint-modeling approaches were unsuccessful in identifying combinations of taxa that were predictive of ABO or secretor status.Conclusions: Despite previous reports, the taxonomic composition of the microbiota does not appear to be strongly associated with ABO or secretor status in 1503 individuals from the United Kingdom. These results highlight the importance of replicating microbiome-associated traits in large, well-powered cohorts to ensure results are robust

Short Term Variability of Evolved Massive Stars with TESS II: A New Class of Cool, Pulsating Supergiants

Massive stars briefly pass through the yellow supergiant (YSG) phase as they evolve redward across the HR diagram and expand into red supergiants (RSGs). Higher-mass stars pass through the YSG phase again as they evolve blueward after experiencing significant RSG mass loss. These post-RSG objects offer us a tantalizing glimpse into which stars end their lives as RSGs, and why. One telltale sign of a post-RSG object may be an instability to pulsations, depending on the star's interior structure. Here we report the discovery of five YSGs with pulsation periods faster than 1 day, found in a sample of 76 cool supergiants observed by \tess at two-minute cadence. These pulsating YSGs are concentrated in a HR diagram region not previously associated with pulsations; we conclude that this is a genuine new class of pulsating star, Fast Yellow Pulsating Supergiants (FYPS). For each FYPS, we extract frequencies via iterative prewhitening and conduct a time-frequency analysis. One FYPS has an extracted frequency that is split into a triplet, and the amplitude of that peak is modulated on the same timescale as the frequency spacing of the triplet; neither rotation nor binary effects are likely culprits. We discuss the evolutionary status of FYPS and conclude that they are candidate post-RSGs. All stars in our sample also show the same stochastic low-frequency variability (SLFV) found in hot OB stars and attributed to internal gravity waves. Finally, we find four $\alpha$ Cygni variables in our sample, of which three are newly discovered.Comment: 29 pages, 13 figures, 8 tables. Accepted for publication in ApJ. Comments welcom

The Properties of Fast Yellow Pulsating Supergiants: FYPS Point the Way to Missing Red Supergiants

Fast yellow pulsating supergiants (FYPS) are a recently-discovered class of evolved massive pulsator. As candidate post-red supergiant objects, and one of the few classes of pulsating evolved massive stars, these objects have incredible potential to change our understanding of the structure and evolution of massive stars. Here we examine the lightcurves of a sample of 126 cool supergiants in the Magellanic Clouds observed by the Transiting Exoplanet Survey Satellite (\tess~) in order to identify pulsating stars. After making quality cuts and filtering out contaminant objects, we examine the distribution of pulsating stars in the Hertzprung-Russel (HR) diagram, and find that FYPS occupy a region above $\log L/L_\odot \gtrsim 5.0$. This luminosity boundary corresponds to stars with initial masses of $\sim$18-20 $M_\odot$, consistent with the most massive red supergiant progenitors of supernovae (SNe) II-P, as well as the observed properties of SNe IIb progenitors. This threshold is in agreement with the picture that FYPS are post-RSG stars. Finally, we characterize the behavior of FYPS pulsations as a function of their location in the HR diagram. We find low frequency pulsations at higher effective temperatures, higher frequency pulsations at lower temperatures, with a transition between the two behaviors at intermediate temperatures. The observed properties of FYPS make them fascinating objects for future theoretical study.Comment: Consistent with published version which contains significantly improved detection and rejection of contaminant objects. Comments welcom

Epigenetic modifications are associated with inter-species gene expression variation in primates

Abstract Background Changes in gene regulation have long been thought to play an important role in evolution and speciation, especially in primates. Over the past decade, comparative genomic studies have revealed extensive inter-species differences in gene expression levels, yet we know much less about the extent to which regulatory mechanisms differ between species. Results To begin addressing this gap, we perform a comparative epigenetic study in primate lymphoblastoid cell lines, to query the contribution of RNA polymerase II and four histone modifications, H3K4me1, H3K4me3, H3K27ac, and H3K27me3, to inter-species variation in gene expression levels. We find that inter-species differences in mark enrichment near transcription start sites are significantly more often associated with inter-species differences in the corresponding gene expression level than expected by chance alone. Interestingly, we also find that first-order interactions among the five marks, as well as chromatin states, do not markedly contribute to the degree of association between the marks and inter-species variation in gene expression levels, suggesting that the marginal effects of the five marks dominate this contribution. Conclusions Our observations suggest that epigenetic modifications are substantially associated with changes in gene expression levels among primates and may represent important molecular mechanisms in primate evolution.http://deepblue.lib.umich.edu/bitstream/2027.42/110207/1/13059_2014_Article_547.pd

No Conclusive Evidence for Transits of Proxima b in MOST photometry

The analysis of Proxima Centauri's radial velocities recently led Anglada-Escud\'e et al. (2016) to claim the presence of a low mass planet orbiting the Sun's nearest star once every 11.2 days. Although the a-priori probability that Proxima b transits its parent star is just 1.5%, the potential impact of such a discovery would be considerable. Independent of recent radial velocity efforts, we observed Proxima Centauri for 12.5 days in 2014 and 31 days in 2015 with the MOST space telescope. We report here that we cannot make a compelling case that Proxima b transits in our precise photometric time series. Imposing an informative prior on the period and phase, we do detect a candidate signal with the expected depth. However, perturbing the phase prior across 100 evenly spaced intervals reveals one strong false-positive and one weaker instance. We estimate a false-positive rate of at least a few percent and a much higher false-negative rate of 20-40%, likely caused by the very high flare rate of Proxima Centauri. Comparing our candidate signal to HATSouth ground-based photometry reveals that the signal is somewhat, but not conclusively, disfavored (1-2 sigmas) leading us to argue that the signal is most likely spurious. We expect that infrared photometric follow-up could more conclusively test the existence of this candidate signal, owing to the suppression of flare activity and the impressive infrared brightness of the parent star.Comment: Accepted to ApJ. Posterior samples, MOST photometry and HATSouth photometry are all available at https://github.com/CoolWorlds/Proxim

Kepler Flares II: The Temporal Morphology of White-Light Flares on GJ 1243

We present the largest sample of flares ever compiled for a single M dwarf, the active M4 star GJ 1243. Over 6100 individual flare events, with energies ranging from $10^{29}$ to $10^{33}$ erg, are found in 11 months of 1-minute cadence data from Kepler. This sample is unique for its completeness and dynamic range. We have developed automated tools for finding flares in short-cadence Kepler light curves, and performed extensive validation and classification of the sample by eye. From this pristine sample of flares we generate a median flare template. This template shows that two exponential cooling phases are present during the white-light flare decay, providing fundamental constraints for models of flare physics. The template is also used as a basis function to decompose complex multi-peaked flares, allowing us to study the energy distribution of these events. Only a small number of flare events are not well fit by our template. We find that complex, multi-peaked flares occur in over 80% of flares with a duration of 50 minutes or greater. The underlying distribution of flare durations for events 10 minutes and longer appears to follow a broken power law. Our results support the idea that sympathetic flaring may be responsible for some complex flare events.Comment: 12 pages, 9 figures, accepted for publication in Ap

Heritable components of the human fecal microbiome are associated with visceral fat

Background: Variation in the human fecal microbiota has previously been associated with body mass index (BMI). Although obesity is a global health burden, the accumulation of abdominal visceral fat is the specific cardio-metabolic disease risk factor. Here, we explore links between the fecal microbiota and abdominal adiposity using body composition as measured by dual-energy X-ray absorptiometry in a large sample of twins from the TwinsUK cohort, comparing fecal 16S rRNA diversity profiles with six adiposity measures.Results: We profile six adiposity measures in 3666 twins and estimate their heritability, finding novel evidence for strong genetic effects underlying visceral fat and android/gynoid ratio. We confirm the association of lower diversity of the fecal microbiome with obesity and adiposity measures, and then compare the association between fecal microbial composition and the adiposity phenotypes in a discovery subsample of twins. We identify associations between the relative abundances of fecal microbial operational taxonomic units (OTUs) and abdominal adiposity measures. Most of these results involve visceral fat associations, with the strongest associations between visceral fat and Oscillospira members. Using BMI as a surrogate phenotype, we pursue replication in independent samples from three population-based cohorts including American Gut, Flemish Gut Flora Project and the extended TwinsUK cohort. Meta-analyses across the replication samples indicate that 8 OTUs replicate at a stringent threshold across all cohorts, while 49 OTUs achieve nominal significance in at least one replication sample. Heritability analysis of the adiposity-associated microbial OTUs prompted us to assess host genetic-microbe interactions at obesity-associated human candidate loci. We observe significant associations of adiposity-OTU abundances with host genetic variants in the FHIT, TDRG1 and ELAVL4 genes, suggesting a potential role for host genes to mediate the link between the fecal microbiome and obesity.Conclusions: Our results provide novel insights into the role of the fecal microbiota in cardio-metabolic disease with clear potential for prevention and novel therapies