Strand-specific RNA sequencing reveals extensive regulated long antisense transcripts that are conserved across yeast species

Background Recent studies in budding yeast have shown that antisense transcription occurs at many loci. However, the functional role of antisense transcripts has been demonstrated only in a few cases and it has been suggested that most antisense transcripts may result from promiscuous bi-directional transcription in a dense genome. Results Here, we use strand-specific RNA sequencing to study anti-sense transcription in Saccharomyces cerevisiae. We detect 1,103 putative antisense transcripts expressed in mid-log phase growth, ranging from 39 short transcripts covering only the 3' UTR of sense genes to 145 long transcripts covering the entire sense open reading frame. Many of these antisense transcripts overlap sense genes that are repressed in mid-log phase and are important in stationary phase, stress response, or meiosis. We validate the differential regulation of 67 antisense transcripts and their sense targets in relevant conditions, including nutrient limitation and environmental stresses. Moreover, we show that several antisense transcripts and, in some cases, their differential expression have been conserved across five species of yeast spanning 150 million years of evolution. Divergence in the regulation of antisense transcripts to two respiratory genes coincides with the evolution of respiro-fermentation. Conclusions Our work provides support for a global and conserved role for antisense transcription in yeast gene regulation.Canadian Friends of the Hebrew UniversityHoward Hughes Medical InstituteHuman Frontier Science Program (Strasbourg, France)Burroughs Wellcome Fund (Career Award at the Scientific Interface)National Institutes of Health (U.S.). Pioneer AwardBroad Institute of MIT and HarvardU.S.-Israel Binational Science Foundation (BSF)National Human Genome Research Institute (U.S.)Alfred P. Sloan Foundatio

Teaching Qualitative Research for Human Services Students: A Three-Phase Model

Qualitative research is an inherent part of the human services profession, since it emphasizes the great and multifaceted complexity characterizing human experience and the sociocultural context in which humans act. In the department of human services at Emek Yezreel College, Israel, we have developed a three-phase model to ensure a relatively intense exposure to and practice in qualitative methodology. While in the first phase students are exposed to the qualitative thinking and writing, they are required in the second phase to take a Qualitative Research Methods course that includes practice. The third and final phase includes conducting a qualitative research seminar. The aim of the present article is to shed light on the dilemmas involved in implementing the three-phase model

Mass Azithromycin Distribution and Community Microbiome: A Cluster-Randomized Trial.

BackgroundMass distributions of oral azithromycin have long been used to eliminate trachoma, and they are now being proposed to reduce childhood mortality. The observed benefit appears to be augmented with each additional treatment, suggesting a possible community-level effect. Here, we assess whether 2 biannual mass treatments of preschool children affect the community's gut microbiome at 6 months after the last distribution.MethodsIn this cluster-randomized controlled trial, children aged 1-60 months in the Dossa region of Niger were randomized at the village level to receive a single dose of azithromycin or placebo every 6 months. Fecal samples were collected 6 months after the second treatment for metagenomic deep sequencing. The prespecified primary outcome was the Euclidean PERMANOVA of the gut microbiome, or effectively the distance between the genus-level centroid at the community level, with the secondary outcome being the Simpson's α diversity.ResultsIn the azithromycin arm, the gut microbial structures were significantly different than in the placebo arm (Euclidean PERMANOVA, P < .001). Further, the diversity of the gut microbiome in the azithromycin arm was significantly lower than in the placebo arm (inverse Simpson's index, P = .005).ConclusionsTwo mass azithromycin administrations, 6 months apart, in preschool children led to long-term alterations of the gut microbiome structure and community diversity. Here, long-term microbial alterations in the community did not imply disease but were associated with an improvement in childhood mortality.Clinical trials registrationNCT02048007

RNA polymerase mapping during stress responses reveals widespread nonproductive transcription in yeast

BACKGROUND: The use of genome-wide RNA abundance profiling by microarrays and deep sequencing has spurred a revolution in our understanding of transcriptional control. However, changes in mRNA abundance reflect the combined effect of changes in RNA production, processing, and degradation, and thus, mRNA levels provide an occluded view of transcriptional regulation. RESULTS: To partially disentangle these issues, we carry out genome-wide RNA polymerase II (PolII) localization profiling in budding yeast in two different stress response time courses. While mRNA changes largely reflect changes in transcription, there remains a great deal of variation in mRNA levels that is not accounted for by changes in PolII abundance. We find that genes exhibiting \u27excess\u27 mRNA produced per PolII are enriched for those with overlapping cryptic transcripts, indicating a pervasive role for nonproductive or regulatory transcription in control of gene expression. Finally, we characterize changes in PolII localization when PolII is genetically inactivated using the rpb1-1 temperature-sensitive mutation. We find that PolII is lost from chromatin after roughly an hour at the restrictive temperature, and that there is a great deal of variability in the rate of PolII loss at different loci. CONCLUSIONS: Together, these results provide a global perspective on the relationship between PolII and mRNA production in budding yeast

A pre-initiation complex at the 3′-end of genes drives antisense transcription independent of divergent sense transcription

The precise nature of antisense transcripts in eukaryotes such as Saccharomyces cerevisiae remains elusive. Here we show that the 3′ regions of genes possess a promoter architecture, including a pre-initiation complex (PIC), which mirrors that at the 5′ region and which is much more pronounced at genes with a defined antisense transcript. Remarkably, for genes with an antisense transcript, average levels of PIC components at the 3′ region are ∼60% of those at the 5′ region. Moreover, at these genes, average levels of nascent antisense transcription are ∼45% of sense transcription. We find that this 3′ promoter architecture persists for highly transcribed antisense transcripts where there are only low levels of transcription in the divergent sense direction, suggesting that the 3′ regions of genes can drive antisense transcription independent of divergent sense transcription. To validate this, we insert short 3′ regions into the middle of other genes and find that they are capable of both initiating antisense transcripts and terminating sense transcripts. Our results suggest that antisense transcription can be regulated independently of divergent sense transcription in a PIC-dependent manner and we propose that regulated production of antisense transcripts represents a fundamental and widespread component of gene regulation

Comprehensive comparative analysis of strand-specific RNA sequencing methods

Strand-specific, massively parallel cDNA sequencing (RNA-seq) is a powerful tool for transcript discovery, genome annotation and expression profiling. There are multiple published methods for strand-specific RNA-seq, but no consensus exists as to how to choose between them. Here we developed a comprehensive computational pipeline to compare library quality metrics from any RNA-seq method. Using the well-annotated Saccharomyces cerevisiae transcriptome as a benchmark, we compared seven library-construction protocols, including both published and our own methods. We found marked differences in strand specificity, library complexity, evenness and continuity of coverage, agreement with known annotations and accuracy for expression profiling. Weighing each method's performance and ease, we identified the dUTP second-strand marking and the Illumina RNA ligation methods as the leading protocols, with the former benefitting from the current availability of paired-end sequencing. Our analysis provides a comprehensive benchmark, and our computational pipeline is applicable for assessment of future protocols in other organisms.Howard Hughes Medical InstituteUnited States-Israel Binational Science Foundatio

Sub-clinical detection of gut microbial biomarkers of obesity and type 2 diabetes

Background: Obesity and type 2 diabetes (T2D) are linked both with host genetics and with environmental factors, including dysbioses of the gut microbiota. However, it is unclear whether these microbial changes precede disease onset. Twin cohorts present a unique genetically-controlled opportunity to study the relationships between lifestyle factors and the microbiome. In particular, we hypothesized that family-independent changes in microbial composition and metabolic function during the sub-clinical state of T2D could be either causal or early biomarkers of progression. Methods: We collected fecal samples and clinical metadata from 20 monozygotic Korean twins at up to two time points, resulting in 36 stool shotgun metagenomes. While the participants were neither obese nor diabetic, they spanned the entire range of healthy to near-clinical values and thus enabled the study of microbial associations during sub-clinical disease while accounting for genetic background. Results: We found changes both in composition and in function of the sub-clinical gut microbiome, including a decrease in Akkermansia muciniphila suggesting a role prior to the onset of disease, and functional changes reflecting a response to oxidative stress comparable to that previously observed in chronic T2D and inflammatory bowel diseases. Finally, our unique study design allowed us to examine the strain similarity between twins, and we found that twins demonstrate strain-level differences in composition despite species-level similarities. Conclusions: These changes in the microbiome might be used for the early diagnosis of an inflamed gut and T2D prior to clinical onset of the disease and will help to advance toward microbial interventions. Electronic supplementary material The online version of this article (doi:10.1186/s13073-016-0271-6) contains supplementary material, which is available to authorized users

Sub-clinical detection of gut microbial biomarkers of obesity and type 2 diabetes

Background: Obesity and type 2 diabetes (T2D) are linked both with host genetics and with environmental factors, including dysbioses of the gut microbiota. However, it is unclear whether these microbial changes precede disease onset. Twin cohorts present a unique genetically-controlled opportunity to study the relationships between lifestyle factors and the microbiome. In particular, we hypothesized that family-independent changes in microbial composition and metabolic function during the sub-clinical state of T2D could be either causal or early biomarkers of progression. Methods: We collected fecal samples and clinical metadata from 20 monozygotic Korean twins at up to two time points, resulting in 36 stool shotgun metagenomes. While the participants were neither obese nor diabetic, they spanned the entire range of healthy to near-clinical values and thus enabled the study of microbial associations during sub-clinical disease while accounting for genetic background. Results: We found changes both in composition and in function of the sub-clinical gut microbiome, including a decrease in Akkermansia muciniphila suggesting a role prior to the onset of disease, and functional changes reflecting a response to oxidative stress comparable to that previously observed in chronic T2D and inflammatory bowel diseases. Finally, our unique study design allowed us to examine the strain similarity between twins, and we found that twins demonstrate strain-level differences in composition despite species-level similarities. Conclusions: These changes in the microbiome might be used for the early diagnosis of an inflamed gut and T2D prior to clinical onset of the disease and will help to advance toward microbial interventions

Cesarean or vaginal birth does not impact the longitudinal development of the gut microbiome in a cohort of exclusively preterm infants

The short and long-term impact of birth mode on the developing gut microbiome in neonates has potential implications for the health of infants. In term infants, the microbiome immediately following birth across multiple body sites corresponds to birth mode, with increased Bacteroides in vaginally delivered infants. We aimed to determine the impact of birth mode of the preterm gut microbiome over the first 100 days of life and following neonatal intensive care unit (NICU) discharge. In total, 867 stool samples from 46 preterm infants (21 cesarean and 25 vaginal), median gestational age 27 weeks, were sequenced (V4 region 16S rRNA gene, Illumina MiSeq). Of these, 776 samples passed quality filtering and were included in the analysis. The overall longitudinal alpha-diversity and within infant beta-diversity was comparable between cesarean and vaginally delivered infants. Vaginally delivered infants kept significantly more OTUs from 2 months of life and following NICU discharge, but OTUs lost, gained, and regained were not different based on birth mode. Furthermore, the temporal progression of dominant genera was comparable between birth modes and no significant difference was found for any genera following adjustment for covariates. Lastly, preterm gut community types (PGCTs) showed some moderate differences in very early life, but progressed toward a comparable pattern by week 5. No PGCT was significantly associated with cesarean or vaginal birth. Unlike term infants, birth mode was not significantly associated with changes in microbial diversity, composition, specific taxa, or overall microbial development in preterm infants. This may result from the dominating effects of NICU exposures including the universal use of antibiotics immediately following birth and/or the lack of Bacteroides colonizing preterm infants

Establishing What Constitutes a Healthy Human Gut Microbiome: State of the Science, Regulatory Considerations, and Future Directions.

On December 17, 2018, the North American branch of the International Life Sciences Institute (ILSI North America) convened a workshop "Can We Begin to Define a Healthy Gut Microbiome Through Quantifiable Characteristics?" with >40 invited academic, government, and industry experts in Washington, DC. The workshop objectives were to 1) develop a collective expert assessment of the state of the evidence on the human gut microbiome and associated human health benefits, 2) see if there was sufficient evidence to establish measurable gut microbiome characteristics that could serve as indicators of "health," 3) identify short- and long-term research needs to fully characterize healthy gut microbiome-host relationships, and 4) publish the findings. Conclusions were as follows: 1) mechanistic links of specific changes in gut microbiome structure with function or markers of human health are not yet established; 2) it is not established if dysbiosis is a cause, consequence, or both of changes in human gut epithelial function and disease; 3) microbiome communities are highly individualized, show a high degree of interindividual variation to perturbation, and tend to be stable over years; 4) the complexity of microbiome-host interactions requires a comprehensive, multidisciplinary research agenda to elucidate relationships between gut microbiome and host health; 5) biomarkers and/or surrogate indicators of host function and pathogenic processes based on the microbiome need to be determined and validated, along with normal ranges, using approaches similar to those used to establish biomarkers and/or surrogate indicators based on host metabolic phenotypes; 6) future studies measuring responses to an exposure or intervention need to combine validated microbiome-related biomarkers and/or surrogate indicators with multiomics characterization of the microbiome; and 7) because static genetic sampling misses important short- and long-term microbiome-related dynamic changes to host health, future studies must be powered to account for inter- and intraindividual variation and should use repeated measures within individuals