Rye: genetic ancestry inference at biobank scale.

Biobank projects are generating genomic data for many thousands of individuals. Computational methods are needed to handle these massive data sets, including genetic ancestry (GA) inference tools. Current methods for GA inference do not scale to biobank-size genomic datasets. We present Rye-a new algorithm for GA inference at biobank scale. We compared the accuracy and runtime performance of Rye to the widely used RFMix, ADMIXTURE and iAdmix programs and applied it to a dataset of 488221 genome-wide variant samples from the UK Biobank. Rye infers GA based on principal component analysis of genomic variant samples from ancestral reference populations and query individuals. The algorithm's accuracy is powered by Metropolis-Hastings optimization and its speed is provided by non-negative least squares regression. Rye produces highly accurate GA estimates for three-way admixed populations-African, European and Native American-compared to RFMix and ADMIXTURE (${R}^2 = \ 0.998 - 1.00$), and shows 50× runtime improvement compared to ADMIXTURE on the UK Biobank dataset. Rye analysis of UK Biobank samples demonstrates how it can be used to infer GA at both continental and subcontinental levels. We discuss user consideration and options for the use of Rye; the program and its documentation are distributed on the GitHub repository: https://github.com/healthdisparities/rye.

Multistate Outbreak of SARS-CoV-2 Infections, Including Vaccine Breakthrough Infections, Associated with Large Public Gatherings, United States.

During July 2021, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) B.1.617.2 variant infections, including vaccine breakthrough infections, occurred after large public gatherings in Provincetown, Massachusetts, USA, prompting a multistate investigation. Public health departments identified primary and secondary cases by using coronavirus disease surveillance data, case investigations, and contact tracing. A primary case was defined as SARS-CoV-2 detected <14 days after travel to or residence in Provincetown during July 3-17. A secondary case was defined as SARS-CoV-2 detected <14 days after close contact with a person who had a primary case but without travel to or residence in Provincetown during July 3-August 10. We identified 1,098 primary cases and 30 secondary cases associated with 26 primary cases among fully and non-fully vaccinated persons. Large gatherings can have widespread effects on SARS-CoV-2 transmission, and fully vaccinated persons should take precautions, such as masking, to prevent SARS-CoV-2 transmission, particularly during substantial or high transmission.

Native American admixture recapitulates population-specific migration and settlement of the continental United States.

European and African descendants settled the continental US during the 17th-19th centuries, coming into contact with established Native American populations. The resulting admixture among these groups yielded a significant reservoir of Native American ancestry in the modern US population. We analyzed the patterns of Native American admixture seen for the three largest genetic ancestry groups in the US population: African descendants, Western European descendants, and Spanish descendants. The three groups show distinct Native American ancestry profiles, which are indicative of their historical patterns of migration and settlement across the country. Native American ancestry in the modern African descendant population does not coincide with local geography, instead forming a single group with origins in the southeastern US, consistent with the Great Migration of the early 20th century. Western European descendants show Native American ancestry that tracks their geographic origins across the US, indicative of ongoing contact during westward expansion, and Native American ancestry can resolve Spanish descendant individuals into distinct local groups formed by more recent migration from Mexico and Puerto Rico. We found an anomalous pattern of Native American ancestry from the US southwest, which most likely corresponds to the Nuevomexicano descendants of early Spanish settlers to the region. We addressed a number of controversies surrounding this population, including the extent of Sephardic Jewish ancestry. Nuevomexicanos are less admixed than nearby Mexican-American individuals, with more European and less Native American and African ancestry, and while they do show demonstrable Sephardic Jewish ancestry, the fraction is no greater than seen for other New World Spanish descendant populations.

Population structure and pharmacogenomic risk stratification in the United States.

BACKGROUND: Pharmacogenomic (PGx) variants mediate how individuals respond to medication, and response differences among racial/ethnic groups have been attributed to patterns of PGx diversity. We hypothesized that genetic ancestry (GA) would provide higher resolution for stratifying PGx risk, since it serves as a more reliable surrogate for genetic diversity than self-identified race/ethnicity (SIRE), which includes a substantial social component. We analyzed a cohort of 8628 individuals from the United States (US), for whom we had both SIRE information and whole genome genotypes, with a focus on the three largest SIRE groups in the US: White, Black (African-American), and Hispanic (Latino). Our approach to the question of PGx risk stratification entailed the integration of two distinct methodologies: population genetics and evidence-based medicine. This integrated approach allowed us to consider the clinical implications for the observed patterns of PGx variation found within and between population groups. RESULTS: Whole genome genotypes were used to characterize individuals' continental ancestry fractions-European, African, and Native American-and individuals were grouped according to their GA profiles. SIRE and GA groups were found to be highly concordant. Continental ancestry predicts individuals' SIRE with > 96% accuracy, and accordingly, GA provides only a marginal increase in resolution for PGx risk stratification. In light of the concordance between SIRE and GA, taken together with the fact that information on SIRE is readily available to clinicians, we evaluated PGx variation between SIRE groups to explore the potential clinical utility of race and ethnicity. PGx variants are highly diverged compared to the genomic background; 82 variants show significant frequency differences among SIRE groups, and genome-wide patterns of PGx variation are almost entirely concordant with SIRE. The vast majority of PGx variation is found within rather than between groups, a well-established fact for almost all genetic variants, which is often taken to argue against the clinical utility of population stratification. Nevertheless, analysis of highly differentiated PGx variants illustrates how SIRE partitions PGx variation based on groups' characteristic ancestry patterns. These cases underscore the extent to which SIRE carries clinically valuable information for stratifying PGx risk among populations, albeit with less utility for predicting individual-level PGx alleles (genotypes), supporting the concept of population pharmacogenomics. CONCLUSIONS: Perhaps most interestingly, we show that individuals who identify as Black or Hispanic stand to gain far more from the consideration of race/ethnicity in treatment decisions than individuals from the majority White population.

Rapid Nanopore Whole-Genome Sequencing for Anthrax Emergency Preparedness.

Human anthrax cases necessitate rapid response. We completed Bacillus anthracis nanopore whole-genome sequencing in our high-containment laboratory from a human anthrax isolate hours after receipt. The de novo assembled genome showed no evidence of known antimicrobial resistance genes or introduced plasmid(s). Same-day genomic characterization enhances public health emergency response.

Ancestry effects on type 2 diabetes genetic risk inference in Hispanic/Latino populations.

BACKGROUND: Hispanic/Latino (HL) populations bear a disproportionately high burden of type 2 diabetes (T2D). The ability to predict T2D genetic risk using polygenic risk scores (PRS) offers great promise for improved screening and prevention. However, there are a number of complications related to the accurate inference of genetic risk across HL populations with distinct ancestry profiles. We investigated how ancestry affects the inference of T2D genetic risk using PRS in diverse HL populations from Colombia and the United States (US). In Colombia, we compared T2D genetic risk for the Mestizo population of Antioquia to the Afro-Colombian population of Chocó, and in the US, we compared European-American versus Mexican-American populations. METHODS: Whole genome sequences and genotypes from the 1000 Genomes Project and the ChocoGen Research Project were used for genetic ancestry inference and for T2D polygenic risk score (PRS) calculation. Continental ancestry fractions for HL genomes were inferred via comparison with African, European, and Native American reference genomes, and PRS were calculated using T2D risk variants taken from multiple genome-wide association studies (GWAS) conducted on cohorts with diverse ancestries. A correction for ancestry bias in T2D risk inference based on the frequencies of ancestral versus derived alleles was developed and applied to PRS calculations in the HL populations studied here. RESULTS: T2D genetic risk in Colombian and US HL populations is positively correlated with African and Native American ancestry and negatively correlated with European ancestry. The Afro-Colombian population of Chocó has higher predicted T2D risk than Antioquia, and the Mexican-American population has higher predicted risk than the European-American population. The inferred relative risk of T2D is robust to differences in the ancestry of the GWAS cohorts used for variant discovery. For trans-ethnic GWAS, population-specific variants and variants with same direction effects across populations yield consistent results. Nevertheless, the control for bias in T2D risk prediction confirms that explicit consideration of genetic ancestry can yield more reliable cross-population genetic risk inferences. CONCLUSIONS: T2D associations that replicate across populations provide for more reliable risk inference, and modeling population-specific frequencies of ancestral and derived risk alleles can help control for biases in PRS estimation.

GlobAl Distribution of GEnetic Traits (GADGET) web server: polygenic trait scores worldwide.

Human populations from around the world show striking phenotypic variation across a wide variety of traits. Genome-wide association studies (GWAS) are used to uncover genetic variants that influence the expression of heritable human traits; accordingly, population-specific distributions of GWAS-implicated variants may shed light on the genetic basis of human phenotypic diversity. With this in mind, we developed the GlobAl Distribution of GEnetic Traits web server (GADGET http://gadget.biosci.gatech.edu). The GADGET web server provides users with a dynamic visual platform for exploring the relationship between worldwide genetic diversity and the genetic architecture underlying numerous human phenotypes. GADGET integrates trait-implicated single nucleotide polymorphisms (SNPs) from GWAS, with population genetic data from the 1000 Genomes Project, to calculate genome-wide polygenic trait scores (PTS) for 818 phenotypes in 2504 individual genomes. Population-specific distributions of PTS are shown for 26 human populations across 5 continental population groups, with traits ordered based on the extent of variation observed among populations. Users of GADGET can also upload custom trait SNP sets to visualize global PTS distributions for their own traits of interest.

On transient climate change at the Cretaceous-Paleogene boundary due to atmospheric soot injections.

Climate simulations that consider injection into the atmosphere of 15,000 Tg of soot, the amount estimated to be present at the Cretaceous-Paleogene boundary, produce what might have been one of the largest episodes of transient climate change in Earth history. The observed soot is believed to originate from global wildfires ignited after the impact of a 10-km-diameter asteroid on the Yucatán Peninsula 66 million y ago. Following injection into the atmosphere, the soot is heated by sunlight and lofted to great heights, resulting in a worldwide soot aerosol layer that lasts several years. As a result, little or no sunlight reaches the surface for over a year, such that photosynthesis is impossible and continents and oceans cool by as much as 28 °C and 11 °C, respectively. The absorption of light by the soot heats the upper atmosphere by hundreds of degrees. These high temperatures, together with a massive injection of water, which is a source of odd-hydrogen radicals, destroy the stratospheric ozone layer, such that Earth's surface receives high doses of UV radiation for about a year once the soot clears, five years after the impact. Temperatures remain above freezing in the oceans, coastal areas, and parts of the Tropics, but photosynthesis is severely inhibited for the first 1 y to 2 y, and freezing temperatures persist at middle latitudes for 3 y to 4 y. Refugia from these effects would have been very limited. The transient climate perturbation ends abruptly as the stratosphere cools and becomes supersaturated, causing rapid dehydration that removes all remaining soot via wet deposition.

A Nationwide Screen of Carbapenem-Resistant Klebsiella pneumoniae Reveals an Isolate with Enhanced Virulence and Clinically Undetected Colistin Heteroresistance.

The convergence of hypervirulence and multidrug resistance in Klebsiella pneumoniae is a significant concern. Here, we report the first screen for hypermucoviscosity, a trait associated with increased virulence, using a U.S. surveillance collection of carbapenem-resistant (CR) K. pneumoniae isolates. We identified one hypermucoviscous isolate, which carried a gene encoding the KPC-3 carbapenemase, among numerous resistance genes. The strain further exhibited colistin heteroresistance undetected by diagnostics. This convergence of diverse resistance mechanisms and increased virulence underscores the need for enhanced K. pneumoniae surveillance.

Atmospheric Acetaldehyde: Importance of Air-Sea Exchange and a Missing Source in the Remote Troposphere.

We report airborne measurements of acetaldehyde (CH3CHO) during the first and second deployments of the National Aeronautics and Space Administration (NASA) Atmospheric Tomography Mission (ATom). The budget of CH3CHO is examined using the Community Atmospheric Model with chemistry (CAM-chem), with a newly-developed online air-sea exchange module. The upper limit of the global ocean net emission of CH3CHO is estimated to be 34 Tg a-1 (42 Tg a-1 if considering bubble-mediated transfer), and the ocean impacts on tropospheric CH3CHO are mostly confined to the marine boundary layer. Our analysis suggests that there is an unaccounted CH3CHO source in the remote troposphere and that organic aerosols can only provide a fraction of this missing source. We propose that peroxyacetic acid (PAA) is an ideal indicator of the rapid CH3CHO production in the remote troposphere. The higher-than-expected CH3CHO measurements represent a missing sink of hydroxyl radicals (and halogen radical) in current chemistry-climate models.

Genetic ancestry, admixture and health determinants in Latin America.

BACKGROUND: Modern Latin American populations were formed via genetic admixture among ancestral source populations from Africa, the Americas and Europe. We are interested in studying how combinations of genetic ancestry in admixed Latin American populations may impact genomic determinants of health and disease. For this study, we characterized the impact of ancestry and admixture on genetic variants that underlie health- and disease-related phenotypes in population genomic samples from Colombia, Mexico, Peru, and Puerto Rico. RESULTS: We analyzed a total of 347 admixed Latin American genomes along with 1102 putative ancestral source genomes from Africans, Europeans, and Native Americans. We characterized the genetic ancestry, relatedness, and admixture patterns for each of the admixed Latin American genomes, finding a spectrum of ancestry proportions within and between populations. We then identified single nucleotide polymorphisms (SNPs) with anomalous ancestry-enrichment patterns, i.e. SNPs that exist in any given Latin American population at a higher frequency than expected based on the population's genetic ancestry profile. For this set of ancestry-enriched SNPs, we inspected their phenotypic impact on disease, metabolism, and the immune system. All four of the Latin American populations show ancestry-enrichment for a number of shared pathways, yielding evidence of similar selection pressures on these populations during their evolution. For example, all four populations show ancestry-enriched SNPs in multiple genes from immune system pathways, such as the cytokine receptor interaction, T cell receptor signaling, and antigen presentation pathways. We also found SNPs with excess African or European ancestry that are associated with ancestry-specific gene expression patterns and play crucial roles in the immune system and infectious disease responses. Genes from both the innate and adaptive immune system were found to be regulated by ancestry-enriched SNPs with population-specific regulatory effects. CONCLUSIONS: Ancestry-enriched SNPs in Latin American populations have a substantial effect on health- and disease-related phenotypes. The concordant impact observed for same phenotypes across populations points to a process of adaptive introgression, whereby ancestry-enriched SNPs with specific functional utility appear to have been retained in modern populations by virtue of their effects on health and fitness.

Genome-wide map of Apn1 binding sites under oxidative stress in Saccharomyces cerevisiae.

The DNA is cells is continuously exposed to reactive oxygen species resulting in toxic and mutagenic DNA damage. Although the repair of oxidative DNA damage occurs primarily through the base excision repair (BER) pathway, the nucleotide excision repair (NER) pathway processes some of the same lesions. In addition, damage tolerance mechanisms, such as recombination and translesion synthesis, enable cells to tolerate oxidative DNA damage, especially when BER and NER capacities are exceeded. Thus, disruption of BER alone or disruption of BER and NER in Saccharomyces cerevisiae leads to increased mutations as well as large-scale genomic rearrangements. Previous studies demonstrated that a particular region of chromosome II is susceptible to chronic oxidative stress-induced chromosomal rearrangements, suggesting the existence of DNA damage and/or DNA repair hotspots. Here we investigated the relationship between oxidative damage and genomic instability utilizing chromatin immunoprecipitation combined with DNA microarray technology to profile DNA repair sites along yeast chromosomes under different oxidative stress conditions. We targeted the major yeast AP endonuclease Apn1 as a representative BER protein. Our results indicate that Apn1 target sequences are enriched for cytosine and guanine nucleotides. We predict that BER protects these sites in the genome because guanines and cytosines are thought to be especially susceptible to oxidative attack, thereby preventing large-scale genome destabilization from chronic accumulation of DNA damage. Information from our studies should provide insight into how regional deployment of oxidative DNA damage management systems along chromosomes protects against large-scale rearrangements. Copyright © 2017 John Wiley & Sons, Ltd.

High-throughput sequencing of two populations of extracellular vesicles provides an mRNA signature that can be detected in the circulation of breast cancer patients.

Extracellular vesicles (EVs) contain a wide range of RNA types with a reported prevalence of non-coding RNA. To date a comprehensive characterization of the protein coding transcripts in EVs is still lacking. We performed RNA-Sequencing (RNA-Seq) of 2 EV populations and identified a small fraction of transcripts that were expressed at significantly different levels in large oncosomes and exosomes, suggesting they may mediate specialized functions. However, these 2 EV populations exhibited a common mRNA signature that, in comparison to their donor cells, was significantly enriched in mRNAs encoding E2F transcriptional targets and histone proteins. These mRNAs are primarily expressed in the S-phase of the cell cycle, suggesting that they may be packaged into EVs during S-phase. In silico analysis using subcellular compartment transcriptome data from the ENCODE cell line compendium revealed that EV mRNAs originate from a cytoplasmic RNA pool. The EV signature was independently identified in plasma of patients with breast cancer by RNA-Seq. Furthermore, several transcripts differentially expressed in EVs from patients versus controls mirrored differential expression between normal and breast cancer tissues. Altogether, this largest high-throughput profiling of EV mRNA demonstrates that EVs carry tumor-specific alterations and can be interrogated as a source of cancer-derived cargo.

Protein body formation in leaves of Nicotiana benthamiana: a concentration-dependent mechanism influenced by the presence of fusion tags.

Protein bodies (PBs) are endoplasmic reticulum (ER) derived organelles originally found in seeds whose function is to accumulate seed storage proteins. It has been shown that PB formation is not limited to seeds and green fluorescent protein (GFP) fused to either elastin-like polypeptide (ELP) or hydrophobin (HFBI) fusion tags induce the formation of PBs in leaves of N. benthamiana. In this study, we compared the ELP- and HFBI-induced PBs and showed that ELP-induced PBs are larger than HFBI-induced PBs. The size of ELP- and HFBI-induced PBs increased over time along with the accumulation levels of their fused protein. Our results show that PB formation is a concentration-dependent mechanism in which proteins accumulating at levels higher than 0.2% of total soluble protein are capable of inducing PBs in vivo. Our results show that the presence of fusion tags is not necessary for the formation of PBs, but affects the distribution pattern and size of PBs. This was confirmed by PBs induced by fluorescent proteins as well as fungal xylanases. We noticed that in the process of PB formation, secretory and ER-resident molecules are passively sequestered into the lumen of PBs. We propose to use this property of PBs as a tool to increase the accumulation levels of erythropoietin and human interleukin-10 by co-expression with PB-inducing proteins.

Hypoxia after transarterial chemoembolization may trigger a progenitor cell phenotype in hepatocellular carcinoma.

AIMS: To test the hypothesis that the hypoxia marker carbonic anhydrase IX (CAIX), and the cholangiocytic/progenitor markers cytokeratin (CK) 19 and epithelial cell adhesion molecule (EpCAM), may be expressed in areas of hypoxia in hepatocellular carcinoma (HCC) after transarterial chemoembolization (TACE). METHODS AND RESULTS: Immunohistochemistry for CAIX, CK19 and EpCAM (BerEP4) was performed in 57 HCCs, including 40 residual/recurrent tumours adjacent to the TACE treatment site and 17 untreated tumours from the same 40 patients. CAIX was exxpressed in 19 of 40 residual/recurrent HCCs and in two of 17 untreated HCCs. The rate of CAIX immunoreactivity in the treated tumours was significantly higher than that in the non-treated tumours (47.5% versus 11.8%, P = 0.015). CK19 and EpCAM were expressed in six of 19 and in seven of 19 CAIX-positive TACE-treated HCCs, respectively, but were not expressed in CAIX-negative tumours or untreated tumours. There were significant associations between CK19 and CAIX immunoreactivity, and between EpCAM and CAIX immunoreactivity (P = 0.007 and P = 0.003, respectively). Double staining of CAIX and CK19 showed co-localization of both proteins in five of six cases. Three of seven EpCAM-positive tumours were also positive for CK19. CONCLUSIONS: Hypoxia may trigger the expression of proteins that are normally associated with cholangiocytic/progenitor cell differentiation, suggesting that TACE paradoxically causes an aggressive tumour phenotype.

Whole-Transcriptome profiling of formalin-fixed, paraffin-embedded renal cell carcinoma by RNA-seq.

BACKGROUND: Formalin-fixed paraffin-embedded (FFPE) tissue samples are routinely archived in the course of patient care and can be linked to clinical outcomes with long-term follow-up. However, FFPE tissues have degraded RNA which poses challenges for analyzing gene expression. Next-generation sequencing (NGS) is rapidly becoming accepted as an effective tool for measuring gene expressions for research and clinical use. However, the feasibility of NGS has not been firmly established when using FFPE tissue. RESULTS: We optimized strategies for whole transcriptome sequencing (RNA-seq) using FFPE tissue. Ribosomal RNA (rRNA) was successfully depleted by competitive hybridization using the Ribo-zero™ Kit (Epicentre Biotechnologies), and rRNA sequence content was less than one percent for each library. Gene expression measured by FFPE RNA-seq was compared to two different standards: RNA-seq from fresh frozen (FF) tissue and quantitative PCR (qPCR). Both FF and FFPE tumors were sequenced on an Illumina Genome Analyzer IIX with an average of 10 million reads. The distribution of FPKMs (fragments per kilobase of exon per million fragments mapped) and number of detected genes were similar between FFPE and FF. RNA-seq expressions from FF and FFPE samples from the same renal cell carcinoma (RCC) correlated highly (r = 0.919 for tumor 1 and r = 0.954 for tumor 2). On hierarchical cluster analysis, samples clustered by patient identity rather than method of preservation. TaqMan qPCR of 424 RCC-related genes correlated highly with FFPE RNA-seq expressions (r = 0.775 for FFPE tumor 1, r = 0.803 for FFPE tumor 2). Expression fold changes were considered, to assess biologic relevance of gene expressions. Expression fold changes between FFPE tumors (tumor 1/tumor 2) correlated well when comparing qPCR and RNA-seq (r = 0.890). Expression fold changes between tumors from different risk groups (our high risk RCC/The Cancer Genome Atlas, TCGA, low risk RCC) also correlated well when comparing RNA-seq from FF and FFPE tumors (r = 0.887). CONCLUSIONS: FFPE RNA-seq provides reliable genes expression data, comparable to that obtained from fresh frozen tissue. It represents a useful tool for discovery and validation of biomarkers.

Production and characterization of in planta transiently produced polygalacturanase from Aspergillus niger and its fusions with hydrophobin or ELP tags.

BACKGROUND: Pectinases play an important role in plant cell wall deconstruction and have potential in diverse industries such as food, wine, animal feed, textile, paper, fuel, and others. The demand for such enzymes is increasing exponentially, as are the efforts to improve their production and to implement their use in several industrial processes. The goal of this study was to examine the potential of producing polygalacturonase I from Aspergillus niger in plants and to investigate the effects of subcellular compartmentalization and protein fusions on its accumulation and activity. RESULTS: Polygalacturonase I from Aspergillus niger (AnPGI) was transiently produced in Nicotiana benthamiana by targeting it to five different cellular compartments: apoplast, endoplasmic reticulum (ER), vacuole, chloroplast and cytosol. Accumulation levels of 2.5%, 3.0%, and 1.9% of total soluble protein (TSP) were observed in the apoplast, ER, and vacuole, respectively, and specific activity was significantly higher in vacuole-targeted AnPGI compared to the same enzyme targeted to the ER or apoplast. No accumulation was found for AnPGI when targeted to the chloroplast or cytosol. Analysis of AnPGI fused with elastin-like polypeptide (ELP) revealed a significant increase in the protein accumulation level, especially when targeted to the vacuole where the protein doubles its accumulation to 3.6% of TSP, while the hydrophobin (HFBI) fusion impaired AnPGI accumulation and both tags impaired activity, albeit to different extents. The recombinant protein showed activity against polygalacturonic acid with optimum conditions at pH 5.0 and temperature from 30 to 50°C, depending on its fusion. In vivo analysis of reducing sugar content revealed a higher release of reducing sugars in plant tissue expressing recombinant AnPGI compared to wild type N. benthamiana leaves. CONCLUSION: Our results demonstrate that subcellular compartmentalization of enzymes has an impact on both the target protein accumulation and its activity, especially in the case of proteins that undergo post-translational modifications, and should be taken into consideration when protein production strategies are designed. Using plants to produce heterologous enzymes for the degradation of a key component of the plant cell wall could reduce the cost of biomass pretreatment for the production of cellulosic biofuels.

Compound cis-regulatory elements with both boundary and enhancer sequences in the human genome.

MOTIVATION: It has been suggested that presumably distinct classes of genomic regulatory elements may actually share common sets of features and mechanisms. However, there has been no genome-wide assessment of the prevalence of this phenomenon. RESULTS: To evaluate this possibility, we performed a bioinformatic screen for the existence of compound regulatory elements in the human genome. We identified numerous such colocated boundary and enhancer elements from human CD4(+) T cells. We report evidence that such compound regulatory elements possess unique chromatin features and facilitate cell type-specific functions related to inflammation and immune response in CD4(+) T cells.

Epigenetic regulation of human cis-natural antisense transcripts.

Mammalian genomes encode numerous cis-natural antisense transcripts (cis-NATs). The extent to which these cis-NATs are actively regulated and ultimately functionally relevant, as opposed to transcriptional noise, remains a matter of debate. To address this issue, we analyzed the chromatin environment and RNA Pol II binding properties of human cis-NAT promoters genome-wide. Cap analysis of gene expression data were used to identify thousands of cis-NAT promoters, and profiles of nine histone modifications and RNA Pol II binding for these promoters in ENCODE cell types were analyzed using chromatin immunoprecipitation followed by sequencing (ChIP-seq) data. Active cis-NAT promoters are enriched with activating histone modifications and occupied by RNA Pol II, whereas weak cis-NAT promoters are depleted for both activating modifications and RNA Pol II. The enrichment levels of activating histone modifications and RNA Pol II binding show peaks centered around cis-NAT transcriptional start sites, and the levels of activating histone modifications at cis-NAT promoters are positively correlated with cis-NAT expression levels. Cis-NAT promoters also show highly tissue-specific patterns of expression. These results suggest that human cis-NATs are actively transcribed by the RNA Pol II and that their expression is epigenetically regulated, prerequisites for a functional potential for many of these non-coding RNAs.

Genetic basis of clarithromycin resistance in Bacillus anthracis.

The high-consequence pathogen Bacillus anthracis causes human anthrax and often results in lethal infections without the rapid administration of effective antimicrobial treatment. Antimicrobial resistance profiling is therefore critical to inform post-exposure prophylaxis and treatment decisions, especially during emergencies such as outbreaks or where intentional release is suspected. Whole-genome sequencing using a rapid long-read sequencer can uncover antimicrobial resistance patterns if genetic markers of resistance are known. To identify genomic markers associated with antimicrobial resistance, we isolated B. anthracis derived from the avirulent Sterne strain with elevated minimal inhibitory concentrations to clarithromycin. Mutants were characterized both phenotypically through broth microdilution susceptibility testing and observations during culturing, as well as genotypically with whole-genome sequencing. We identified two different in-frame insertions in the L22 ribosomal protein-encoding gene rplV, which were subsequently confirmed to be involved in clarithromycin resistance through the reversion of the mutant gene to the parent (drug-susceptible) sequence. Detection of the rplV insertions was possible with rapid long-read sequencing, with a time-to-answer within 3 h. The mutations associated with clarithromycin resistance described here will be used in conjunction with known genetic markers of resistance for other antimicrobials to strengthen the prediction of antimicrobial resistance in B. anthracis.IMPORTANCEThe disease anthrax, caused by the pathogen Bacillus anthracis, is extremely deadly if not treated quickly and appropriately. Clarithromycin is an antibiotic recommended for the treatment and post-exposure prophylaxis of anthrax by the Centers for Disease Control and Prevention; however, little is known about the ability of B. anthracis to develop resistance to clarithromycin or the mechanism of that resistance. The characterization of clarithromycin-resistant isolates presented here provides valuable information for researchers and clinicians in the event of a release of the resistant strain. Additionally, knowledge of the genetic basis of resistance provides a foundation for susceptibility prediction through rapid genome sequencing to inform timely treatment decisions.