Tiny Hidden Genes within Our Microbiome.

Exploration of tiny protein-coding sequences within the human microbiome reveals thousands of conserved gene families that have been overlooked by traditional analyses. These small proteins may play key roles in the crosstalk among bacteria within the microbiome and in interactions with their human hosts.

Chimeric CRISPR-CasX enzymes and guide RNAs for improved genome editing activity.

A compact protein with a size of <1,000 amino acids, the CRISPR-associated protein CasX is a fundamentally distinct RNA-guided nuclease when compared to Cas9 and Cas12a. Although it can induce RNA-guided genome editing in mammalian cells, the activity of CasX is less robust than that of the widely used S. pyogenes Cas9. Here, we show that structural features of two CasX homologs and their guide RNAs affect the R-loop complex assembly and DNA cleavage activity. Cryo-EM-based structural engineering of either the CasX protein or the guide RNA produced two new CasX genome editors (DpbCasX-R3-v2 and PlmCasX-R1-v2) with significantly improved DNA manipulation efficacy. These results advance both the mechanistic understanding of CasX and its application as a genome-editing tool.

A scoutRNA Is Required for Some Type V CRISPR-Cas Systems.

CRISPR-Cas12c/d proteins share limited homology with Cas12a and Cas9 bacterial CRISPR RNA (crRNA)-guided nucleases used widely for genome editing and DNA detection. However, Cas12c (C2c3)- and Cas12d (CasY)-catalyzed DNA cleavage and genome editing activities have not been directly observed. We show here that a short-complementarity untranslated RNA (scoutRNA), together with crRNA, is required for Cas12d-catalyzed DNA cutting. The scoutRNA differs in secondary structure from previously described tracrRNAs used by CRISPR-Cas9 and some Cas12 enzymes, and in Cas12d-containing systems, scoutRNA includes a conserved five-nucleotide sequence that is essential for activity. In addition to supporting crRNA-directed DNA recognition, biochemical and cell-based experiments establish scoutRNA as an essential cofactor for Cas12c-catalyzed pre-crRNA maturation. These results define scoutRNA as a third type of transcript encoded by a subset of CRISPR-Cas genomic loci and explain how Cas12c/d systems avoid requirements for host factors including ribonuclease III for bacterial RNA-mediated adaptive immunity.

A Functional Mini-Integrase in a Two-Protein-type V-C CRISPR System.

CRISPR-Cas immunity requires integration of short, foreign DNA fragments into the host genome at the CRISPR locus, a site consisting of alternating repeat sequences and foreign-derived spacers. In most CRISPR systems, the proteins Cas1 and Cas2 form the integration complex and are both essential for DNA acquisition. Most type V-C and V-D systems lack the cas2 gene and have unusually short CRISPR repeats and spacers. Here, we show that a mini-integrase comprising the type V-C Cas1 protein alone catalyzes DNA integration with a preference for short (17- to 19-base-pair) DNA fragments. The mini-integrase has weak specificity for the CRISPR array. We present evidence that the Cas1 proteins form a tetramer for integration. Our findings support a model of a minimal integrase with an internal ruler mechanism that favors shorter repeats and spacers. This minimal integrase may represent the function of the ancestral Cas1 prior to Cas2 adoption.

GeNLP: a web tool for NLP-based exploration and prediction of microbial gene function.

SUMMARY: GeNLP is a web application that enables exploring microbial gene "semantics" and predictions of uncharacterized gene families based on their genomic context. It utilizes a pre-trained language model to uncover gene relationships and allows users to access and utilize the data as well as make their own predictions through an interactive interface. AVAILABILITY AND IMPLEMENTATION: The web application is accessible from all browsers at: http://gnlp.bursteinlab.org/. All source codes are freely available from GitHub under the MIT license here: https://github.com/burstein-lab/genomic-nlp-server.

RNA Targeting by Functionally Orthogonal Type VI-A CRISPR-Cas Enzymes.

CRISPR adaptive immunity pathways protect prokaryotic cells against foreign nucleic acids using CRISPR RNA (crRNA)-guided nucleases. In type VI-A CRISPR-Cas systems, the signature protein Cas13a (formerly C2c2) contains two separate ribonuclease activities that catalyze crRNA maturation and ssRNA degradation. The Cas13a protein family occurs across different bacterial phyla and varies widely in both protein sequence and corresponding crRNA sequence conservation. Although grouped phylogenetically together, we show that the Cas13a enzyme family comprises two distinct functional groups that recognize orthogonal sets of crRNAs and possess different ssRNA cleavage specificities. These functional distinctions could not be bioinformatically predicted, suggesting more subtle co-evolution of Cas13a enzymes. Additionally, we find that Cas13a pre-crRNA processing is not essential for ssRNA cleavage, although it enhances ssRNA targeting for crRNAs encoded internally within the CRISPR array. We define two Cas13a protein subfamilies that can operate in parallel for RNA detection both in bacteria and for diagnostic applications.

Phenotypic and genotypic analysis of antimicrobial resistance in Nocardia species.

BACKGROUND: Antimicrobial resistance is common in Nocardia species but data regarding the molecular mechanisms beyond their resistance traits are limited. Our study aimed to determine the species distribution, the antimicrobial susceptibility profiles, and investigate the associations between the resistance traits and their genotypic determinants. METHODS: The study included 138 clinical strains of Nocardia from nine Israeli microbiology laboratories. MIC values of 12 antimicrobial agents were determined using broth microdilution. WGS was performed on 129 isolates of the eight predominant species. Bioinformatic analysis included phylogeny and determination of antimicrobial resistance genes and mutations. RESULTS: Among the isolates, Nocardia cyriacigeorgica was the most common species (36%), followed by Nocardia farcinica (16%), Nocardia wallacei (13%), Nocardia abscessus (9%) and Nocardia brasiliensis (8%). Linezolid was active against all isolates, followed by trimethoprim/sulfamethoxazole (93%) and amikacin (91%). Resistance to other antibiotics was species-specific, often associated with the presence of resistance genes or mutations: (1) aph(2″) in N. farcinica and N. wallacei (resistance to tobramycin); (ii) blaAST-1 in N. cyriacigeorgica and Nocardia neocaledoniensis (resistance to amoxicillin/clavulanate); (iii) blaFAR-1 in N. farcinica (resistance to ceftriaxone); (iv) Ser83Ala substitution in the gyrA gene in four species (resistance to ciprofloxacin); and (v) the 16S rRNA m1A1408 methyltransferase in N. wallacei isolates (correlating with amikacin resistance). CONCLUSIONS: Our study provides a comprehensive understanding of Nocardia species diversity, antibiotic resistance patterns, and the molecular basis of antimicrobial resistance. Resistance appears to follow species-related patterns, suggesting a lesser role for de novo evolution or transmission of antimicrobial resistance.

New CRISPR-Cas systems from uncultivated microbes.

CRISPR-Cas systems provide microbes with adaptive immunity by employing short DNA sequences, termed spacers, that guide Cas proteins to cleave foreign DNA. Class 2 CRISPR-Cas systems are streamlined versions, in which a single RNA-bound Cas protein recognizes and cleaves target sequences. The programmable nature of these minimal systems has enabled researchers to repurpose them into a versatile technology that is broadly revolutionizing biological and clinical research. However, current CRISPR-Cas technologies are based solely on systems from isolated bacteria, leaving the vast majority of enzymes from organisms that have not been cultured untapped. Metagenomics, the sequencing of DNA extracted directly from natural microbial communities, provides access to the genetic material of a huge array of uncultivated organisms. Here, using genome-resolved metagenomics, we identify a number of CRISPR-Cas systems, including the first reported Cas9 in the archaeal domain of life, to our knowledge. This divergent Cas9 protein was found in little-studied nanoarchaea as part of an active CRISPR-Cas system. In bacteria, we discovered two previously unknown systems, CRISPR-CasX and CRISPR-CasY, which are among the most compact systems yet discovered. Notably, all required functional components were identified by metagenomics, enabling validation of robust in vivo RNA-guided DNA interference activity in Escherichia coli. Interrogation of environmental microbial communities combined with in vivo experiments allows us to access an unprecedented diversity of genomes, the content of which will expand the repertoire of microbe-based biotechnologies.

Internal Medicine Residents' Experience Performing Routine Assessment of What Matters Most to Patients Upon Hospital Admission.

PROBLEM: Failure to elicit patients' values, goals, and priorities can result in missed opportunities to provide patient-centered care. Little is known about resident physicians' direct experience of eliciting patients' values, goals, and priorities and integrating them into routine hospital care. INTERVENTION: In 2017, we asked resident physicians on general internal medicine wards rotations to elicit and document a "Personal History" from patients upon hospital admission, in addition to a traditional social history. We defined a Personal History as documenting "what matters most to the patient and why." The purpose of the Personal History was to understand and consider patients' values, goals, and priorities. We then conducted qualitative interviews of the resident physicians to understand their experiences eliciting and integrating patients' values, goals, and priorities in routine hospital care. CONTEXT: We performed this exploratory intervention at a large high-volume urban hospital. Two teams from general medicine wards participated in the Personal History intervention. We conducted voluntary interviews of eligible residents (n = 14/15; 93%) about their experience after they completed their general wards rotations. Using the coproduction model, our aim was to explore how patients' self-expertise can be combined with physicians' medical expertise to achieve patient-centered care. IMPACT: Four major themes were identified: 1) Taking a Personal History had value, and eliciting patients' self-expertise had the potential to change medical decision making, 2) Situational and relational factors created barriers to obtaining a Personal History, 3) Variability in buy-in with the proposed intervention affected effort, and 4) Meaningful Personal History taking could be an adaptive and longitudinal process. Perceived benefits included improved rapport with patients, helpful for patients with complex medical history, and improved physician-patient communication. Barriers included patient distress, lack of rapport, and responses from patients which did not add new insights. Accountability from attending physicians affected resident effort. Suggested future applications were for patients with serious illness, integration into electronic health records, and skills taught in medical education. LESSONS LEARNED: Resident physicians had generally positive views of eliciting a Personal History from patients upon admission to the hospital. Overall, many residents conveyed the perceived ability to elicit and consider patient's values, goals, and priorities in certain situations (e.g., patient not in distress, adequate rapport, lack of competing priorities such as medical emergencies or overwhelming workloads). External factors, such as electronic health record design and accountability from attending physicians, may further promote residents' efforts to routinely incorporate patients' values, goals, and priorities in clinical care. Increasing familiarity among both resident physicians and patients in routinely discussing patients' values, goals, and priorities may facilitate patient-centered practice.

Association of Primary Care Physician Compensation Incentives and Quality of Care in the United States, 2012-2016.

BACKGROUND: Physician compensation incentives may have positive or negative effects on clinical quality. OBJECTIVE: To assess the association between various physician compensation incentives on technical indicators of primary care quality. DESIGN: Cross-sectional, nationally representative retrospective analysis. PARTICIPANTS: Visits by adults to primary care physicians in the National Ambulatory Medical Care Survey from 2012-2016. We analyzed 49,580 sampled visits, representing 1.45 billion primary care visits. MAIN MEASURES: We assessed the association between 5 compensation incentives - quality measure performance, patient experience scores, individual productivity, practice financial performance, or practice efficiency - and 10 high-value and 7 low-value care measures as well as high-value and low-value care composites. KEY RESULTS: Quality measure performance was an incentive in 22% of visits; patient experience scores, 17%; individual productivity, 57%; practice financial performance, 63%; and practice efficiency, 12%. In adjusted models, none of the compensation incentives were consistently associated with individual high- and low-value measures. None of the compensation incentives were associated with high- or low-value care composites. For example, quality measure performance compensation was not significantly associated with high-value care (visits with quality incentive, 47% of eligible measures met; without quality incentive, 43%; adjusted odds ratio [aOR], 1.02; 95% confidence interval [CI], 0.91 to 1.15) or low-value care (aOR, 0.99; 95% CI, 0.82-1.19). Physician compensation incentives that might be expected to increase low-value care did not: patient experience (aOR for low-value care composite, 0.83; 95% CI, 0.65-1.05), individual productivity (aOR, 1.03; 95% CI, 0.88-1.22), and practice financial performance (aOR, 1.05; 95% CI, 0.81-1.36). CONCLUSION: In this retrospective, cross-sectional, nationally representative analysis of care in the United States, physician compensation incentives were not generally associated with more or less high- or low-value care.

A Scoping Review on the Concept of Physician Caring.

BACKGROUND: Physicians' interest in the health and well-being of their patients is a tenet of medical practice. Physicians' ability to act upon this interest by caring for and about their patients is central to high-quality clinical medicine and may affect burnout. To date, a strong theoretical and empirical understanding of physician caring does not exist. To establish a practical, evidence-based approach to improve health care delivery and potentially address physician burnout, we sought to identify and synthesize existing conceptual models, frameworks, and definitions of physician caring. METHODS: We performed a scoping review on physician caring. In November 2019 and September 2020, we searched PubMed MEDLINE, Embase, PsycINFO, CINAHL, and CENTRAL Register of Controlled Trials to identify conceptual models, frameworks, and definitions of physician caring. Eligible articles involved discussion or study of care or caring among medical practitioners. We created a content summary and performed thematic analysis of extracted data. RESULTS: Of 11,776 articles, we reviewed the full text of 297 articles; 61 articles met inclusion criteria. Commonly identified concepts referenced Peabody's "secret of care" and the ethics of care. In bioethics, caring is described as a virtue. Contradictions exist among concepts of caring, such as whether caring is an attitude, emotion, or behavior, and the role of relationship development. Thematic analysis of all concepts and definitions identified six aspects of physician caring: (1) relational aspects, (2) technical aspects, (3) physician attitudes and characteristics, (4) agency, (5) reciprocity, and (6) physician self-care. DISCUSSION: Caring is instrumental to clinical medicine. However, scientific understanding of what constitutes caring from physicians is limited by contradictions across concepts. A unifying concept of physician caring does not yet exist. This review proposes six aspects of physician caring which can be used to develop evidence-based approaches to improve health care delivery and potentially mitigate physician burnout.

Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection.

Bacterial adaptive immune systems use CRISPRs (clustered regularly interspaced short palindromic repeats) and CRISPR-associated (Cas) proteins for RNA-guided nucleic acid cleavage. Although most prokaryotic adaptive immune systems generally target DNA substrates, type III and VI CRISPR systems direct interference complexes against single-stranded RNA substrates. In type VI systems, the single-subunit C2c2 protein functions as an RNA-guided RNA endonuclease (RNase). How this enzyme acquires mature CRISPR RNAs (crRNAs) that are essential for immune surveillance and how it carries out crRNA-mediated RNA cleavage remain unclear. Here we show that bacterial C2c2 possesses a unique RNase activity responsible for CRISPR RNA maturation that is distinct from its RNA-activated single-stranded RNA degradation activity. These dual RNase functions are chemically and mechanistically different from each other and from the crRNA-processing behaviour of the evolutionarily unrelated CRISPR enzyme Cpf1 (ref. 11). The two RNase activities of C2c2 enable multiplexed processing and loading of guide RNAs that in turn allow sensitive detection of cellular transcripts.

Identical bacterial populations colonize premature infant gut, skin, and oral microbiomes and exhibit different in situ growth rates.

The initial microbiome impacts the health and future development of premature infants. Methodological limitations have led to gaps in our understanding of the habitat range and subpopulation complexity of founding strains, as well as how different body sites support microbial growth. Here, we used metagenomics to reconstruct genomes of strains that colonized the skin, mouth, and gut of two hospitalized premature infants during the first month of life. Seven bacterial populations, considered to be identical given whole-genome average nucleotide identity of >99.9%, colonized multiple body sites, yet none were shared between infants. Gut-associated Citrobacter koseri genomes harbored 47 polymorphic sites that we used to define 10 subpopulations, one of which appeared in the gut after 1 wk but did not spread to other body sites. Differential genome coverage was used to measure bacterial population replication rates in situ. In all cases where the same bacterial population was detected in multiple body sites, replication rates were faster in mouth and skin compared to the gut. The ability of identical strains to colonize multiple body sites underscores the habit flexibility of initial colonists, whereas differences in microbial replication rates between body sites suggest differences in host control and/or resource availability. Population genomic analyses revealed microdiversity within bacterial populations, implying initial inoculation by multiple individual cells with distinct genotypes. Overall, however, the overlap of strains across body sites implies that the premature infant microbiome can exhibit very low microbial diversity.

Accurate, multi-kb reads resolve complex populations and detect rare microorganisms.

Accurate evaluation of microbial communities is essential for understanding global biogeochemical processes and can guide bioremediation and medical treatments. Metagenomics is most commonly used to analyze microbial diversity and metabolic potential, but assemblies of the short reads generated by current sequencing platforms may fail to recover heterogeneous strain populations and rare organisms. Here we used short (150-bp) and long (multi-kb) synthetic reads to evaluate strain heterogeneity and study microorganisms at low abundance in complex microbial communities from terrestrial sediments. The long-read data revealed multiple (probably dozens of) closely related species and strains from previously undescribed Deltaproteobacteria and Aminicenantes (candidate phylum OP8). Notably, these are the most abundant organisms in the communities, yet short-read assemblies achieved only partial genome coverage, mostly in the form of short scaffolds (N50 = ∼ 2200 bp). Genome architecture and metabolic potential for these lineages were reconstructed using a new synteny-based method. Analysis of long-read data also revealed thousands of species whose abundances were <0.1% in all samples. Most of the organisms in this "long tail" of rare organisms belong to phyla that are also represented by abundant organisms. Genes encoding glycosyl hydrolases are significantly more abundant than expected in rare genomes, suggesting that rare species may augment the capability for carbon turnover and confer resilience to changing environmental conditions. Overall, the study showed that a diversity of closely related strains and rare organisms account for a major portion of the communities. These are probably common features of many microbial communities and can be effectively studied using a combination of long and short reads.

Genomic resolution of a cold subsurface aquifer community provides metabolic insights for novel microbes adapted to high CO2 concentrations.

As in many deep underground environments, the microbial communities in subsurface high-CO2 ecosystems remain relatively unexplored. Recent investigations based on single-gene assays revealed a remarkable variety of organisms from little studied phyla in Crystal Geyser (Utah, USA), a site where deeply sourced CO2 -saturated fluids are erupted at the surface. To provide genomic resolution of the metabolisms of these organisms, we used a novel metagenomic approach to recover 227 high-quality genomes from 150 microbial species affiliated with 46 different phylum-level lineages. Bacteria from two novel phylum-level lineages have the capacity for CO2 fixation. Analyses of carbon fixation pathways in all studied organisms revealed that the Wood-Ljungdahl pathway and the Calvin-Benson-Bassham Cycle occurred with the highest frequency, whereas the reverse TCA cycle was little used. We infer that this, and selection for form II RuBisCOs, are adaptions to high CO2 -concentrations. However, many autotrophs can also grow mixotrophically, a strategy that confers metabolic versatility. The assignment of 156 hydrogenases to 90 different organisms suggests that H2 is an important inter-species energy currency even under gaseous CO2 -saturation. Overall, metabolic analyses at the organism level provided insight into the biochemical cycles that support subsurface life under the extreme condition of CO2 saturation.

ZBTB16: a novel sensitive and specific biomarker for yolk sac tumor.

Although the function of zinc finger and BTB domain containing 16 (ZBTB16) in spermatogenesis is well documented, expression of ZBTB16 in germ cell tumors has not yet been studied. The aim of this study was to investigate the immunohistochemical expression and diagnostic utility of ZBTB16 in germ cell tumors. A total of 67 adult germ cell tumors were studied (62 testicular germ cell tumors, 2 ovarian yolk sac tumors, 1 mediastinal yolk sac tumor, and 2 retroperitoneal metastatic yolk sac tumors). The 62 testicular primary germ cell tumors are as follows: 34 pure germ cell tumors (20 seminomas, 8 embryonal carcinomas, 2 teratomas, 1 choriocarcinoma, 1 carcinoid, and 2 spermatocytic tumors) and 28 mixed germ cell tumors (composed of 13 embryonal carcinomas, 15 yolk sac tumors, 15 teratomas, 7 seminomas, and 3 choriocarcinomas in various combinations). Thirty-five cases contained germ cell neoplasia in situ. Yolk sac tumor was consistently reactive for ZBTB16. Among the 15 testicular yolk sac tumors in mixed germ cell tumors, all displayed moderate to diffuse ZBTB16 staining. ZBTB16 reactivity was present regardless of the histologic patterns of yolk sac tumor and ZBTB16 was able to pick up small foci of yolk sac tumor intermixed/embedded in other germ cell tumor subtype elements. Diffuse ZBTB16 immunoreactivity was also observed in 2/2 metastatic yolk sac tumors, 1/1 mediastinal yolk sac tumor, 2/2 ovarian yolk sac tumors, 2/2 spermatocytic tumors, 1/1 carcinoid, and the spermatogonial cells. All the other non-yolk sac germ cell tumors were nonreactive, including seminoma (n=27), embryonal carcinoma (n=21), teratoma (n=17), choriocarcinoma (n=4), and germ cell neoplasia in situ (n=35). The sensitivity and specificity of ZBTB16 in detecting yolk sac tumor among the germ cell tumors was 100% (20/20) and 96% (66/69), respectively. In conclusion, ZBTB16 is a highly sensitive and specific marker for yolk sac tumor.

DNA motifs determining the efficiency of adaptation into the Escherichia coli CRISPR array.

Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated proteins constitute a recently identified prokaryotic defense system against invading nucleic acids. DNA segments, termed protospacers, are integrated into the CRISPR array in a process called adaptation. Here, we establish a PCR-based assay that enables evaluating the adaptation efficiency of specific spacers into the type I-E Escherichia coli CRISPR array. Using this assay, we provide direct evidence that the protospacer adjacent motif along with the first base of the protospacer (5'-AAG) partially affect the efficiency of spacer acquisition. Remarkably, we identified a unique dinucleotide, 5'-AA, positioned at the 3' end of the spacer, that enhances efficiency of the spacer's acquisition. Insertion of this dinucleotide increased acquisition efficiency of two different spacers. DNA sequencing of newly adapted CRISPR arrays revealed that the position of the newly identified motif with respect to the 5'-AAG is important for affecting acquisition efficiency. Analysis of approximately 1 million spacers showed that this motif is overrepresented in frequently acquired spacers compared with those acquired rarely. Our results represent an example of a short nonprotospacer adjacent motif sequence that affects acquisition efficiency and suggest that other as yet unknown motifs affect acquisition efficiency in other CRISPR systems as well.

Computational modeling and experimental validation of the Legionella and Coxiella virulence-related type-IVB secretion signal.

Legionella and Coxiella are intracellular pathogens that use the virulence-related Icm/Dot type-IVB secretion system to translocate effector proteins into host cells during infection. These effectors were previously shown to contain a C-terminal secretion signal required for their translocation. In this research, we implemented a hidden semi-Markov model to characterize the amino acid composition of the signal, thus providing a comprehensive computational model for the secretion signal. This model accounts for dependencies among sites and captures spatial variation in amino acid composition along the secretion signal. To validate our model, we predicted and synthetically constructed an optimal secretion signal whose sequence is different from that of any known effector. We show that this signal efficiently translocates into host cells in an Icm/Dot-dependent manner. Additionally, we predicted in silico and experimentally examined the effects of mutations in the secretion signal, which provided innovative insights into its characteristics. Some effectors were found to lack a strong secretion signal according to our model. We demonstrated that these effectors were highly dependent on the IcmS-IcmW chaperons for their translocation, unlike effectors that harbor a strong secretion signal. Furthermore, our model is innovative because it enables searching ORFs for secretion signals on a genomic scale, which led to the identification and experimental validation of 20 effectors from Legionella pneumophila, Legionella longbeachae, and Coxiella burnetii. Our combined computational and experimental methodology is general and can be applied to the identification of a wide spectrum of protein features that lack sequence conservation but have similar amino acid characteristics.

Changes in exon-intron structure during vertebrate evolution affect the splicing pattern of exons.

Exon-intron architecture is one of the major features directing the splicing machinery to the short exons that are located within long flanking introns. However, the evolutionary dynamics of exon-intron architecture and its impact on splicing is largely unknown. Using a comparative genomic approach, we analyzed 17 vertebrate genomes and reconstructed the ancestral motifs of both 3' and 5' splice sites, as also the ancestral length of exons and introns. Our analyses suggest that vertebrate introns increased in length from the shortest ancestral introns to the longest primate introns. An evolutionary analysis of splice sites revealed that weak splice sites act as a restrictive force keeping introns short. In contrast, strong splice sites allow recognition of exons flanked by long introns. Reconstruction of the ancestral state suggests these phenomena were not prevalent in the vertebrate ancestor, but appeared during vertebrate evolution. By calculating evolutionary rate shifts in exons, we identified cis-acting regulatory sequences that became fixed during the transition from early vertebrates to mammals. Experimental validations performed on a selection of these hexamers confirmed their regulatory function. We additionally revealed many features of exons that can discriminate alternative from constitutive exons. These features were integrated into a machine-learning approach to predict whether an exon is alternative. Our algorithm obtains very high predictive power (AUC of 0.91), and using these predictions we have identified and successfully validated novel alternatively spliced exons. Overall, we provide novel insights regarding the evolutionary constraints acting upon exons and their recognition by the splicing machinery.

CoPAP: Coevolution of presence-absence patterns.

Evolutionary analysis of phyletic patterns (phylogenetic profiles) is widely used in biology, representing presence or absence of characters such as genes, restriction sites, introns, indels and methylation sites. The phyletic pattern observed in extant genomes is the result of ancestral gain and loss events along the phylogenetic tree. Here we present CoPAP (coevolution of presence-absence patterns), a user-friendly web server, which performs accurate inference of coevolving characters as manifested by co-occurring gains and losses. CoPAP uses state-of-the-art probabilistic methodologies to infer coevolution and allows for advanced network analysis and visualization. We developed a platform for comparing different algorithms that detect coevolution, which includes simulated data with pairs of coevolving sites and independent sites. Using these simulated data we demonstrate that CoPAP performance is higher than alternative methods. We exemplify CoPAP utility by analyzing coevolution among thousands of bacterial genes across 681 genomes. Clusters of coevolving genes that were detected using our method largely coincide with known biosynthesis pathways and cellular modules, thus exhibiting the capability of CoPAP to infer biologically meaningful interactions. CoPAP is freely available for use at http://copap.tau.ac.il/.