Variability and bias in microbiome metagenomic sequencing: an interlaboratory study comparing experimental protocols.

Several studies have documented the significant impact of methodological choices in microbiome analyses. The myriad of methodological options available complicate the replication of results and generally limit the comparability of findings between independent studies that use differing techniques and measurement pipelines. Here we describe the Mosaic Standards Challenge (MSC), an international interlaboratory study designed to assess the impact of methodological variables on the results. The MSC did not prescribe methods but rather asked participating labs to analyze 7 shared reference samples (5 × human stool samples and 2 × mock communities) using their standard laboratory methods. To capture the array of methodological variables, each participating lab completed a metadata reporting sheet that included 100 different questions regarding the details of their protocol. The goal of this study was to survey the methodological landscape for microbiome metagenomic sequencing (MGS) analyses and the impact of methodological decisions on metagenomic sequencing results. A total of 44 labs participated in the MSC by submitting results (16S or WGS) along with accompanying metadata; thirty 16S rRNA gene amplicon datasets and 14 WGS datasets were collected. The inclusion of two types of reference materials (human stool and mock communities) enabled analysis of both MGS measurement variability between different protocols using the biologically-relevant stool samples, and MGS bias with respect to ground truth values using the DNA mixtures. Owing to the compositional nature of MGS measurements, analyses were conducted on the ratio of Firmicutes: Bacteroidetes allowing us to directly apply common statistical methods. The resulting analysis demonstrated that protocol choices have significant effects, including both bias of the MGS measurement associated with a particular methodological choices, as well as effects on measurement robustness as observed through the spread of results between labs making similar methodological choices. In the analysis of the DNA mock communities, MGS measurement bias was observed even when there was general consensus among the participating laboratories. This study was the result of a collaborative effort that included academic, commercial, and government labs. In addition to highlighting the impact of different methodological decisions on MGS result comparability, this work also provides insights for consideration in future microbiome measurement study design.

High-throughput genetic manipulation of multicellular organisms using a machine-vision guided embryonic microinjection robot.

Microinjection is a technique used for transgenesis, mutagenesis, cell labeling, cryopreservation, and in vitro fertilization in multiple single and multicellular organisms. Microinjection requires specialized skills and involves rate-limiting and labor-intensive preparatory steps. Here, we constructed a machine-vision guided generalized robot that fully automates the process of microinjection in fruit fly (Drosophila melanogaster) and zebrafish (Danio rerio) embryos. The robot uses machine learning models trained to detect embryos in images of agar plates and identify specific anatomical locations within each embryo in 3D space using dual view microscopes. The robot then serially performs a microinjection in each detected embryo. We constructed and used three such robots to automatically microinject tens of thousands of Drosophila and zebrafish embryos. We systematically optimized robotic microinjection for each species and performed routine transgenesis with proficiency comparable to highly skilled human practitioners while achieving up to 4× increases in microinjection throughput in Drosophila. The robot was utilized to microinject pools of over 20,000 uniquely barcoded plasmids into 1,713 embryos in 2 days to rapidly generate more than 400 unique transgenic Drosophila lines. This experiment enabled a novel measurement of the number of independent germline integration events per successfully injected embryo. Finally, we showed that robotic microinjection of cryoprotective agents in zebrafish embryos significantly improves vitrification rates and survival of cryopreserved embryos post-thaw as compared to manual microinjection. We anticipate that the robot can be used to carry out microinjection for genome-wide manipulation and cryopreservation at scale in a wide range of organisms.

Rational probe design for efficient rRNA depletion and improved metatranscriptomic analysis of human microbiomes.

The microbiota that colonize the human gut and other tissues are dynamic, varying both in composition and functional state between individuals and over time. Gene expression measurements can provide insights into microbiome composition and function. However, efficient and unbiased removal of microbial ribosomal RNA (rRNA) presents a barrier to acquiring metatranscriptomic data. Here we describe a probe set that achieves efficient enzymatic rRNA removal of complex human-associated microbial communities. We demonstrate that the custom probe set can be further refined through an iterative design process to efficiently deplete rRNA from a range of human microbiome samples. Using synthetic nucleic acid spike-ins, we show that the rRNA depletion process does not introduce substantial quantitative error in gene expression profiles. Successful rRNA depletion allows for efficient characterization of taxonomic and functional profiles, including during the development of the human gut microbiome. The pan-human microbiome enzymatic rRNA depletion probes described here provide a powerful tool for studying the transcriptional dynamics and function of the human microbiome.

Toward invasive mussel genetic biocontrol: Approaches, challenges, and perspectives.

Invasive freshwater mussels, such as the zebra (Dreissena polymorpha), quagga (Dreissena rostriformis bugensis), and golden (Limnoperna fortunei) mussel have spread outside their native ranges throughout many regions of the North American, South American, and European continents in recent decades, damaging infrastructure and the environment. This review describes ongoing efforts by multiple groups to develop genetic biocontrol methods for invasive mussels. First, we provide an overview of genetic biocontrol strategies that have been applied in other invasive or pest species. Next, we summarize physical and chemical methods that are currently in use for invasive mussel control. We then describe the multidisciplinary approaches our groups are employing to develop genetic biocontrol tools for invasive mussels. Finally, we discuss the challenges and limitations of applying genetic biocontrol tools to invasive mussels. Collectively, we aim to openly share information and combine expertise to develop practical tools to enable the management of invasive freshwater mussels.

Methods in Nutrition & Gut Microbiome Research: An American Society for Nutrition Satellite Session [13 October 2022].

The microbial cells colonizing the human body form an ecosystem that is integral to the regulation and maintenance of human health. Elucidation of specific associations between the human microbiome and health outcomes is facilitating the development of microbiome-targeted recommendations and treatments (e.g., fecal microbiota transplant; pre-, pro-, and post-biotics) to help prevent and treat disease. However, the potential of such recommendations and treatments to improve human health has yet to be fully realized. Technological advances have led to the development and proliferation of a wide range of tools and methods to collect, store, sequence, and analyze microbiome samples. However, differences in methodology at each step in these analytic processes can lead to variability in results due to the unique biases and limitations of each component. This technical variability hampers the detection and validation of associations with small to medium effect sizes. Therefore, the American Society for Nutrition (ASN) Nutritional Microbiology Group Engaging Members (GEM), sponsored by the Institute for the Advancement of Food and Nutrition Sciences (IAFNS), hosted a satellite session on methods in nutrition and gut microbiome research to review currently available methods for microbiome research, best practices, as well as tools and standards to aid in comparability of methods and results. This manuscript summarizes the topics and research discussed at the session. Consideration of the guidelines and principles reviewed in this session will increase the accuracy, precision, and comparability of microbiome research and ultimately the understanding of the associations between the human microbiome and health.

No apparent role for the Wari insulator in transcriptional regulation of the endogenous white gene of Drosophila melanogaster.

Chromatin insulators have been proposed to play an important role in chromosome organization and local regulatory interactions. In Drosophila , one of these insulators is known as Wari. It is located immediately downstream of the 3' end of the white transcription unit. Wari has been proposed to interact with the white promoter region, thereby facilitating recycling of the RNA polymerase machinery. We have tested this model by deleting the Wari insulator at the endogenous white locus and could not detect a significant effect on eye pigmentation.

The genome of the zebra mussel, Dreissena polymorpha: a resource for comparative genomics, invasion genetics, and biocontrol.

The zebra mussel, Dreissena polymorpha, continues to spread from its native range in Eurasia to Europe and North America, causing billions of dollars in damage and dramatically altering invaded aquatic ecosystems. Despite these impacts, there are few genomic resources for Dreissena or related bivalves. Although the D. polymorpha genome is highly repetitive, we have used a combination of long-read sequencing and Hi-C-based scaffolding to generate a high-quality chromosome-scale genome assembly. Through comparative analysis and transcriptomics experiments, we have gained insights into processes that likely control the invasive success of zebra mussels, including shell formation, synthesis of byssal threads, and thermal tolerance. We identified multiple intact steamer-like elements, a retrotransposon that has been linked to transmissible cancer in marine clams. We also found that D. polymorpha have an unusual 67 kb mitochondrial genome containing numerous tandem repeats, making it the largest observed in Eumetazoa. Together these findings create a rich resource for invasive species research and control efforts.

Multiscale, multi-perspective imaging assisted robotic microinjection of 3D biological structures.

Microinjection is a widely used technique employed by biologists with applications in transgenesis, cryopreservation, mutagenesis, labeling/dye injection and in-vitro fertilization. However, microinjection is an extremely laborious manual procedure, which makes it a critical bottleneck in the field and thus ripe for automation. Here, we present a computer-guided robot that automates the targeted microinjection of Drosophila melanogaster and zebrafish (Danio rerio) embryos, two important model organisms in biological research. The robot uses a series of cameras to image an agar plate containing embryos at multiple magnifications and perspectives. This imaging is combined with machine learning and computer vision algorithms to pinpoint a location on the embryo for targeted microinjection with microscale precision. We demonstrate the utility of this microinjection robot to successfully microinject Drosophila melanogaster and zebrafish embryos. Results obtained indicate that the robotic microinjection approach can significantly increase the throughput of microinjection as compared to manual microinjection while maintaining survival rates comparable to human operators. In the future, this robotic platform can be used to perform high throughput microinjection experiments and can be extended to automatically microinject a host of organisms such as roundworms (Caenorhabditis elegans), mosquito (Culicidae) embryos, sea urchins (Echinoidea) and frog (Xenopus) oocytes.

CLAMP regulates zygotic genome activation in Drosophila embryos.

Embryonic patterning is critically dependent on zygotic genome activation (ZGA). In Drosophila melanogaster embryos, the pioneer factor Zelda directs ZGA, possibly in conjunction with other factors. Here, we have explored the novel involvement of Chromatin-Linked Adapter for MSL Proteins (CLAMP) during ZGA. CLAMP binds thousands of sites genome-wide throughout early embryogenesis. Interestingly, CLAMP relocates to target promoter sequences across the genome when ZGA is initiated. Although there is a considerable overlap between CLAMP and Zelda binding sites, the proteins display distinct temporal dynamics. To assess whether CLAMP occupancy affects gene expression, we analyzed transcriptomes of embryos zygotically compromised for either clamp or zelda and found that transcript levels of many zygotically activated genes are similarly affected. Importantly, compromising either clamp or zelda disrupted the expression of critical segmentation and sex determination genes bound by CLAMP (and Zelda). Furthermore, clamp knockdown embryos recapitulate other phenotypes observed in Zelda-depleted embryos, including nuclear division defects, centrosome aberrations, and a disorganized actomyosin network. Based on these data, we propose that CLAMP acts in concert with Zelda to regulate early zygotic transcription.

Saliva Testing Is Accurate for Early-Stage and Presymptomatic COVID-19.

Although nasopharyngeal samples have been considered the gold standard for COVID-19 testing, variability in viral load across different anatomical sites could cause nasopharyngeal samples to be less sensitive than saliva or nasal samples in certain cases. Self-collected samples have logistical advantages over nasopharyngeal samples, making them amenable to population-scale screening. To evaluate sampling alternatives for population screening, we collected nasopharyngeal, saliva, and nasal samples from two cohorts with varied levels and types of symptoms. In a mixed cohort of 60 symptomatic and asymptomatic participants, we found that saliva had 88% concordance with nasopharyngeal samples when tested in the same testing lab (n = 41) and 68% concordance when tested in different testing labs (n = 19). In a second cohort of 20 participants hospitalized for COVID-19, saliva had 74% concordance with nasopharyngeal samples tested in the same testing lab but detected virus in two participants that tested negative with nasopharyngeal samples on the same day. Medical record review showed that the saliva-based testing sensitivity was related to the timing of symptom onset and disease stage. We find that no sample site will be perfectly sensitive for COVID-19 testing in all situations, and the significance of negative results will always need to be determined in the context of clinical signs and symptoms. Saliva retained high clinical sensitivity for early-stage and presymptomatic COVID-19 while allowing easier collection, minimizing the exposure of health care workers, and need for personal protective equipment and making it a viable option for population-scale testing. IMPORTANCE Methods for COVID-19 detection are necessary for public health efforts to monitor the spread of disease. Nasopharyngeal samples have been considered the best approach for COVID-19 testing. However, alternative samples like self-collected saliva offer advantages for population-scale screening. Meta-analyses of recent studies suggest that saliva is useful for detecting SARS-CoV-2; however, differences in disease prevalence, sample collection, and analysis methods still confound strong conclusions on the utility of saliva compared to nasopharyngeal samples. Here, we find that the sensitivity of saliva testing is related to both the timing of the sample collection relative to symptom onset and the disease stage. Importantly, several clinical vignettes in our cohorts highlight the challenges of medical decision making with limited knowledge of the associations between laboratory test data and the natural biology of infection.

Dissecting and tuning primer editing by proofreading polymerases.

Proofreading polymerases have 3' to 5' exonuclease activity that allows the excision and correction of mis-incorporated bases during DNA replication. In a previous study, we demonstrated that in addition to correcting substitution errors and lowering the error rate of DNA amplification, proofreading polymerases can also edit PCR primers to match template sequences. Primer editing is a feature that can be advantageous in certain experimental contexts, such as amplicon-based microbiome profiling. Here we develop a set of synthetic DNA standards to report on primer editing activity and use these standards to dissect this phenomenon. The primer editing standards allow next-generation sequencing-based enzymological measurements, reveal the extent of editing, and allow the comparison of different polymerases and cycling conditions. We demonstrate that proofreading polymerases edit PCR primers in a concentration-dependent manner, and we examine whether primer editing exhibits any sequence specificity. In addition, we use these standards to show that primer editing is tunable through the incorporation of phosphorothioate linkages. Finally, we demonstrate the ability of primer editing to robustly rescue the drop-out of taxa with 16S rRNA gene-targeting primer mismatches using mock communities and human skin microbiome samples.

Comprehensive Genomic Discovery of Non-Coding Transcriptional Enhancers in the African Malaria Vector Anopheles coluzzii.

Almost all regulation of gene expression in eukaryotic genomes is mediated by the action of distant non-coding transcriptional enhancers upon proximal gene promoters. Enhancer locations cannot be accurately predicted bioinformatically because of the absence of a defined sequence code, and thus functional assays are required for their direct detection. Here we used a massively parallel reporter assay, Self-Transcribing Active Regulatory Region sequencing (STARR-seq), to generate the first comprehensive genome-wide map of enhancers in Anopheles coluzzii, a major African malaria vector in the Gambiae species complex. The screen was carried out by transfecting reporter libraries created from the genomic DNA of 60 wild A. coluzzii from Burkina Faso into A. coluzzii 4a3A cells, in order to functionally query enhancer activity of the natural population within the homologous cellular context. We report a catalog of 3,288 active genomic enhancers that were significant across three biological replicates, 74% of them located in intergenic and intronic regions. The STARR-seq enhancer screen is chromatin-free and thus detects inherent activity of a comprehensive catalog of enhancers that may be restricted in vivo to specific cell types or developmental stages. Testing of a validation panel of enhancer candidates using manual luciferase assays confirmed enhancer function in 26 of 28 (93%) of the candidates over a wide dynamic range of activity from two to at least 16-fold activity above baseline. The enhancers occupy only 0.7% of the genome, and display distinct composition features. The enhancer compartment is significantly enriched for 15 transcription factor binding site signatures, and displays divergence for specific dinucleotide repeats, as compared to matched non-enhancer genomic controls. The genome-wide catalog of A. coluzzii enhancers is publicly available in a simple searchable graphic format. This enhancer catalogue will be valuable in linking genetic and phenotypic variation, in identifying regulatory elements that could be employed in vector manipulation, and in better targeting of chromosome editing to minimize extraneous regulation influences on the introduced sequences. Importance: Understanding the role of the non-coding regulatory genome in complex disease phenotypes is essential, but even in well-characterized model organisms, identification of regulatory regions within the vast non-coding genome remains a challenge. We used a large-scale assay to generate a genome wide map of transcriptional enhancers. Such a catalogue for the important malaria vector, Anopheles coluzzii, will be an important research tool as the role of non-coding regulatory variation in differential susceptibility to malaria infection is explored and as a public resource for research on this important insect vector of disease.

A rapid, cost-effective tailed amplicon method for sequencing SARS-CoV-2.

BACKGROUND: The global COVID-19 pandemic has led to an urgent need for scalable methods for clinical diagnostics and viral tracking. Next generation sequencing technologies have enabled large-scale genomic surveillance of SARS-CoV-2 as thousands of isolates are being sequenced around the world and deposited in public data repositories. A number of methods using both short- and long-read technologies are currently being applied for SARS-CoV-2 sequencing, including amplicon approaches, metagenomic methods, and sequence capture or enrichment methods. Given the small genome size, the ability to sequence SARS-CoV-2 at scale is limited by the cost and labor associated with making sequencing libraries. RESULTS: Here we describe a low-cost, streamlined, all amplicon-based method for sequencing SARS-CoV-2, which bypasses costly and time-consuming library preparation steps. We benchmark this tailed amplicon method against both the ARTIC amplicon protocol and sequence capture approaches and show that an optimized tailed amplicon approach achieves comparable amplicon balance, coverage metrics, and variant calls to the ARTIC v3 approach. CONCLUSIONS: The tailed amplicon method we describe represents a cost-effective and highly scalable method for SARS-CoV-2 sequencing.

Influence of genetic polymorphism on transcriptional enhancer activity in the malaria vector Anopheles coluzzii.

Enhancers are cis-regulatory elements that control most of the developmental and spatial gene expression in eukaryotes. Genetic variation of enhancer sequences is known to influence phenotypes, but the effect of enhancer variation upon enhancer functional activity and downstream phenotypes has barely been examined in any species. In the African malaria vector, Anopheles coluzzii, we identified candidate enhancers in the proximity of genes relevant for immunity, insecticide resistance, and development. The candidate enhancers were functionally validated using luciferase reporter assays, and their activity was found to be essentially independent of their physical orientation, a typical property of enhancers. All of the enhancers segregated genetically polymorphic alleles, which displayed significantly different levels of functional activity. Deletion mutagenesis and functional testing revealed a fine structure of positive and negative regulatory elements that modulate activity of the enhancer core. Enhancer polymorphisms segregate in wild A. coluzzii populations in West Africa. Thus, enhancer variants that modify target gene expression leading to likely phenotypic consequences are frequent in nature. These results demonstrate the existence of naturally polymorphic A. coluzzii enhancers, which may help explain important differences between individuals or populations for malaria transmission efficiency and vector adaptation to the environment.

Genomewide Assessment of Mycobacterium tuberculosis Conditionally Essential Metabolic Pathways.

A better understanding of essential cellular functions in pathogenic bacteria is important for the development of more effective antimicrobial agents. We performed a comprehensive identification of essential genes in Mycobacterium tuberculosis, the major causative agent of tuberculosis, using a combination of transposon insertion sequencing (Tn-seq) and comparative genomic analysis. To identify conditionally essential genes by Tn-seq, we used media with different nutrient compositions. Although many conditional gene essentialities were affected by the presence of relevant nutrient sources, we also found that the essentiality of genes in a subset of metabolic pathways was unaffected by metabolite availability. Comparative genomic analysis revealed that not all essential genes identified by Tn-seq were fully conserved within the M. tuberculosis complex, including some existing antitubercular drug target genes. In addition, we utilized an available M. tuberculosis genome-scale metabolic model, iSM810, to predict M. tuberculosis gene essentiality in silico Comparing the sets of essential genes experimentally identified by Tn-seq to those predicted in silico reveals the capabilities and limitations of gene essentiality predictions, highlighting the complexity of M. tuberculosis essential metabolic functions. This study provides a promising platform to study essential cellular functions in M. tuberculosis IMPORTANCE Mycobacterium tuberculosis causes 10 million cases of tuberculosis (TB), resulting in over 1 million deaths each year. TB therapy is challenging because it requires a minimum of 6 months of treatment with multiple drugs. Protracted treatment times and the emergent spread of drug-resistant M. tuberculosis necessitate the identification of novel targets for drug discovery to curb this global health threat. Essential functions, defined as those indispensable for growth and/or survival, are potential targets for new antimicrobial drugs. In this study, we aimed to define gene essentialities of M. tuberculosis on a genomewide scale to comprehensively identify potential targets for drug discovery. We utilized a combination of experimental (functional genomics) and in silico approaches (comparative genomics and flux balance analysis). Our functional genomics approach identified sets of genes whose essentiality was affected by nutrient availability. Comparative genomics revealed that not all essential genes were fully conserved within the M. tuberculosis complex. Comparing sets of essential genes identified by functional genomics to those predicted by flux balance analysis highlighted gaps in current knowledge regarding M. tuberculosis metabolic capabilities. Thus, our study identifies numerous potential antitubercular drug targets and provides a comprehensive picture of the complexity of M. tuberculosis essential cellular functions.

Measuring sequencer size bias using REcount: a novel method for highly accurate Illumina sequencing-based quantification.

Quantification of DNA sequence tags from engineered constructs such as plasmids, transposons, or other transgenes underlies many functional genomics measurements. Typically, such measurements rely on PCR followed by next-generation sequencing. However, PCR amplification can introduce significant quantitative error. We describe REcount, a novel PCR-free direct counting method. Comparing measurements of defined plasmid pools to droplet digital PCR data demonstrates that REcount is highly accurate and reproducible. We use REcount to provide new insights into clustering biases due to molecule length across different Illumina sequencers and illustrate the impacts on interpretation of next-generation sequencing data and the economics of data generation.

Publisher Correction: Diverse populations of local interneurons integrate into the Drosophila adult olfactory circuit.

The original version of this Article contained errors in Figs. 4 and 6. In Fig. 4, panel a, text labels UAS-FLP and LexAop2>stop>myr::smGdP-HA were shifted upwards during typesetting of the figure, and in Fig. 6, panel h, the number 15 was incorrectly placed on the heat map scale. These have now been corrected in both the PDF and HTML versions of the Article.

Evaluating the Information Content of Shallow Shotgun Metagenomics.

Although microbial communities are associated with human, environmental, plant, and animal health, there exists no cost-effective method for precisely characterizing species and genes in such communities. While deep whole-metagenome shotgun (WMS) sequencing provides high taxonomic and functional resolution, it is often prohibitively expensive for large-scale studies. The prevailing alternative, 16S rRNA gene amplicon (16S) sequencing, often does not resolve taxonomy past the genus level and provides only moderately accurate predictions of the functional profile; thus, there is currently no widely accepted approach to affordable, high-resolution, taxonomic, and functional microbiome analysis. To address this technology gap, we evaluated the information content of shallow shotgun sequencing with as low as 0.5 million sequences per sample as an alternative to 16S sequencing for large human microbiome studies. We describe a library preparation protocol enabling shallow shotgun sequencing at approximately the same per-sample cost as 16S sequencing. We analyzed multiple real and simulated biological data sets, including two novel human stool samples with ultradeep sequencing of 2.5 billion sequences per sample, and found that shallow shotgun sequencing recovers more-accurate species-level taxonomic and functional profiles of the human microbiome than 16S sequencing. We discuss the inherent limitations of shallow shotgun sequencing and note that 16S sequencing remains a valuable and important method for taxonomic profiling of novel environments. Although deep WMS sequencing remains the gold standard for high-resolution microbiome analysis, we recommend that researchers consider shallow shotgun sequencing as a useful alternative to 16S sequencing for large-scale human microbiome research studies where WMS sequencing may be cost-prohibitive. IMPORTANCE A common refrain in recent microbiome-related academic meetings is that the field needs to move away from broad taxonomic surveys using 16S sequencing and toward more powerful longitudinal studies using shotgun sequencing. However, performing deep shotgun sequencing in large longitudinal studies remains prohibitively expensive for all but the most well-funded research labs and consortia, which leads many researchers to choose 16S sequencing for large studies, followed by deep shotgun sequencing on a subset of targeted samples. Here, we show that shallow- or moderate-depth shotgun sequencing may be used by researchers to obtain species-level taxonomic and functional data at approximately the same cost as amplicon sequencing. While shallow shotgun sequencing is not intended to replace deep shotgun sequencing for strain-level characterization, we recommend that microbiome scientists consider using shallow shotgun sequencing instead of 16S sequencing for large-scale human microbiome studies.

Comprehensive Functional Analysis of the Enterococcus faecalis Core Genome Using an Ordered, Sequence-Defined Collection of Insertional Mutations in Strain OG1RF.

Enterococcus faecalis is a common commensal bacterium in animal gastrointestinal (GI) tracts and a leading cause of opportunistic infections of humans in the modern health care setting. E. faecalis OG1RF is a plasmid-free strain that contains few mobile elements yet retains the robust survival characteristics, intrinsic antibiotic resistance, and virulence traits characteristic of most E. faecalis genotypes. To facilitate interrogation of the core enterococcal genetic determinants for competitive fitness in the GI tract, biofilm formation, intrinsic antimicrobial resistance, and survival in the environment, we generated an arrayed, sequence-defined set of chromosomal transposon insertions in OG1RF. We used an orthogonal pooling strategy in conjunction with Illumina sequencing to identify a set of mutants with unique, single Himar-based transposon insertions. The mutants contained insertions in 1,926 of 2,651 (72.6%) annotated open reading frames and in the majority of hypothetical protein-encoding genes and intergenic regions greater than 100 bp in length, which could encode small RNAs. As proof of principle of the usefulness of this arrayed transposon library, we created a minimal input pool containing 6,829 mutants chosen for maximal genomic coverage and used an approach that we term SMarT (sequence-defined mariner technology) transposon sequencing (TnSeq) to identify numerous genetic determinants of bile resistance in E. faecalis OG1RF. These included several genes previously associated with bile acid resistance as well as new loci. Our arrayed library allows functional screening of a large percentage of the genome with a relatively small number of mutants, reducing potential effects of bottlenecking, and enables immediate recovery of mutants following competitions. IMPORTANCE The robust ability of Enterococcus faecalis to survive outside the host and to spread via oral-fecal transmission and its high degree of intrinsic and acquired antimicrobial resistance all complicate the treatment of hospital-acquired enterococcal infections. The conserved E. faecalis core genome serves as an important genetic scaffold for evolution of this bacterium in the modern health care setting and also provides interesting vaccine and drug targets. We used an innovative pooling/sequencing strategy to map a large collection of arrayed transposon insertions in E. faecalis OG1RF and generated an arrayed library of defined mutants covering approximately 70% of the OG1RF genome. Then, we performed high-throughput transposon sequencing experiments using this library to determine core genomic determinants of bile resistance in OG1RF. This collection is a valuable resource for comprehensive, functional enterococcal genomics using both traditional and high-throughput approaches and enables immediate recovery of mutants of interest.

Diverse populations of local interneurons integrate into the Drosophila adult olfactory circuit.

Drosophila olfactory local interneurons (LNs) in the antennal lobe are highly diverse and variable. How and when distinct types of LNs emerge, differentiate, and integrate into the olfactory circuit is unknown. Through systematic developmental analyses, we found that LNs are recruited to the adult olfactory circuit in three groups. Group 1 LNs are residual larval LNs. Group 2 are adult-specific LNs that emerge before cognate sensory and projection neurons establish synaptic specificity, and Group 3 LNs emerge after synaptic specificity is established. Group 1 larval LNs are selectively reintegrated into the adult circuit through pruning and re-extension of processes to distinct regions of the antennal lobe, while others die during metamorphosis. Precise temporal control of this pruning and cell death shapes the global organization of the adult antennal lobe. Our findings provide a road map to understand how LNs develop and contribute to constructing the olfactory circuit.