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Streptococcus suis is an emerging zoonotic agent responsible for high-mortality outbreaks among the human population in China. In this species, the ABC transporter SatAB mediates fluoroquinolone resistance when overexpressed. Here, we describe and characterize satR, an open reading frame (ORF) encoding a MarR superfamily regulator that acts as a repressor of satAB. satR is cotranscribed with satAB, and its interruption entails the overexpression of the pump, leading to a clinically relevant in- crease in resistance to fluoroquinolones., Universidad Complutense de Madrid, Ministerio de Ciencia e Innovación, Comunidad de Madrid, Depto. de Sanidad Animal, Fac. de Veterinaria, TRUE, pub

Improved sequencing technologies have profoundly altered global views of fungal diversity and evolution. High-throughput sequencing methods are critical for studying fungi due to the cryptic, symbiotic nature of many species, particularly those that are difficult to culture. However, the low coverage genome sequencing (LCGS) approach to phylogenomic inference has not been widely applied to fungi. Here we analyzed 171 Kickxellomycotina fungi using LCGS methods to obtain hundreds of marker genes for robust phylogenomic reconstruction. Additionally, we mined our LCGS data for a set of nine rDNA and protein coding genes to enable analyses across species for which no LCGS data were obtained. The main goals of this study were to: 1) evaluate the quality and utility of LCGS data for both phylogenetic reconstruction and functional annotation, 2) test relationships among clades of Kickxellomycotina, and 3) perform comparative functional analyses between clades to gain insight into putative trophic modes. In opposition to previous studies, our nine-gene analyses support two clades of arthropod gut dwelling species and suggest a possible single evolutionary event leading to this symbiotic lifestyle. Furthermore, we resolve the mycoparasitic Dimargaritales as the earliest diverging clade in the subphylum and find four major clades of Coemansia species. Finally, functional analyses illustrate clear variation in predicted carbohydrate active enzymes and secondary metabolites (SM) based on ecology, that is biotroph versus saprotroph. Saprotrophic Kickxellales broadly lack many known pectinase families compared with saprotrophic Mucoromycota and are depauperate for SM but have similar numbers of predicted chitinases as mycoparasitic.

Violacein is a water-insoluble violet pigment produced by various Gram-negative bacteria. The compound and the bacteria that produce it have been gaining attention due to the antimicrobial and proposed antitumour properties of violacein and the possibility that strains producing it may have broad industrial uses. Bacteria that produce violacein have been isolated from diverse environments including fresh and ocean waters, glaciers, tropical soils, trees, fish and the skin of amphibians. We report here the isolation and characterization of six violacein-producing bacterial strains and three non-violacein-producing close relatives, each isolated from either an aquatic environment or moist food materials in northern California, USA. For each isolate, we characterized traditional phenotypes, generated and analysed draft genome sequences, and carried out multiple types of taxonomic, phylogenetic and phylogenomic analyses. Based on these analyses we assign putative identifications to the nine isolates, which include representatives of the genera Chromobacterium, Aquitalea, Iodobacter, Duganella, Massilia and Janthinobacterium. In addition, we discuss the utility of various metrics for taxonomic assignment in these groups including average nucleotide identity, whole genome phylogenetic analysis and extent of recent homologous recombination using the software program PopCOGenT.

Different plant species within the grasses were parallel targets of domestication, giving rise to crops with distinct evolutionary histories and traits1. Key traits that distinguish these species are mediated by specialized cell types2. Here we compare the transcriptomes of root cells in three grass species-Zea mays, Sorghum bicolor and Setaria viridis. We show that single-cell and single-nucleus RNA sequencing provide complementary readouts of cell identity in dicots and monocots, warranting a combined analysis. Cell types were mapped across species to identify robust, orthologous marker genes. The comparative cellular analysis shows that the transcriptomes of some cell types diverged more rapidly than those of others-driven, in part, by recruitment of gene modules from other cell types. The data also show that a recent whole-genome duplication provides a rich source of new, highly localized gene expression domains that favour fast-evolving cell types. Together, the cell-by-cell comparative analysis shows how fine-scale cellular profiling can extract conserved modules from a pan transcriptome and provide insight on the evolution of cells that mediate key functions in crops.

Poa pratensis, commonly known as Kentucky bluegrass, is a popular cool-season grass species used as turf in lawns and recreation areas globally. Despite its substantial economic value, a reference genome had not previously been assembled due to the genome's relatively large size and biological complexity that includes apomixis, polyploidy, and interspecific hybridization. We report here a fortuitous de novo assembly and annotation of a P. pratensis genome. Instead of sequencing the genome of a C4 grass, we accidentally sampled and sequenced tissue from a weedy P. pratensis whose stolon was intertwined with that of the C4 grass. The draft assembly consists of 6.09 Gbp with an N50 scaffold length of 65.1 Mbp, and a total of 118 scaffolds, generated using PacBio long reads and Bionano optical map technology. We annotated 256K gene models and found 58% of the genome to be composed of transposable elements. To demonstrate the applicability of the reference genome, we evaluated population structure and estimated genetic diversity in P. pratensis collected from three North American prairies, two in Manitoba, Canada and one in Colorado, USA. Our results support previous studies that found high genetic diversity and population structure within the species. The reference genome and annotation will be an important resource for turfgrass breeding and study of bluegrasses.

The Stellers jay is a familiar bird of western forests from Alaska south to Nicaragua. Here, we report a draft reference assembly for the species generated from PacBio HiFi long-read and Omni-C chromatin-proximity sequencing data as part of the California Conservation Genomics Project (CCGP). Sequenced reads were assembled into 352 scaffolds totaling 1.16 Gb in length. Assembly metrics indicate a highly contiguous and complete assembly with a contig N50 of 7.8 Mb, scaffold N50 of 25.8 Mb, and BUSCO completeness score of 97.2%. Repetitive elements span 16.6% of the genome including nearly 90% of the W chromosome. Compared with high-quality assemblies from other members of the family Corvidae, the Stellers jay genome contains a larger proportion of repetitive elements than 4 crow species (Corvus), but a lower proportion of repetitive elements than the California scrub-jay (Aphelocoma californica). This reference genome will serve as an essential resource for future studies on speciation, local adaptation, phylogeography, and conservation genetics in this species of significant biological interest.

Spray-induced gene silencing (SIGS) is an emerging tool for crop pest protection. It utilizes exogenously applied double-stranded RNA to specifically reduce pest target gene expression using endogenous RNA interference machinery. In this study, SIGS methods were developed and optimized for powdery mildew fungi, which are widespread obligate biotrophic fungi that infect agricultural crops, using the known azole-fungicide target cytochrome P450 51 (CYP51) in the Golovinomyces orontii-Arabidopsis thaliana pathosystem. Additional screening resulted in the identification of conserved gene targets and processes important to powdery mildew proliferation: apoptosis-antagonizing transcription factor in essential cellular metabolism and stress response; lipid catabolism genes lipase a, lipase 1, and acetyl-CoA oxidase in energy production; and genes involved in manipulation of the plant host via abscisic acid metabolism (9-cis-epoxycarotenoid dioxygenase, xanthoxin dehydrogenase, and a putative abscisic acid G-protein coupled receptor) and secretion of the effector protein, effector candidate 2. Powdery mildew is the dominant disease impacting grapes and extensive powdery mildew resistance to applied fungicides has been reported. We therefore developed SIGS for the Erysiphe necator-Vitis vinifera system and tested six successful targets identified using the G. orontii-A. thaliana system. For all targets tested, a similar reduction in powdery mildew disease was observed between systems. This indicates screening of broadly conserved targets in the G. orontii-A. thaliana pathosystem identifies targets and processes for the successful control of other powdery mildew fungi. The efficacy of SIGS on powdery mildew fungi makes SIGS an exciting prospect for commercial powdery mildew control.

Wine cultivars are available to growers in multiple clonal selections with agronomic and enological differences. Phenotypic differences between clones originated from somatic mutations that accrued over thousands of asexual propagation cycles. Genetic diversity between grape cultivars remains unexplored, and tools to discriminate unequivocally clones have been lacking. This study aimed to uncover genetic variations among a group of clonal selections of 4 important Vitis vinifera cultivars: Cabernet sauvignon, Sauvignon blanc, Chardonnay, and Merlot, and use this information to develop genetic markers to discriminate the clones of these cultivars. We sequenced with short-read sequencing technology the genomes of 18 clones, including biological replicates for a total of 46 genomes. Sequences were aligned to their respective cultivars reference genome for variant calling. We used reference genomes of Cabernet sauvignon, Chardonnay, and Merlot and developed a de novo genome assembly of Sauvignon blanc using long-read sequencing. On average, 4 million variants were detected for each clone, with 74.2% being single nucleotide variants and 25.8% being small insertions or deletions (InDel). The frequency of these variants was consistent across all clones. From these variants, we validated 46 clonal markers using high-throughput amplicon sequencing for 77.7% of the evaluated clones, most of them small InDel. These results represent an advance in grapevine genotyping strategies and will benefit the viticulture industry for the characterization and identification of the plant material.

MOTIVATION: Double-stranded RNAs (dsRNAs) are potent triggers of innate immune responses upon recognition by cytosolic dsRNA sensor proteins. Identification of endogenous dsRNAs helps to better understand the dsRNAome and its relevance to innate immunity related to human diseases. RESULTS: Here, we report dsRID (double-stranded RNA identifier), a machine-learning-based method to predict dsRNA regions in silico, leveraging the power of long-read RNA-sequencing (RNA-seq) and molecular traits of dsRNAs. Using models trained with PacBio long-read RNA-seq data derived from Alzheimers disease (AD) brain, we show that our approach is highly accurate in predicting dsRNA regions in multiple datasets. Applied to an AD cohort sequenced by the ENCODE consortium, we characterize the global dsRNA profile with potentially distinct expression patterns between AD and controls. Together, we show that dsRID provides an effective approach to capture global dsRNA profiles using long-read RNA-seq data. AVAILABILITY AND IMPLEMENTATION: Software implementation of dsRID, and genomic coordinates of regions predicted by dsRID in all samples are available at the GitHub repository: https://github.com/gxiaolab/dsRID.

The impact of genetic regulatory variation active in early pancreatic development on adult pancreatic disease and traits is not well understood. Here, we generate a panel of 107 fetal-like iPSC-derived pancreatic progenitor cells (iPSC-PPCs) from whole genome-sequenced individuals and identify 4065 genes and 4016 isoforms whose expression and/or alternative splicing are affected by regulatory variation. We integrate eQTLs identified in adult islets and whole pancreas samples, which reveal 1805 eQTL associations that are unique to the fetal-like iPSC-PPCs and 1043 eQTLs that exhibit regulatory plasticity across the fetal-like and adult pancreas tissues. Colocalization with GWAS risk loci for pancreatic diseases and traits show that some putative causal regulatory variants are active only in the fetal-like iPSC-PPCs and likely influence disease by modulating expression of disease-associated genes in early development, while others with regulatory plasticity likely exert their effects in both the fetal and adult pancreas by modulating expression of different disease genes in the two developmental stages.

Coccidioidomycosis is a common fungal disease that is endemic to arid and semi-arid regions of both American continents. Coccidioides immitis and Coccidioides posadasii are the etiological agents of the disease, also known as Valley Fever. For several decades, the C. posadasii strain Silveira has been used widely in vaccine studies, is the source strain for production of diagnostic antigens, and is a widely used experimental strain for functional studies. In 2009, the genome was sequenced using Sanger sequencing technology, and a draft assembly and annotation were made available. In this study, the genome of the Silveira strain was sequenced using single molecule real-time sequencing PacBio technology, assembled into chromosomal-level contigs, genotyped, and the genome was reannotated using sophisticated and curated in silico tools. This high-quality genome sequencing effort has improved our understanding of chromosomal structure, gene set annotation, and lays the groundwork for identification of structural variants (e.g. transversions, translocations, and copy number variants), assessment of gene gain and loss, and comparison of transposable elements in future phylogenetic and population genomics studies.

Structural variants (SVs) are implicated in the etiology of Mendelian diseases but have been systematically underascertained owing to sequencing technology limitations. Long-read sequencing enables comprehensive detection of SVs, but approaches for prioritization of candidate SVs are needed. Structural variant Annotation and analysis (SvAnna) assesses all classes of SVs and their intersection with transcripts and regulatory sequences, relating predicted effects on gene function with clinical phenotype data. SvAnna places 87% of deleterious SVs in the top ten ranks. The interpretable prioritizations offered by SvAnna will facilitate the widespread adoption of long-read sequencing in diagnostic genomics. SvAnna is available at https://github.com/TheJacksonLaboratory/SvAnn a .

Regulation of bacteriophage gene expression involves repressor proteins that bind and downregulate early lytic promoters. A large group of mycobacteriophages code for repressors that are unusual in also terminating transcription elongation at numerous binding sites (stoperators) distributed across the phage genome. Here we provide the X-ray crystal structure of a mycobacteriophage immunity repressor bound to DNA, which reveals the binding of a monomer to an asymmetric DNA sequence using two independent DNA binding domains. The structure is supported by small-angle X-ray scattering, DNA binding, molecular dynamics, and in vivo immunity assays. We propose a model for how dual DNA binding domains facilitate regulation of both transcription initiation and elongation, while enabling evolution of other superinfection immune specificities.

Eight new species of Isodiametridae (Acoela) are presented and phylogenetic relationships of the family using 18S and COI sequence data reconstructed. Four species of Baltalimania, two species of Praeaphanostoma and two species of Pseudoposthia are described that all can be distinguished both morphologically and through differences in the DNA sequences as supported by mPTP analysis. The addition of eight new species increases the number of nominal species of Isodiametridae worldwide by 10% and within Sweden by 31%, but also highlights several taxonomic problems. Several species are now confirmed as isodiametrids that are otherwise incongruent with the family's diagnostic characters. Generic keys are presented for Baltalimania and Praeaphanostoma.

Structural variants (SVs) are implicated in the etiology of Mendelian diseases but have been systematically underascertained owing to sequencing technology limitations. Long-read sequencing enables comprehensive detection of SVs, but approaches for prioritization of candidate SVs are needed. Structural variant Annotation and analysis (SvAnna) assesses all classes of SVs and their intersection with transcripts and regulatory sequences, relating predicted effects on gene function with clinical phenotype data. SvAnna places 87% of deleterious SVs in the top ten ranks. The interpretable prioritizations offered by SvAnna will facilitate the widespread adoption of long-read sequencing in diagnostic genomics. SvAnna is available at https://github.com/TheJacksonLaboratory/SvAnn a .

Regulation of bacteriophage gene expression involves repressor proteins that bind and downregulate early lytic promoters. A large group of mycobacteriophages code for repressors that are unusual in also terminating transcription elongation at numerous binding sites (stoperators) distributed across the phage genome. Here we provide the X-ray crystal structure of a mycobacteriophage immunity repressor bound to DNA, which reveals the binding of a monomer to an asymmetric DNA sequence using two independent DNA binding domains. The structure is supported by small-angle X-ray scattering, DNA binding, molecular dynamics, and in vivo immunity assays. We propose a model for how dual DNA binding domains facilitate regulation of both transcription initiation and elongation, while enabling evolution of other superinfection immune specificities.

BackgroundThe eukaryotic parasite Plasmodium falciparum causes millions of malarial infections annually while drug resistance to common anti-malarials is further confounding eradication efforts. Translation is an attractive therapeutic target that will benefit from a deeper mechanistic understanding. As the rate limiting step of translation, initiation is a primary driver of translational efficiency. It is a complex process regulated by both cis and trans acting factors, providing numerous potential targets. Relative to model organisms and humans, P. falciparum mRNAs feature unusual 5' untranslated regions suggesting cis-acting sequence complexity in this parasite may act to tune levels of protein synthesis through their effects on translational efficiency.MethodsHere, in vitro translation is deployed to compare the role of cis-acting regulatory sequences in P. falciparum and humans. Using parasite mRNAs with high or low translational efficiency, the presence, position, and termination status of upstream "AUG"s, in addition to the base composition of the 5' untranslated regions, were characterized.ResultsThe density of upstream "AUG"s differed significantly among the most and least efficiently translated genes in P. falciparum, as did the average "GC" content of the 5' untranslated regions. Using exemplars from highly translated and poorly translated mRNAs, multiple putative upstream elements were interrogated for impact on translational efficiency. Upstream "AUG"s were found to repress translation to varying degrees, depending on their position and context, while combinations of upstream "AUG"s had non-additive effects. The base composition of the 5' untranslated regions also impacted translation, but to a lesser degree. Surprisingly, the effects of cis-acting sequences were remarkably conserved between P. falciparum and humans.ConclusionsWhile translational regulation is inherently complex, this work contributes toward a more comprehensive unde

The major biological methyl donor, S-adenosylmethionine (adoMet) synthesis occurs mainly in the liver. Methionine adenosyltransferase 1A (MAT1A) and glycine N-methyltransferase (GNMT) are two key enzymes involved in the functional implications of that variation. We collected 42 RNA-seq data from paired hepatocellular carcinoma (HCC) and its adjacent normal liver tissue from the Cancer Genome Atlas (TCGA). There was no mutation found in MAT1A or GNMT RNA in the 42 HCC patients. The 11,799 genes were annotated in the RNA-Seq data, and their expression levels were used to investigate the phenotypes of low MAT1A and low GNMT by Gene Set Enrichment Analysis (GSEA). The REACTOME_TRANSLATION gene set was enriched and visualized in a heatmap along with corresponding differences in gene expression between low MAT1A versus high MAT1A and low GNMT versus high GNMT. We identified 43 genes of the REACTOME_TRANSLATION gene set that are powerful prognosis factors in HCC. The significantly predicted genes were referred into eukaryotic translation initiation (EIF3B, EIF3K), eukaryotic translation elongation (EEF1D), and ribosomal proteins (RPs). Cell models expressing various MAT1A and GNMT proved that simultaneous restoring the expression of MAT1A and GNMT decreased cell proliferation, invasion, as well as the REACTOME_TRANSLATION gene EEF1D, consistent with a better prognosis in human HCC. We demonstrated new findings that downregulation or defect in MAT1A and GNMT genes can enrich the protein-associated translation process that may account for poor HCC prognosis. This is the first study demonstrated that MAT1A and GNMT, the 2 key enzymes involved in methionine cycle, could attenuate the function of ribosome translation. We propose a potential novel mechanism by which the diminished GNMT and MAT1A expression may confer poor prognosis for HCC.

HIV-1-infected cells that persist despite antiretroviral therapy (ART) are frequently considered "transcriptionally silent," but active viral gene expression may occur in some cells, challenging the concept of viral latency. Applying an assay for profiling the transcriptional activity and the chromosomal locations of individual proviruses, we describe a global genomic and epigenetic map of transcriptionally active and silent proviral species and evaluate their longitudinal evolution in persons receiving suppressive ART. Using genome-wide epigenetic reference data, we show that proviral transcriptional activity is associated with activating epigenetic chromatin features in linear proximity of integration sites and in their inter- and intrachromosomal contact regions. Transcriptionally active proviruses were actively selected against during prolonged ART; however, this pattern was violated by large clones of virally infected cells that may outcompete negative selection forces through elevated intrinsic proliferative activity. Our results suggest that transcriptionally active proviruses are dynamically evolving under selection pressure by host factors.

Coccidioidomycosis is a common fungal disease that is endemic to arid and semi-arid regions of both American continents. Coccidioides immitis and Coccidioides posadasii are the etiological agents of the disease, also known as Valley Fever. For several decades, the C. posadasii strain Silveira has been used widely in vaccine studies, is the source strain for production of diagnostic antigens, and is a widely used experimental strain for functional studies. In 2009, the genome was sequenced using Sanger sequencing technology, and a draft assembly and annotation were made available. In this study, the genome of the Silveira strain was sequenced using single molecule real-time sequencing PacBio technology, assembled into chromosomal-level contigs, genotyped, and the genome was reannotated using sophisticated and curated in silico tools. This high-quality genome sequencing effort has improved our understanding of chromosomal structure, gene set annotation, and lays the groundwork for identification of structural variants (e.g. transversions, translocations, and copy number variants), assessment of gene gain and loss, and comparison of transposable elements in future phylogenetic and population genomics studies.

The cellular response to stress is an important determinant of disease pathogenesis. Uncovering the molecular fingerprints of distinct stress responses may identify novel biomarkers and key signaling pathways for different diseases. Emerging evidence shows that transfer RNA-derived small RNAs (tDRs) play pivotal roles in stress responses. However, RNA modifications present on tDRs are barriers to accurately quantifying tDRs using traditional small RNA sequencing. Here, AlkB-facilitated methylation sequencing is used to generate a comprehensive landscape of cellular and extracellular tDR abundances in various cell types during different stress responses. Extracellular tDRs are found to have distinct fragmentation signatures from intracellular tDRs and these tDR signatures are better indicators of different stress responses than miRNAs. These distinct extracellular tDR fragmentation patterns and signatures are also observed in plasma from patients on cardiopulmonary bypass. It is additionally demonstrated that angiogenin and RNASE1 are themselves regulated by stressors and contribute to the stress-modulated abundance of sub-populations of cellular and extracellular tDRs. Finally, a sub-population of extracellular tDRs is identified for which AGO2 appears to be required for their expression. Together, these findings provide a detailed profile of stress-responsive tDRs and provide insight about tDR biogenesis and stability in response to cellular stressors.

Over the last 25 years, biology has entered the genomic era and is becoming a science of 'big data'. Most interpretations of genomic analyses rely on accurate functional annotations of the proteins encoded by more than 500 000 genomes sequenced to date. By different estimates, only half the predicted sequenced proteins carry an accurate functional annotation, and this percentage varies drastically between different organismal lineages. Such a large gap in knowledge hampers all aspects of biological enterprise and, thereby, is standing in the way of genomic biology reaching its full potential. A brainstorming meeting to address this issue funded by the National Science Foundation was held during 3-4 February 2022. Bringing together data scientists, biocurators, computational biologists and experimentalists within the same venue allowed for a comprehensive assessment of the current state of functional annotations of protein families. Further, major issues that were obstructing the field were identified and discussed, which ultimately allowed for the proposal of solutions on how to move forward.

The giant ciliate Stentor coeruleus is a classical model system for studying regeneration and morphogenesis in a single cell. The anterior of the cell is marked by an array of cilia, known as the oral apparatus, which can be induced to shed and regenerate in a series of reproducible morphological steps, previously shown to require transcription. If a cell is cut in half, each half regenerates an intact cell. We used RNA sequencing (RNAseq) to assay the dynamic changes in Stentor's transcriptome during regeneration, after both oral apparatus shedding and bisection, allowing us to identify distinct temporal waves of gene expression including kinases, RNA -binding proteins, centriole biogenesis factors, and orthologs of human ciliopathy genes. By comparing transcriptional profiles of different regeneration events, we identified distinct modules of gene expression corresponding to oral apparatus regeneration, posterior holdfast regeneration, and recovery after wounding. By measuring gene expression after blocking translation, we show that the sequential waves of gene expression involve a cascade mechanism in which later waves of expression are triggered by translation products of early-expressed genes. Among the early-expressed genes, we identified an E2F transcription factor and the RNA-binding protein Pumilio as potential regulators of regeneration based on the expression pattern of their predicted target genes. RNAi-mediated knockdown experiments indicate that Pumilio is required for regenerating oral structures of the correct size. E2F is involved in the completion of regeneration but is dispensable for earlier steps. This work allows us to classify regeneration genes into groups based on their potential role for regeneration in distinct cell regeneration paradigms, and provides insight into how a single cell can coordinate complex morphogenetic pathways to regenerate missing structures.

Life on Earth has evolved from initial simplicity to the astounding complexity we experience today. Bacteria and archaea have largely excelled in metabolic diversification, but eukaryotes additionally display abundant morphological innovation. How have these innovations come about and what constraints are there on the origins of novelty and the continuing maintenance of biodiversity on Earth? The history of life and the code for the working parts of cells and systems are written in the genome. The Earth BioGenome Project has proposed that the genomes of all extant, named eukaryotes-about 2 million species-should be sequenced to high quality to produce a digital library of life on Earth, beginning with strategic phylogenetic, ecological, and high-impact priorities. Here we discuss why we should sequence all eukaryotic species, not just a representative few scattered across the many branches of the tree of life. We suggest that many questions of evolutionary and ecological significance will only be addressable when whole-genome data representing divergences at all of the branchings in the tree of life or all species in natural ecosystems are available. We envisage that a genomic tree of life will foster understanding of the ongoing processes of speciation, adaptation, and organismal dependencies within entire ecosystems. These explorations will resolve long-standing problems in phylogenetics, evolution, ecology, conservation, agriculture, bioindustry, and medicine.

Nascent RNA-sequencing tracks transcription at nucleotide resolution. The genomic distribution of engaged transcription complexes, in turn, uncovers functional genomic regions. Here, we provide analytical steps to (1) identify transcribed regulatory elements de novo genome-wide, (2) quantify engaged transcription complexes at enhancers, promoter-proximal regions, divergent transcripts, gene bodies, and termination windows, and (3) measure distribution of transcription machineries and regulatory proteins across functional genomic regions. This protocol tracks engaged transcription complexes across functional genomic regions demonstrated in human K562 erythroleukemia cells. For complete details on the use and execution of this protocol, please refer to Vihervaara et al. (2021)., QC 20220930

Small RNAs (sRNAs) are known to regulate pathogenic plant-microbe interactions. Emerging evidence from the study of these model systems suggests that microRNAs (miRNAs) can be translocated between microbes and plants to facilitate symbiosis. The roles of sRNAs in mutualistic mycorrhizal fungal interactions, however, are largely unknown. In this study, we characterized miRNAs encoded by the ectomycorrhizal fungus Pisolithus microcarpus and investigated their expression during mutualistic interaction with Eucalyptus grandis. Using sRNA sequencing data and in situ miRNA detection, a novel fungal miRNA, Pmic_miR-8, was found to be transported into E. grandis roots after interaction with P. microcarpus Further characterization experiments demonstrate that inhibition of Pmic_miR-8 negatively impacts the maintenance of mycorrhizal roots in E. grandis, while supplementation of Pmic_miR-8 led to deeper integration of the fungus into plant tissues. Target prediction and experimental testing suggest that Pmic_miR-8 may target the host NB-ARC domain containing transcripts, suggesting a potential role for this miRNA in subverting host signaling to stabilize the symbiotic interaction. Altogether, we provide evidence of previously undescribed cross-kingdom sRNA transfer from ectomycorrhizal fungi to plant roots, shedding light onto the involvement of miRNAs during the developmental process of mutualistic symbioses.

PurposeThere is limited payer coverage for genome sequencing (GS) relative to exome sequencing (ES) in the U.S. Our objective was to assess payers' considerations for coverage of GS versus coverage of ES and requirements payers have for coverage of GS. The study was conducted by the NIH-funded Clinical Sequencing Evidence-Generating Research Consortium (CSER).MethodsWe conducted semi-structured interviews with representatives of private payer organizations (payers, N = 12) on considerations and evidentiary and other needs for coverage of GS and ES. Data were analyzed using thematic analysis.ResultsWe described four categories of findings and solutions: demonstrated merits of GS versus ES, enhanced methods for evidence generation, consistent laboratory processes/sequencing methods, and enhanced implementation/care delivery. Payers see advantages to GS vs. ES and are open to broader GS coverage but need more proof of these advantages to consider them in coverage decision-making. Next steps include establishing evidence of benefits in specific clinical scenarios, developing quality standards, ensuring transparency of laboratory methods, developing clinical centers of excellence, and incorporating the role of genetic professionals.ConclusionBy comparing coverage considerations for GS and ES, we identified a path forward for coverage of GS. Future research should explicitly address payers' conditions for coverage.

A global international initiative, such as the Earth BioGenome Project (EBP), requires both agreement and coordination on standards to ensure that the collective effort generates rapid progress toward its goals. To this end, the EBP initiated five technical standards committees comprising volunteer members from the global genomics scientific community: Sample Collection and Processing, Sequencing and Assembly, Annotation, Analysis, and IT and Informatics. The current versions of the resulting standards documents are available on the EBP website, with the recognition that opportunities, technologies, and challenges may improve or change in the future, requiring flexibility for the EBP to meet its goals. Here, we describe some highlights from the proposed standards, and areas where additional challenges will need to be met.

BackgroundSeahorses, seadragons, pygmy pipehorses, and pipefishes (Syngnathidae, Syngnathiformes) are among the most recognizable groups of fishes because of their derived morphology, unusual life history, and worldwide distribution. Despite previous phylogenetic studies and recent new species descriptions of syngnathids, the evolutionary relationships among several major groups within this family remain unresolved.ResultsHere, we provide a reconstruction of syngnathid phylogeny based on genome-wide sampling of 1314 ultraconserved elements (UCEs) and expanded taxon sampling to assess the current taxonomy and as a basis for macroevolutionary insights. We sequenced a total of 244 new specimens across 117 species and combined with published UCE data for a total of 183 species of Syngnathidae, about 62% of the described species diversity, to compile the most data-rich phylogeny to date. We estimated divergence times using 14 syngnathiform fossils, including nine fossils with newly proposed phylogenetic affinities, to better characterize current and historical biogeographical patterns, and to reconstruct diversification through time. We present a phylogenetic hypothesis that is well-supported and provides several notable insights into syngnathid evolution. We found nine non-monophyletic genera, evidence for seven cryptic species, five potentially invalid synonyms, and identified a novel sister group to the seahorses, the Indo-Pacific pipefishes Halicampus macrorhynchus and H. punctatus. In addition, the morphologically distinct southwest Pacific seahorse Hippocampus jugumus was recovered as the sister to all other non-pygmy seahorses. As found in many other groups, a high proportion of syngnathid lineages appear to have originated in the Central Indo-Pacific and subsequently dispersed to adjoining regions. Conversely, we also found an unusually high subsequent return of lineages from southern Australasia to the Central Indo-Pacific. Diversification rates rose abrupt

Tobamoviruses are agriculturally relevant viruses that cause crop losses and have infected plants in many regions of the world. These viruses are frequently found in municipal wastewater, likely coming from human diet and industrial waste across wastewater catchment areas. As part of a large wastewater-based epidemiology study across Southern California, we analyzed RNA sequence data from 275 influent wastewater samples obtained from eight wastewater treatment plants with a catchment area of approximately 16 million people from July 2020 to August 2021. We assembled 1,083 high-quality genomes, enumerated viral sequencing reads, and detected thousands of single nucleotide variants from eight common tobamoviruses: bell pepper mottle virus, cucumber green mottle mosaic virus, pepper mild mottle virus, tobacco mild green mosaic virus, tomato brown rugose fruit virus, tomato mosaic virus, tomato mottle mosaic virus, and tropical soda apple mosaic virus. We show that single nucleotide variants had amino acid-altering consequences along with synonymous mutations, which represents potential evolution with functional consequences in genomes of these viruses. Our study shows the importance of wastewater sequencing to monitor the genomic diversity of these plant-infecting viruses, and we suggest that our data could be used to continue tracking the genomic variability of such pathogens. IMPORTANCE Diseases caused by viruses in the genus Tobamovirus cause crop losses around the world. As with other viruses, mutation occurring in the virus's genomes can have functional consequences and may alter viral infectivity. Many of these plant-infecting viruses have been found in wastewater, likely coming from human consumption of infected plants and produce. By sequencing RNA extracted from influent wastewater obtained from eight wastewater treatment plants in Southern California, we assembled high-quality viral genomes and detected thousands of single nucleotide variants from eight tobam

Human herpes simplex virus 2 (HSV-2) is a ubiquitous, slowly evolving DNA virus. HSV-2 has two primary lineages, one found in West and Central Africa and the other found worldwide. Competing hypotheses have been proposed to explain how HSV-2 migrated out-of-Africa (i)HSV-2 followed human migration out-of-Africa 50-100 thousand years ago, or (ii)HSV-2 migrated via the trans-Atlantic slave trade 150-500 years ago. Limited geographic sampling and lack of molecular clock signal has precluded robust comparison. Here, we analyze newly sequenced HSV-2 genomes from Africa to resolve geography and timing of divergence events within HSV-2. Phylogeographic analysis consistently places the ancestor of worldwide dispersal in East Africa, though molecular clock is too slow to be detected using available data. Rates 4.2 × 10-8-5.6 × 10-8 substitutions/site/year, consistent with previous age estimates, suggest a worldwide dispersal 22-29 thousand years ago. Thus, HSV-2 likely migrated with humans from East Africa and dispersed after the Last Glacial Maximum.

Evolutionary biologists have long sought to disentangle phylogenetic relationships among taxa spanning the tree of life, an increasingly important task as anthropogenic influences accelerate population declines and species extinctions, particularly in insects. Phylogenetic analyses are commonly used to identify unique evolutionary lineages, to clarify taxonomic designations of the focal taxa, and to inform conservation decisions. Advances in DNA sequencing techniques have increasingly facilitated the ability of researchers to apply genomic methods to phylogenetic analyses, even for non-model organisms. Stoneflies are non-model insects that are important bioindicators of the quality of freshwater habitats and landscape disturbance as they spend the immature stages of their life cycles in fresh water, and the adult stages in terrestrial environments. Phylogenetic relationships within the stonefly genus Suwallia (Insecta: Plecoptera: Chloroperlidae) are poorly understood, and have never been assessed using molecular data. We used DNA sequence data from genome-wide ultraconserved element loci to generate the first molecular phylogeny for the group and assess its monophyly. We found that Palearctic and Nearctic Suwallia do not form reciprocally monophyletic clades, and that a biogeographic history including dispersal, vicariance, and founder event speciation via jump dispersal best explains the geographic distribution of this group. Our results also strongly suggest that Neaviperla forcipata (Neave, 1929) is nested within Suwallia, and the concept of the genus Suwallia should be revised to include it. Thus, we formally propose a new taxonomic combination wherein Neaviperla forcipata (Neave, 1929) is reclassified as Suwallia forcipata (Neave, 1929). Moreover, some Suwallia species (e.g., S. amoenacolens, S. kerzhneri, S. marginata, S. pallidula, and S. starki) exhibit pronounced cryptic diversity that is worthy of further investigation. These findings provide a first glim

Subviral agents are nucleic acids which lack the features for classification as a virus. Tombusvirus-like associated RNAs (tlaRNAs) are subviral positive-sense, single-stranded RNAs that replicate autonomously, yet depend on a coinfecting virus for encapsidation and transmission. TlaRNAs produce abundant subgenomic RNA (sgRNA) upon infection. Here, we investigate how the well-studied tlaRNA, ST9, produces sgRNA and its function. We found ST9 is a noncoding RNA, due to its lack of protein coding capacity. We used resistance assays with eukaryotic Exoribonuclease-1 (XRN1) to investigate sgRNA production via incomplete degradation of genomic RNA. The ST9 3' untranslated region stalled XRN1 very near the 5' sgRNA end. Thus, the XRN family of enzymes drives sgRNA accumulation in ST9-infected tissue by incomplete degradation of ST9 RNA. This work suggests tlaRNAs are not just parasites of viruses with compatible capsids, but also mutually beneficial partners that influence host cell RNA biology.

The mitochondrial genome (mtDNA) is an important source of disease-causing genetic variability, but existing sequencing methods limit understanding, precluding phased measurement of mutations and clear detection of large sporadic deletions. We adapted a method for amplification-free sequence enrichment using Cas9 cleavage to obtain full length nanopore reads of mtDNA. We then utilized the long reads to phase mutations in a patient with an mtDNA-linked syndrome and demonstrated that this method can map age-induced mtDNA deletions. We believe this method will offer deeper insight into our understanding of mtDNA variation.

While genome recoding using quadruplet codons to incorporate non-proteinogenic amino acids is attractive for biotechnology and bioengineering purposes, the mechanism through which such codons are translated is poorly understood. Here we investigate translation of quadruplet codons by a +1-frameshifting tRNA, SufB2, that contains an extra nucleotide in its anticodon loop. Natural post-transcriptional modification of SufB2 in cells prevents it from frameshifting using a quadruplet-pairing mechanism such that it preferentially employs a triplet-slippage mechanism. We show that SufB2 uses triplet anticodon-codon pairing in the 0-frame to initially decode the quadruplet codon, but subsequently shifts to the +1-frame during tRNA-mRNA translocation. SufB2 frameshifting involves perturbation of an essential ribosome conformational change that facilitates tRNA-mRNA movements at a late stage of the translocation reaction. Our results provide a molecular mechanism for SufB2-induced +1 frameshifting and suggest that engineering of a specific ribosome conformational change can improve the efficiency of genome recoding.

Viral genome sequencing has guided our understanding of the spread and extent of genetic diversity of SARS-CoV-2 during the COVID-19 pandemic. SARS-CoV-2 viral genomes are usually sequenced from nasopharyngeal swabs of individual patients to track viral spread. Recently, RT-qPCR of municipal wastewater has been used to quantify the abundance of SARS-CoV-2 in several regions globally. However, metatranscriptomic sequencing of wastewater can be used to profile the viral genetic diversity across infected communities. Here, we sequenced RNA directly from sewage collected by municipal utility districts in the San Francisco Bay Area to generate complete and nearly complete SARS-CoV-2 genomes. The major consensus SARS-CoV-2 genotypes detected in the sewage were identical to clinical genomes from the region. Using a pipeline for single nucleotide variant calling in a metagenomic context, we characterized minor SARS-CoV-2 alleles in the wastewater and detected viral genotypes which were also found within clinical genomes throughout California. Observed wastewater variants were more similar to local California patient-derived genotypes than they were to those from other regions within the United States or globally. Additional variants detected in wastewater have only been identified in genomes from patients sampled outside California, indicating that wastewater sequencing can provide evidence for recent introductions of viral lineages before they are detected by local clinical sequencing. These results demonstrate that epidemiological surveillance through wastewater sequencing can aid in tracking exact viral strains in an epidemic context.

APOBEC mutagenesis, a major driver of cancer evolution, is known for targeting TpC sites in DNA. Recently, we showed that APOBEC3A (A3A) targets DNA hairpin loops. Here, we show that DNA secondary structure is in fact an orthogonal influence on A3A substrate optimality and, surprisingly, can override the TpC sequence preference. VpC (non-TpC) sites in optimal hairpins can outperform TpC sites as mutational hotspots. This expanded understanding of APOBEC mutagenesis illuminates the genomic Twin Paradox, a puzzling pattern of closely spaced mutation hotspots in cancer genomes, in which one is a canonical TpC site but the other is a VpC site, and double mutants are seen only in trans, suggesting a two-hit driver event. Our results clarify this paradox, revealing that both hotspots in these twins are optimal A3A substrates. Our findings reshape the notion of a mutation signature, highlighting the additive roles played by DNA sequence and DNA structure.

Given the global impact and severity of COVID-19, there is a pressing need for a better understanding of the SARS-CoV-2 genome and mutations. Multi-strain sequence alignments of coronaviruses (CoV) provide important information for interpreting the genome and its variation. We apply a comparative genomics method, ConsHMM, to the multi-strain alignments of CoV to annotate every base of the SARS-CoV-2 genome with conservation states based on sequence alignment patterns among CoV. The learned conservation states show distinct enrichment patterns for genes, protein domains, and other regions of interest. Certain states are strongly enriched or depleted of SARS-CoV-2 mutations, which can be used to predict potentially consequential mutations. We expect the conservation states to be a resource for interpreting the SARS-CoV-2 genome and mutations.

Platelet aggregation at the site of atherosclerotic vascular injury is the underlying pathophysiology of myocardial infarction and stroke. To build upon prior GWAS, here we report on 16 loci identified through a whole genome sequencing (WGS) approach in 3,855 NHLBI Trans-Omics for Precision Medicine (TOPMed) participants deeply phenotyped for platelet aggregation. We identify the RGS18 locus, which encodes a myeloerythroid lineage-specific regulator of G-protein signaling that co-localizes with expression quantitative trait loci (eQTL) signatures for RGS18 expression in platelets. Gene-based approaches implicate the SVEP1 gene, a known contributor of coronary artery disease risk. Sentinel variants at RGS18 and PEAR1 are associated with thrombosis risk and increased gastrointestinal bleeding risk, respectively. Our WGS findings add to previously identified GWAS loci, provide insights regarding the mechanism(s) by which genetics may influence cardiovascular disease risk, and underscore the importance of rare variant and regulatory approaches to identifying loci contributing to complex phenotypes.

Viruses are integral components of all ecosystems and microbiomes on Earth. Through pervasive infections of their cellular hosts, viruses can reshape microbial community structure and drive global nutrient cycling. Over the past decade, viral sequences identified from genomes and metagenomes have provided an unprecedented view of viral genome diversity in nature. Since 2016, the IMG/VR database has provided access to the largest collection of viral sequences obtained from (meta)genomes. Here, we present the third version of IMG/VR, composed of 18 373 cultivated and 2 314 329 uncultivated viral genomes (UViGs), nearly tripling the total number of sequences compared to the previous version. These clustered into 935 362 viral Operational Taxonomic Units (vOTUs), including 188 930 with two or more members. UViGs in IMG/VR are now reported as single viral contigs, integrated proviruses or genome bins, and are annotated with a new standardized pipeline including genome quality estimation using CheckV, taxonomic classification reflecting the latest ICTV update, and expanded host taxonomy prediction. The new IMG/VR interface enables users to efficiently browse, search, and select UViGs based on genome features and/or sequence similarity. IMG/VR v3 is available at https://img.jgi.doe.gov/vr, and the underlying data are available to download at https://genome.jgi.doe.gov/portal/IMG_VR.

Under the Baltimore nucleic acid-based virus classification scheme, the herpesvirus human cytomegalovirus (HCMV) is a Class I virus, meaning that it contains a double-stranded DNA genome-and no RNA. Here, we report sub-particle cryoEM reconstructions of HCMV virions at 2.9 Å resolution revealing structures resembling non-coding transfer RNAs (tRNAs) associated with the virion's capsid-bound tegument protein, pp150. Through deep sequencing, we show that these RNA sequences match human tRNAs, and we built atomic models using the most abundant tRNA species. Based on our models, tRNA recruitment is mediated by the electrostatic interactions between tRNA phosphate groups and the helix-loop-helix motif of HCMV pp150. The specificity of these interactions may explain the absence of such tRNA densities in murine cytomegalovirus and other human herpesviruses.

Using the Cap Analysis of Gene Expression (CAGE) technology, the FANTOM5 consortium provided one of the most comprehensive maps of transcription start sites (TSSs) in several species. Strikingly, ~72% of them could not be assigned to a specific gene and initiate at unconventional regions, outside promoters or enhancers. Here, we probe these unassigned TSSs and show that, in all species studied, a significant fraction of CAGE peaks initiate at microsatellites, also called short tandem repeats (STRs). To confirm this transcription, we develop Cap Trap RNA-seq, a technology which combines cap trapping and long read MinION sequencing. We train sequence-based deep learning models able to predict CAGE signal at STRs with high accuracy. These models unveil the importance of STR surrounding sequences not only to distinguish STR classes, but also to predict the level of transcription initiation. Importantly, genetic variants linked to human diseases are preferentially found at STRs with high transcription initiation level, supporting the biological and clinical relevance of transcription initiation at STRs. Together, our results extend the repertoire of non-coding transcription associated with DNA tandem repeats and complexify STR polymorphism.

RNAcentral is a comprehensive database of non-coding RNA (ncRNA) sequences that provides a single access point to 44 RNA resources and >18 million ncRNA sequences from a wide range of organisms and RNA types. RNAcentral now also includes secondary (2D) structure information for >13 million sequences, making RNAcentral the world's largest RNA 2D structure database. The 2D diagrams are displayed using R2DT, a new 2D structure visualization method that uses consistent, reproducible and recognizable layouts for related RNAs. The sequence similarity search has been updated with a faster interface featuring facets for filtering search results by RNA type, organism, source database or any keyword. This sequence search tool is available as a reusable web component, and has been integrated into several RNAcentral member databases, including Rfam, miRBase and snoDB. To allow for a more fine-grained assignment of RNA types and subtypes, all RNAcentral sequences have been annotated with Sequence Ontology terms. The RNAcentral database continues to grow and provide a central data resource for the RNA community. RNAcentral is freely available at https://rnacentral.org.

Ultraconserved enhancer sequences show perfect conservation between human and rodent genomes, suggesting that their functions are highly sensitive to mutation. However, current models of enhancer function do not sufficiently explain this extreme evolutionary constraint. We subjected 23 ultraconserved enhancers to different levels of mutagenesis, collectively introducing 1,547 mutations, and examined their activities in transgenic mouse reporter assays. Overall, we find that the regulatory properties of ultraconserved enhancers are robust to mutation. Upon mutagenesis, nearly all (19/23, 83%) still functioned as enhancers at one developmental stage, as did most of those tested again later in development (5/9, 56%). Replacement of endogenous enhancers with mutated alleles in mice corroborated results of transgenic assays, including the functional resilience of ultraconserved enhancers to mutation. Our findings show that the currently known activities of ultraconserved enhancers do not necessarily require the perfect conservation observed in evolution and suggest that additional regulatory or other functions contribute to their sequence constraint.

Expression of a few master transcription factors can reprogram the epigenetic landscape and three-dimensional chromatin topology of differentiated cells and achieve pluripotency. During reprogramming, thousands of long-range chromatin contacts are altered, and changes in promoter association with enhancers dramatically influence transcription. Molecular participants at these sites have been identified, but how this re-organization might be orchestrated is not known. Biomolecular condensation is implicated in subcellular organization, including the recruitment of RNA polymerase in transcriptional activation. Here, we show that reprogramming factor KLF4 undergoes biomolecular condensation even in the absence of its intrinsically disordered region. Liquid-liquid condensation of the isolated KLF4 DNA binding domain with a DNA fragment from the NANOG proximal promoter is enhanced by CpG methylation of a KLF4 cognate binding site. We propose KLF4-mediated condensation as one mechanism for selectively organizing and re-organizing the genome based on the local sequence and epigenetic state.

Studies of acute myeloid leukemia rely on DNA sequencing and immunophenotyping by flow cytometry as primary tools for disease characterization. However, leukemia tumor heterogeneity complicates integration of DNA variants and immunophenotypes from separate measurements. Here we introduce DAb-seq, a technology for simultaneous capture of DNA genotype and cell surface phenotype from single cells at high throughput, enabling direct profiling of proteogenomic states in tens of thousands of cells. To demonstrate the approach, we analyze the disease of three patients with leukemia over multiple treatment timepoints and disease recurrences. We observe complex genotype-phenotype dynamics that illustrate the subtlety of the disease process and the degree of incongruity between blast cell genotype and phenotype in different clinical scenarios. Our results highlight the importance of combined single-cell DNA and protein measurements to fully characterize the heterogeneity of leukemia.

Under the Baltimore nucleic acid-based virus classification scheme, the herpesvirus human cytomegalovirus (HCMV) is a Class I virus, meaning that it contains a double-stranded DNA genome-and no RNA. Here, we report sub-particle cryoEM reconstructions of HCMV virions at 2.9 Å resolution revealing structures resembling non-coding transfer RNAs (tRNAs) associated with the virion's capsid-bound tegument protein, pp150. Through deep sequencing, we show that these RNA sequences match human tRNAs, and we built atomic models using the most abundant tRNA species. Based on our models, tRNA recruitment is mediated by the electrostatic interactions between tRNA phosphate groups and the helix-loop-helix motif of HCMV pp150. The specificity of these interactions may explain the absence of such tRNA densities in murine cytomegalovirus and other human herpesviruses.

APOBEC mutagenesis, a major driver of cancer evolution, is known for targeting TpC sites in DNA. Recently, we showed that APOBEC3A (A3A) targets DNA hairpin loops. Here, we show that DNA secondary structure is in fact an orthogonal influence on A3A substrate optimality and, surprisingly, can override the TpC sequence preference. VpC (non-TpC) sites in optimal hairpins can outperform TpC sites as mutational hotspots. This expanded understanding of APOBEC mutagenesis illuminates the genomic Twin Paradox, a puzzling pattern of closely spaced mutation hotspots in cancer genomes, in which one is a canonical TpC site but the other is a VpC site, and double mutants are seen only in trans, suggesting a two-hit driver event. Our results clarify this paradox, revealing that both hotspots in these twins are optimal A3A substrates. Our findings reshape the notion of a mutation signature, highlighting the additive roles played by DNA sequence and DNA structure.

Viruses are integral components of all ecosystems and microbiomes on Earth. Through pervasive infections of their cellular hosts, viruses can reshape microbial community structure and drive global nutrient cycling. Over the past decade, viral sequences identified from genomes and metagenomes have provided an unprecedented view of viral genome diversity in nature. Since 2016, the IMG/VR database has provided access to the largest collection of viral sequences obtained from (meta)genomes. Here, we present the third version of IMG/VR, composed of 18 373 cultivated and 2 314 329 uncultivated viral genomes (UViGs), nearly tripling the total number of sequences compared to the previous version. These clustered into 935 362 viral Operational Taxonomic Units (vOTUs), including 188 930 with two or more members. UViGs in IMG/VR are now reported as single viral contigs, integrated proviruses or genome bins, and are annotated with a new standardized pipeline including genome quality estimation using CheckV, taxonomic classification reflecting the latest ICTV update, and expanded host taxonomy prediction. The new IMG/VR interface enables users to efficiently browse, search, and select UViGs based on genome features and/or sequence similarity. IMG/VR v3 is available at https://img.jgi.doe.gov/vr, and the underlying data are available to download at https://genome.jgi.doe.gov/portal/IMG_VR.

The establishment of cardiac function in the developing embryo is essential to ensure blood flow and, therefore, growth and survival of the animal. The molecular mechanisms controlling normal cardiac rhythm remain to be fully elucidated. From a forward genetic screen, we identified a unique mutant, grime, that displayed a specific cardiac arrhythmia phenotype. We show that loss-of-function mutations in tmem161b are responsible for the phenotype, identifying Tmem161b as a regulator of cardiac rhythm in zebrafish. To examine the evolutionary conservation of this function, we generated knockout mice for Tmem161b. Tmem161b knockout mice are neonatal lethal and cardiomyocytes exhibit arrhythmic calcium oscillations. Mechanistically, we find that Tmem161b is expressed at the cell membrane of excitable cells and live imaging shows it is required for action potential repolarization in the developing heart. Electrophysiology on isolated cardiomyocytes demonstrates that Tmem161b is essential to inhibit Ca2+ and K+ currents in cardiomyocytes. Importantly, Tmem161b haploinsufficiency leads to cardiac rhythm phenotypes, implicating it as a candidate gene in heritable cardiac arrhythmia. Overall, these data describe Tmem161b as a highly conserved regulator of cardiac rhythm that functions to modulate ion channel activity in zebrafish and mice.