High-throughput Genomic Approaches Research Articles

DDK-Linker: a network-based strategy identifies disease signals by linking high-throughput omics datasets to disease knowledge.

The high-throughput genomic and proteomic scanning approaches allow investigators to measure the quantification of genome-wide genes (or gene products) for certain disease conditions, which plays an essential role in promoting the discovery of disease mechanisms. The high-throughput approaches often generate a large gene list of interest (GOIs), such as differentially expressed genes/proteins. However, researchers have to perform manual triage and validation to explore the most promising, biologically plausible linkages between the known disease genes and GOIs (disease signals) for further study. Here, to address this challenge, we proposed a network-based strategy DDK-Linker to facilitate the exploration of disease signals hidden in omics data by linking GOIs to disease knowns genes. Specifically, it reconstructed gene distances in the protein-protein interaction (PPI) network through six network methods (random walk with restart, Deepwalk, Node2Vec, LINE, HOPE, Laplacian) to discover disease signals in omics data that have shorter distances to disease genes. Furthermore, benefiting from the establishment of knowledge base we established, the abundant bioinformatics annotations were provided for each candidate disease signal. To assist in omics data interpretation and facilitate the usage, we have developed this strategy into an application that users can access through a website or download the R package. We believe DDK-Linker will accelerate the exploring of disease genes and drug targets in a variety of omics data, such as genomics, transcriptomics and proteomics data, and provide clues for complex disease mechanism and pharmacological research. DDK-Linker is freely accessible at http://ddklinker.ncpsb.org.cn/.

ENFOQUES GENÓMICOS Y TRANSCRIPTÓMICOS PARA ESTUDIAR ÁRBOLES MADERABLES: PERSPECTIVAS PARA EL ESTUDIO DE CEDRO ROJO (Cedrela odorata L.)

<p><strong>Introduction</strong>: The high-throughput genomic and transcriptomic approach has been developed and implemented to address the main challenges that the timber forestry sector faces, such as population growth, climate change, deforestation and the loss of forest ecosystem services. <strong>Objective:</strong> To carry out a bibliographic review focused on the genomes and transcriptomes of timber trees reported in the databases, with special attention to red cedar (<em>Cedrela odorata</em> L.), due to its importance as precious wood in Mexico. <strong>Methodology:</strong> A literature review was carried out, directed at studying timber trees with genomic and transcriptomic strategies in different databases such as Pubmed, Scopus, Google Scholar, ScienceDirect, Wiley Online Library, MDPI and Scielo to identify the timber species that have been reported genomes and transcriptomes. The structure of the review was the genomics of timber trees, the transcriptomics of wood, and the potential species for study due to their importance and finally, the databases for consultation. Subsequently, a bibliometric study was carried out with the bibliometrix library in R Studio. <strong>Main results</strong>: The first genome to be assembled at the chromosome level was the black cottonwood. Among the timber trees, the genomes of black cottonwood, desert poplar, eucalyptus and oak with a length of 392, 496, 691 and 789 Mb have been reported. Through study of the transcriptome, it has been possible to identify genes related to formation of the wood in a hybrid poplar (<em>Populus alba</em> L.<em> × P. glandulosa</em>) and <em>P. tremula</em> L. and with drought tolerance in <em>Pinus massoniana</em> and <em>Pinus pinaster</em> Aiton. In red cedar (<em>Cedrela odorata</em> L.), the transcriptome was obtained by sequencing a single leaf, identifying 52,181 gene models. In the NCBI, EMBL-EBI, TreeGenes, PLAZA databases and the hardwood genomics website it is possible to find information related to the genomics and transcriptomics of timber species. <strong>Implications:</strong> More research is required in omics in timber, particularly in red cedar, since the search on these topics yielded little information. <strong>Conclusion</strong>: Through the bibliographic review in databases, the timber trees that have a described genome and transcriptome were identified. This information can be used for the assembly and annotation of new genomes, identification of genes and molecular markers, among other applications.</p>

Disentangling the complexity of psoriasis in the post-genome-wide association era.

In recent years, genome-wide association studies (GWAS) have been instrumental in unraveling the genetic architecture of complex diseases, including psoriasis. The application of large-scale GWA studies in psoriasis has illustrated several associated loci that participate in the cutaneous inflammation, however explaining a fraction of the disease heritability. With the advent of high-throughput sequencing technologies and functional genomics approaches, the post-GWAS era aims to unravel the functional mechanisms underlying the inter-individual variability in psoriasis patients. In this review, we present the key advances of psoriasis GWAS in under-represented populations, rare, non-coding and structural variants and epistatic phenomena that orchestrate the interplay between different cell types. We further review the gene-gene and gene-environment interactions contributing to the disease predisposition and development of comorbidities through Mendelian randomization studies and pleiotropic effects of psoriasis-associated loci. We finally examine the holistic approaches conducted in psoriasis through system genetics and state-of-the-art transcriptomic analyses, discussing their potential implication in the expanding field of precision medicine and characterization of comorbidities.

Reviewing PTBP1 Domain Modularity in the Pre-Genomic Era: A Foundation to Guide the Next Generation of Exploring PTBP1 Structure-Function Relationships.

Polypyrimidine tract binding protein 1 (PTBP1) is one of the most well-described RNA binding proteins, known initially for its role as a splicing repressor before later studies revealed its numerous roles in RNA maturation, stability, and translation. While PTBP1's various biological roles have been well-described, it remains unclear how its four RNA recognition motif (RRM) domains coordinate these functions. The early PTBP1 literature saw extensive effort placed in detailing structures of each of PTBP1's RRMs, as well as their individual RNA sequence and structure preferences. However, limitations in high-throughput and high-resolution genomic approaches (i.e., next-generation sequencing had not yet been developed) precluded the functional translation of these findings into a mechanistic understanding of each RRM's contribution to overall PTBP1 function. With the emergence of new technologies, it is now feasible to begin elucidating the individual contributions of each RRM to PTBP1 biological functions. Here, we review all the known literature describing the apo and RNA bound structures of each of PTBP1's RRMs, as well as the emerging literature describing the dependence of specific RNA processing events on individual RRM domains. Our goal is to provide a framework of the structure-function context upon which to facilitate the interpretation of future studies interrogating the dynamics of PTBP1 function.

MECHANISMS OF DEVELOPMENT AND REGENERATION IN HYDRA

Abstract Hydra vulgaris is a small and simple aquatic animal capable of whole-body regeneration and has negligible senescence. The entire animal, including the nervous system, is composed of about 25 cell types, and can regenerate from a fragment of tissue as small as ~300 cells. In addition, all cell types are continually renewed in the uninjured adult as part of normal homeostasis; every differentiated cell type is replaced approximately every 20 days, which likely contributes to its lack of aging. The remarkable features of Hydra are enabled by three distinct populations of stem cells that support the three lineages that make up the adult Hydra – the ectodermal epithelial lineage, the endodermal epithelial lineage, and the interstitial lineage (includes the neurons). A major goal of our laboratory is to understand the gene regulatory networks that control the specification of all Hydra cell types in the uninjured (homeostatic) state and then understand how injury triggers these differentiation pathways at unexpected locations during regeneration. Using high throughput genomics approaches such as scRNA-seq, ATAC-seq, and Cut&Tag, we have transcriptionally defined every cell type in Hydra and identified putative transcriptional regulators for each cell type. This includes the 11 neuronal subtypes that comprise the nerve net that spans the entire length of the Hydra body. We are currently leveraging these data to conduct functional testing of key putative regulators and to identify injury inputs into cell specification events during regeneration.

MicroRNA Expression Profiling Using Agilent One-Color Microarray.

MicroRNA (miRNA) expression profiling is an important tool to identify miRNA regulation in physiological or pathological states. This technique has a large number of molecular diagnostic applications, including cancer, cardiovascular and autoimmune diseases, and forensics. To date, a multitude of high-throughput genomic approaches have been developed. Here, we focus on miRNA expression profiling by microarray using SurePrint technology, providing a description of both the workflow and methods for expression profiling by Agilent One-Color Microarray.

The Enigmatic Etiology of Oculo-Auriculo-Vertebral Spectrum (OAVS): An Exploratory Gene Variant Interaction Approach in Candidate Genes.

The clinical diagnosis of oculo-auriculo-vertebral spectrum (OAVS) is established when microtia is present in association with hemifacial hypoplasia (HH) and/or ocular, vertebral, and/or renal malformations. Genetic and non-genetic factors have been associated with microtia/OAVS. Although the etiology remains unknown in most patients, some cases may have an autosomal dominant, autosomal recessive, or multifactorial inheritance. Among the possible genetic factors, gene-gene interactions may play important roles in the etiology of complex diseases, but the literature lacks related reports in OAVS patients. Therefore, we performed a gene-variant interaction analysis within five microtia/OAVS candidate genes (HOXA2, TCOF1, SALL1, EYA1 and TBX1) in 49 unrelated OAVS Mexican patients (25 familial and 24 sporadic cases). A statistically significant intergenic interaction (p-value < 0.001) was identified between variants p.(Pro1099Arg) TCOF1 (rs1136103) and p.(Leu858=) SALL1 (rs1965024). This intergenic interaction may suggest that the products of these genes could participate in pathways related to craniofacial alterations, such as the retinoic acid (RA) pathway. The absence of clearly pathogenic variants in any of the analyzed genes does not support a monogenic etiology for microtia/OAVS involving these genes in our patients. Our findings could suggest that in addition to high-throughput genomic approaches, future gene-gene interaction analyses could contribute to improving our understanding of the etiology of microtia/OAVS.

Highly rapid and diverse sex chromosome evolution in the Odorrana frog species complex.

Sex chromosomes in poikilothermal vertebrates are characterized by rapid and diverse evolution at the species or population level. Our previous study revealed that the Taiwanese frog Odorrana swinhoana (2n=26) has a unique system of multiple sex chromosomes created by three sequential translocations among chromosomes 1, 3, and 7. To reveal the evolutionary history of sex chromosomes in the Odorrana species complex, we first identified the original, homomorphic sex chromosomes, prior to the occurrence of translocations, in the ancestral-type population of O. swinhoana. Then, we extended the investigation to a closely related Japanese species, Odorrana utsunomiyaorum, which is distributed on two small islands. We used a high-throughput nuclear genomic approach to analyze single-nucleotide polymorphisms and identify the sex-linked markers. Those isolated from the O. swinhoana ancestral-type population were found to be aligned to chromosome 1 and showed male heterogamety. In contrast, almost all the sex-linked markers isolated from O. utsunomiyaorum were heterozygous in females and homozygous in males and were aligned to chromosome 9. Morphologically, we confirmed chromosome 9 to be heteromorphic in females, showing a ZZ-ZW sex determination system, in which the W chromosomes were heterochromatinized in a stripe pattern along the chromosome axis. These results indicated that after divergence of the two species, the ancestral homomorphic sex chromosome 1 underwent highly rapid and diverse evolution, i.e., sequential translocations with two autosomes in O. swinhoana, and turnover to chromosome 9 in O. utsunomiyaorum, with a transition from XY to ZW heterogamety and change to heteromorphy.

Tissue stem cells: survival of the fittest

Barrier epithelial tissues such as the skin, lung and gut are the first line of defense between our body and the outside world. As such, their stem cells must continually generate and rejuvenate their tissue, but also self‐renew to maintain the reservoir of stem cells. When the barrier has been breached, for example by wounding, the stem cells must not only repair the damaged tissue but also call to the immune system to help guard against pathogen entry. All the while the stem cells must protect themselves from a variety of assaults, including not only injury but also mechanical stress, noxious chemicals, ultraviolet radiation, and allergens that stimulate an inflammatory response. How do stem cells equip themselves to respond to these different stresses? My laboratory uses mice as a model organism and the skin epithelium as our tissue to explore the molecular mechanisms involved. We use high throughput genetic and genomic approaches to learn at a molecular level how the stem cells balance growth and differentiation in normal tissue homeostasis, and how they change their behavior and interactions with their neighbors (the stem cell ‘niche’) in response to changes in their environment. Our global objective is to apply our knowledge of the basic science of epithelial stem cells to unfold new avenues for therapeutics to treat inflammatory conditions and cancers.

Super-resolution Chromatin Visualization Using a CombinedMethod of Fluorescence In Situ Hybridization and Structured Illumination Microscopy in Solanum lycopersicum.

Chromatin organization influences gene and transposon expression, and regulates various cellular processes. Higher order chromatin structure has been widely studied using genomic approaches and microscopy imageanalyses. Chromosome conformation capture and sequencing the junction of DNA fragments enables the study of both chromatin interaction and chromosome folding. However, certain cell types are embedded in other cell types which complicate the process of studying them using high-throughput genomic approaches. To overcome this limitation, high-resolution microscopy techniques are now available to investigate chromatin organization in single cells. In this chapter, we provide a detailed protocol to prepare chromosome spreading from tomato nuclei, to label genomic loci by fluorescence in situ hybridization, and to visualize these locations at high resolution with Structured Illumination microscopy.

Virus-Induced Gene Silencing in Wheat and Related Monocot Species.

Advances made in genome sequencing projects and structural genomics are generating large repertoire of candidate genes in plants associated with specific agronomic traits. Rapid and high-throughput functional genomics approaches are therefore needed to validate the biological function of these genes especially for agronomically important crops beyond the few model plant species. This can be achieved by utilizing available gene knockout or transgenic methodologies, but these can take considerable time and effort particularly in crops with large and complex genomes such as wheat. Therefore, any tool that expedites the validation of gene function is of particular benefit especially in cereal crop plants that are genetically difficult to transform. One such reverse genetics tool is virus-induced gene silencing (VIGS) which relies on the plants' natural antiviral RNA silencing defence mechanism. VIGS is used to downregulate target gene expression in a transient manner which persists long enough to determine its effect on a specific trait. VIGS based on Barley stripe mosaic virus (BSMV) is rapid, powerful, efficient, and relatively inexpensive tool for the analysis of gene function in cereal species. Here we present detailed protocols for BSMV-mediated VIGS for robust gene silencing in bread wheat and related species.

The nedd-8 activating enzyme gene underlies genetic resistance to infectious pancreatic necrosis virus in Atlantic salmon

Genetic resistance to infectious pancreatic necrosis virus (IPNV) in Atlantic salmon is a rare example of a trait where a single locus (QTL) explains almost all of the genetic variation. Genetic marker tests based on this QTL on salmon chromosome 26 have been widely applied in selective breeding to markedly reduce the incidence of the disease. In the current study, whole genome sequencing and functional annotation approaches were applied to characterise genes and variants in the QTL region. This was complemented by an analysis of differential expression between salmon fry of homozygous resistant and homozygous susceptible genotypes challenged with IPNV. These analyses pointed to the NEDD-8 activating enzyme 1 (nae1) gene as a putative functional candidate underlying the QTL effect. The role of nae1 in IPN resistance was further assessed via CRISPR-Cas9 knockout of the nae1 gene and chemical inhibition of the nae1 protein activity in Atlantic salmon cell lines, both of which resulted in highly significant reduction in productive IPNV replication. In contrast, CRISPR-Cas9 knockout of a candidate gene previously purported to be a cellular receptor for the virus (cdh1) did not have a major impact on productive IPNV replication. These results suggest that nae1 is the causative gene underlying the major QTL affecting resistance to IPNV in salmon, provide further evidence for the critical role of neddylation in host-pathogen interactions, and highlight the value in combining high-throughput genomics approaches with targeted genome editing to understand the genetic basis of disease resistance.

Penile Cancer-Derived Cells Molecularly Characterized as Models to Guide Targeted Therapies.

Penile cancer (PeCa) is a common disease in poor and developing countries, showing high morbidity rates. Despite the recent progress in understanding the molecular events involved in PeCa, the lack of well-characterized in vitro models precludes new advances in anticancer drug development. Here we describe the establishment of five human primary penile cancer-derived cell cultures, including two epithelial and three cancer-associated fibroblast (CAF) cells. Using high-throughput genomic approaches, we found that the epithelial PeCa derived- cells recapitulate the molecular alterations of their primary tumors and present the same deregulated signaling pathways. The differentially expressed genes and proteins identified are components of key oncogenic pathways, including EGFR and PI3K/AKT/mTOR. We showed that epithelial PeCa derived cells presented a good response to cisplatin, a common therapeutic approach used in PeCa patients. The growth of a PeCa-derived cell overexpressing EGFR was inhibited by EGFR inhibitors (cetuximab, gefitinib, and erlotinib). We also identified CAF signature markers in three PeCa-derived cells with fibroblast-like morphology, indicating that those cells are suitable models for PeCa microenvironment studies. We thus demonstrate the utility of PeCa cell models to dissect mechanisms that promote penile carcinogenesis, which are useful models to evaluate therapeutic approaches for the disease.

New trends in bioassessment of aquatic ecosystems: from organisms to DNA-based metrics

Traditionally, the biological quality of aquatic ecosystems is assessed using selected groups of organisms that can be identified morphologically. Recent advances in high-throughput genomic approaches offered new opportunities to monitor biodiversity and assess ecological status using DNA barcoding and metabarcoding. The DNA-based tools have been used in three different ways: (1) to replace morphological identification of biological quality elements in existing biotic indices, (2) to develop new molecular indices based on morphologically inconspicuous groups of potential environmental indicators, and (3) to predict biotic indices from environmental DNA datasets using machine learning methods (Pawlowski et al. 2018). The next steps need to take advantages and challenges of these different approaches into account in view of their future application in routine bioassessment.The Working Group 2 of DNAqua-Net, Biotic Indices & Metrics, has worked with several task forces tackling different organism groups (fish, macroinvertebrates, diatoms, bacteria, protists, meiofauna), because challenges have been shown to be quite different dependent on the target organisms Kahlert et al. 2019. For the fish the eDNA-metabarcoding methods are well developed and give very good results in terms of species detection. The important question is to see if the semi-quantitative data retrieved from the metabarcoding (proportion in eDNA sequences) could be translated to proportions in biomass/numbers that are now used in many indices. The fish researchers are trying to fit these data in, but some correction factors might be needed to correct for differences between molecular and conventional methodsRegarding the macroinvertebrates, much discussion regarding index development was focusing on the importance of abundance measurements, and it was tested how existing indices would perform if barcoding data would be used instead of morphological data. Still discussion is ongoing on several technical issues, including the use of preservative for DNA extraction from bulk samples, the choice of primers for PCR amplification and the incompleteness of reference databases which impedes the correct assignment of eDNA sequences. Also minimum standards for routine operation are still missing.The diatom group has worked much on practical issues, starting a large initiative to compare diatom metabarcoding protocols used in routine freshwater biomonitoring for standardization (Bailet et al. 2019, Keck et al. 2018, Vasselon et al. 2017). With diatoms, all three approaches to develop molecular indices have been tested and seem promising, i.e. using existing indices with taxa names derived by matching sequences with reference databases, developing new indices based on molecular data only with traditional fixed scores, and using machine-learning techniques (Bailet et al. 2020, Vasselon et al. 2018, Tapolczai et al. 2019, Keck et al. 2018) The micro- and meiobiota group has worked towards an inclusion of microorganisms into aquatic assessment, because the microbial community dynamics are a missing link important for our understanding of rapid changes in the structure and function of aquatic ecosystems, and should be addressed in the future environmental monitoring of freshwater ecosystems (Sagova-Mareckova et al. 2021). Another focus was on how sediment DNA analysis can be integrated into stated goals of routine monitoring applications. It has been an interesting journey, and we WG2 coordinators would like to thank all the people for their engagement! Keep up the good work!

Catalyzing Clinical Implementation of Pharmacogenomics and Personalized Medicine Interventions in Africa

Pharmacogenomics is considered to be the low-hanging fruit in the tree of genomic medicine with numerous examples of its successful implementation in the clinic. In this perspective, we provide details about the potential clinical application of pharmacogenomics in African populations by using relevant drug cases and high-throughput genomics approaches; involving numerous countries and stakeholders; and most importantly exploiting the existing knowledge of respective large-scale initiatives. We emphasize on the necessity of constructing appropriate frameworks for government policies in African countries. We also provide input about different initiatives in the field of genomics medicine implementation in Africa, not only for their potential for synergy and collaboration among them, but also as models for replication in other regions worldwide, aiming for healthcare improvement.

Local, but not long-distance dispersal of penguin ticks between two sub-Antarctic islands

Advances in high throughput genomic approaches are enabling the accurate appraisal of movements of diverse species, previously considered intractable. The impact of long-distance dispersal and distribution changes on species interactions (such as host-parasite interactions) is of particular importance as attempts are made to project how ecosystems will shift under environmental change. The sub-Antarctic region, comprising isolated islands separated by hundreds to thousands of kilometres of open ocean, presents an ideal model system for studying long-distance dispersal, distribution, and ecosystem change. Here we used genomic methods to determine the extent of movement of penguin ticks (Ixodes uriae) among different host species, and among penguin colonies at small (within an island) and large (among islands separated by >6000 km) scales, in the sub-Antarctic region. Our results suggest that I. uriae ticks may be readily shared between distantly related penguin species with similar phenology, but indicate that – as inferred by previous research – ticks are less likely to be shared between flighted and non-flighted sea- birds. We also find evidence for small-scale movements of penguin ticks with their hosts, but no evidence for movements between islands separated by thousands of kilometers of open ocean. These inferred limitations to penguin tick movement could be the result of restricted host movements or the inability of penguin ticks to survive extended trips at sea. Our findings help elucidate parasite-host dynamics, with implications for host health and persistence in a region experiencing rapid environmental change.

Abstract IA04: Stem cells: Coping with stress and cancer

Abstract Adult tissue stem cells have the ability to self-renew long term and differentiate into one or more tissues. Many stem cells are used sparingly to replenish cells during normal homeostasis. However, even stem cells that are quiescent must be able to respond quickly to injury in order to fuel rapid tissue regeneration. How stem cells balance self-renewal and differentiation is of fundamental importance to our understanding of normal tissue maintenance and wound repair. The regulatory circuitry governing this normal balancing act must be intricately regulated in normal homeostasis and then transiently altered to cope with injury responses. Increasing evidence suggests that the mechanism goes awry in inflammation and becomes hijacked in cancers. Skin epithelium is an excellent model system to understand how stem cells remain quiescent during times of minimal wear and tear, how these cells become mobilized during the cyclical bouts of hair growth and wound repair, and how the normal process of stem cell activation goes awry in cancer and inflammation. We have identified and characterized at a molecular level the skin’s stem cells and shown that they reside in distinct niches that impart to the stem cells their behavior both in task and in the molecular properties they display. We use high-throughput genetic and genomic approaches to dissect at a molecular level how stem cell interactions with their niches differ in homeostasis, wound repair, and inflammation, and how heterogeneity in the tumor microenvironment can confer on stem cells resistance to chemotherapy and immunotherapy. Our global objective is to apply our knowledge of the basic science of epithelial stem cells to unfold new avenues for therapeutics. Citation Format: Elaine Fuchs. Stem cells: Coping with stress and cancer [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr IA04.

A Novel Screening Approach for the Dissection of Cellular Regulatory Networks of NF-κB Using Arrayed CRISPR gRNA Libraries.

CRISPR/Cas9 is increasingly being used as a tool to prosecute functional genomic screens. However, it is not yet possible to apply the approach at scale across a full breadth of cell types and endpoints. In order to address this, we developed a novel and robust workflow for array-based lentiviral CRISPR/Cas9 screening. We utilized a β-lactamase reporter gene assay to investigate mediators of TNF-α-mediated NF-κB signaling. The system was adapted for CRISPR/Cas9 through the development of a cell line stably expressing Cas9 and application of a lentiviral gRNA library comprising mixtures of four gRNAs per gene. We screened a 743-gene kinome library whereupon hits were independently ranked by percent inhibition, Z' score, strictly standardized mean difference, and T statistic. A consolidated and optimized ranking was generated using Borda-based methods. Screening data quality was above acceptable limits (Z' ≥ 0.5). In order to determine the contribution of individual gRNAs and to better understand false positives and negatives, a subset of gRNAs, against 152 genes, were profiled in singlicate format. We highlight the use of known reference genes and high-throughput, next-generation amplicon and RNA sequencing to assess screen data quality. Screening with singlicate gRNAs was more successful than screening with mixtures at identifying genes with known regulatory roles in TNF-α-mediated NF-κB signaling and was found to be superior to previous RNAi-based methods. These results add to the available data on TNF-α-mediated NF-κB signaling and establish a high-throughput functional genomic screening approach, utilizing a vector-based arrayed gRNA library, applicable across a wide variety of endpoints and cell types at a genome-wide scale.

Small nucleolar RNAs: continuing identification of novel members and increasing diversity of their molecular mechanisms of action.

Identified five decades ago amongst the most abundant cellular RNAs, small nucleolar RNAs (snoRNAs) were initially described as serving as guides for the methylation and pseudouridylation of ribosomal RNA through direct base pairing. In recent years, however, increasingly powerful high-throughput genomic approaches and strategies have led to the discovery of many new members of the family and surprising diversity in snoRNA functionality and mechanisms of action. SnoRNAs are now known to target RNAs of many biotypes for a wider range of modifications, interact with diverse binding partners, compete with other binders for functional interactions, recruit diverse players to targets and affect protein function and accessibility through direct interaction. This mini-review presents the continuing characterization of the snoRNome through the identification of new snoRNA members and the discovery of their mechanisms of action, revealing a highly versatile noncoding family playing central regulatory roles and connecting the main cellular processes.

Old Trade, New Tricks: Insights into the Spontaneous Mutation Process from the Partnering of Classical Mutation Accumulation Experiments with High-Throughput Genomic Approaches.

Mutations spawn genetic variation which, in turn, fuels evolution. Hence, experimental investigations into the rate and fitness effects of spontaneous mutations are central to the study of evolution. Mutation accumulation (MA) experiments have served as a cornerstone for furthering our understanding of spontaneous mutations for four decades. In the pregenomic era, phenotypic measurements of fitness-related traits in MA lines were used to indirectly estimate key mutational parameters, such as the genomic mutation rate, new mutational variance per generation, and the average fitness effect of mutations. Rapidly emerging next-generating sequencing technology has supplanted this phenotype-dependent approach, enabling direct empirical estimates of the mutation rate and a more nuanced understanding of the relative contributions of different classes of mutations to the standing genetic variation. Whole-genome sequencing of MA lines bears immense potential to provide a unified account of the evolutionary process at multiple levels—the genetic basis of variation, and the evolutionary dynamics of mutations under the forces of selection and drift. In this review, we have attempted to synthesize key insights into the spontaneous mutation process that are rapidly emerging from the partnering of classical MA experiments with high-throughput sequencing, with particular emphasis on the spontaneous rates and molecular properties of different mutational classes in nuclear and mitochondrial genomes of diverse taxa, the contribution of mutations to the evolution of gene expression, and the rate and stability of transgenerational epigenetic modifications. Future advances in sequencing technologies will enable greater species representation to further refine our understanding of mutational parameters and their functional consequences.