Whole-body imaging with single-cell resolution by tissue decolorization.

The development of whole-body imaging at single-cell resolution enables system-level approaches to studying cellular circuits in organisms. Previous clearing methods focused on homogenizing mismatched refractive indices of individual tissues, enabling reductions in opacity but falling short of achieving transparency. Here, we show that an aminoalcohol decolorizes blood by efficiently eluting the heme chromophore from hemoglobin. Direct transcardial perfusion of an aminoalcohol-containing cocktail that we previously termed CUBIC coupled with a 10 day to 2 week clearing protocol decolorized and rendered nearly transparent almost all organs of adult mice as well as the entire body of infant and adult mice. This CUBIC-perfusion protocol enables rapid whole-body and whole-organ imaging at single-cell resolution by using light-sheet fluorescent microscopy. The CUBIC protocol is also applicable to 3D pathology, anatomy, and immunohistochemistry of various organs. These results suggest that whole-body imaging of colorless tissues at high resolution will contribute to organism-level systems biology.

Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis.

Systems-level identification and analysis of cellular circuits in the brain will require the development of whole-brain imaging with single-cell resolution. To this end, we performed comprehensive chemical screening to develop a whole-brain clearing and imaging method, termed CUBIC (clear, unobstructed brain imaging cocktails and computational analysis). CUBIC is a simple and efficient method involving the immersion of brain samples in chemical mixtures containing aminoalcohols, which enables rapid whole-brain imaging with single-photon excitation microscopy. CUBIC is applicable to multicolor imaging of fluorescent proteins or immunostained samples in adult brains and is scalable from a primate brain to subcellular structures. We also developed a whole-brain cell-nuclear counterstaining protocol and a computational image analysis pipeline that, together with CUBIC reagents, enable the visualization and quantification of neural activities induced by environmental stimulation. CUBIC enables time-course expression profiling of whole adult brains with single-cell resolution.

Circadian ribosome profiling reveals a role for the Period2 upstream open reading frame in sleep.

Many mammalian proteins have circadian cycles of production and degradation, and many of these rhythms are altered posttranscriptionally. We used ribosome profiling to examine posttranscriptional control of circadian rhythms by quantifying RNA translation in the liver over a 24-h period from circadian-entrained mice transferred to constant darkness conditions and by comparing ribosome binding levels to protein levels for 16 circadian proteins. We observed large differences in ribosome binding levels compared to protein levels, and we observed delays between peak ribosome binding and peak protein abundance. We found extensive binding of ribosomes to upstream open reading frames (uORFs) in circadian mRNAs, including the core clock gene Period2 (Per2). An increase in the number of uORFs in the 5'UTR was associated with a decrease in ribosome binding in the main coding sequence and a reduction in expression of synthetic reporter constructs. Mutation of the Per2 uORF increased luciferase and fluorescence reporter expression in 3T3 cells and increased luciferase expression in PER2:LUC MEF cells. Mutation of the Per2 uORF in mice increased Per2 mRNA expression, enhanced ribosome binding on Per2, and reduced total sleep time compared to that in wild-type mice. These results suggest that uORFs affect mRNA posttranscriptionally, which can impact physiological rhythms and sleep.

Role of the bicarbonate transporter SLC4γ in stony-coral skeleton formation and evolution.

Coral reefs are highly diverse ecosystems of immense ecological, economic, and aesthetic importance built on the calcium-carbonate-based skeletons of stony corals. The formation of these skeletons is threatened by increasing ocean temperatures and acidification, and a deeper understanding of the molecular mechanisms involved may assist efforts to mitigate the effects of such anthropogenic stressors. In this study, we focused on the role of the predicted bicarbonate transporter SLC4γ, which was suggested in previous studies to be a product of gene duplication and to have a role in coral-skeleton formation. Our comparative-genomics study using 30 coral species and 15 outgroups indicates that SLC4γ is present throughout the stony corals, but not in their non-skeleton-forming relatives, and apparently arose by gene duplication at the onset of stony-coral evolution. Our expression studies show that SLC4γ, but not the closely related and apparently ancestral SLC4ß, is highly upregulated during coral development coincident with the onset of skeleton deposition. Moreover, we show that juvenile coral polyps carrying CRISPR/Cas9-induced mutations in SLC4γ are defective in skeleton formation, with the severity of the defect in individual animals correlated with their frequencies of SLC4γ mutations. Taken together, the results suggest that the evolution of the stony corals involved the neofunctionalization of the newly arisen SLC4γ for a unique role in the provision of concentrated bicarbonate for calcium-carbonate deposition. The results also demonstrate the feasibility of reverse-genetic studies of ecologically important traits in adult corals.

Reduced thermal tolerance in a coral carrying CRISPR-induced mutations in the gene for a heat-shock transcription factor.

Reef-building corals are keystone species that are threatened by anthropogenic stresses including climate change. To investigate corals' responses to stress and other aspects of their biology, numerous genomic and transcriptomic studies have been performed, generating many hypotheses about the roles of particular genes and molecular pathways. However, it has not generally been possible to test these hypotheses rigorously because of the lack of genetic tools for corals or closely related cnidarians. CRISPR technology seems likely to alleviate this problem. Indeed, we show here that microinjection of single-guide RNA/Cas9 ribonucleoprotein complexes into fertilized eggs of the coral Acropora millepora can produce a sufficiently high frequency of mutations to detect a clear phenotype in the injected generation. Based in part on experiments in a sea-anemone model system, we targeted the gene encoding Heat Shock Transcription Factor 1 (HSF1) and obtained larvae in which >90% of the gene copies were mutant. The mutant larvae survived well at 27 °C but died rapidly at 34 °C, a temperature that did not produce detectable mortality over the duration of the experiment in wild-type (WT) larvae or larvae injected with Cas9 alone. We conclude that HSF1 function (presumably its induction of genes in response to heat stress) plays an important protective role in corals. More broadly, we conclude that CRISPR mutagenesis in corals should allow wide-ranging and rigorous tests of gene function in both larval and adult corals.

Comprehensive, integrated, and phased whole-genome analysis of the primary ENCODE cell line K562.

K562 is widely used in biomedical research. It is one of three tier-one cell lines of ENCODE and also most commonly used for large-scale CRISPR/Cas9 screens. Although its functional genomic and epigenomic characteristics have been extensively studied, its genome sequence and genomic structural features have never been comprehensively analyzed. Such information is essential for the correct interpretation and understanding of the vast troves of existing functional genomics and epigenomics data for K562. We performed and integrated deep-coverage whole-genome (short-insert), mate-pair, and linked-read sequencing as well as karyotyping and array CGH analysis to identify a wide spectrum of genome characteristics in K562: copy numbers (CN) of aneuploid chromosome segments at high-resolution, SNVs and indels (both corrected for CN in aneuploid regions), loss of heterozygosity, megabase-scale phased haplotypes often spanning entire chromosome arms, structural variants (SVs), including small and large-scale complex SVs and nonreference retrotransposon insertions. Many SVs were phased, assembled, and experimentally validated. We identified multiple allele-specific deletions and duplications within the tumor suppressor gene FHIT Taking aneuploidy into account, we reanalyzed K562 RNA-seq and whole-genome bisulfite sequencing data for allele-specific expression and allele-specific DNA methylation. We also show examples of how deeper insights into regulatory complexity are gained by integrating genomic variant information and structural context with functional genomics and epigenomics data. Furthermore, using K562 haplotype information, we produced an allele-specific CRISPR targeting map. This comprehensive whole-genome analysis serves as a resource for future studies that utilize K562 as well as a framework for the analysis of other cancer genomes.

Haplotype-resolved and integrated genome analysis of the cancer cell line HepG2.

HepG2 is one of the most widely used human cancer cell lines in biomedical research and one of the main cell lines of ENCODE. Although the functional genomic and epigenomic characteristics of HepG2 are extensively studied, its genome sequence has never been comprehensively analyzed and higher order genomic structural features are largely unknown. The high degree of aneuploidy in HepG2 renders traditional genome variant analysis methods challenging and partially ineffective. Correct and complete interpretation of the extensive functional genomics data from HepG2 requires an understanding of the cell line's genome sequence and genome structure. Using a variety of sequencing and analysis methods, we identified a wide spectrum of genome characteristics in HepG2: copy numbers of chromosomal segments at high resolution, SNVs and Indels (corrected for aneuploidy), regions with loss of heterozygosity, phased haplotypes extending to entire chromosome arms, retrotransposon insertions and structural variants (SVs) including complex and somatic genomic rearrangements. A large number of SVs were phased, sequence assembled and experimentally validated. We re-analyzed published HepG2 datasets for allele-specific expression and DNA methylation and assembled an allele-specific CRISPR/Cas9 targeting map. We demonstrate how deeper insights into genomic regulatory complexity are gained by adopting a genome-integrated framework.

A benchmark of computational CRISPR-Cas9 guide design methods.

The popularity of CRISPR-based gene editing has resulted in an abundance of tools to design CRISPR-Cas9 guides. This is also driven by the fact that designing highly specific and efficient guides is a crucial, but not trivial, task in using CRISPR for gene editing. Here, we thoroughly analyse the performance of 18 design tools. They are evaluated based on runtime performance, compute requirements, and guides generated. To achieve this, we implemented a method for auditing system resources while a given tool executes, and tested each tool on datasets of increasing size, derived from the mouse genome. We found that only five tools had a computational performance that would allow them to analyse an entire genome in a reasonable time, and without exhausting computing resources. There was wide variation in the guides identified, with some tools reporting every possible guide while others filtered for predicted efficiency. Some tools also failed to exclude guides that would target multiple positions in the genome. We also considered two collections with over a thousand guides each, for which experimental data is available. There is a lot of variation in performance between the datasets, but the relative order of the tools is partially conserved. Importantly, the most striking result is a lack of consensus between the tools. Our results show that CRISPR-Cas9 guide design tools need further work in order to achieve rapid whole-genome analysis and that improvements in guide design will likely require combining multiple approaches.

Improving CRISPR guide design with consensus approaches.

BACKGROUND: CRISPR-based systems are playing an important role in modern genome engineering. A large number of computational methods have been developed to assist in the identification of suitable guides. However, there is only limited overlap between the guides that each tool identifies. This can motivate further development, but also raises the question of whether it is possible to combine existing tools to improve guide design. RESULTS: We considered nine leading guide design tools, and their output when tested using two sets of guides for which experimental validation data is available. We found that consensus approaches were able to outperform individual tools. The best performance (with a precision of up to 0.912) was obtained when combining four of the tools and accepting all guides selected by at least three of them. CONCLUSIONS: These results can be used to improve CRISPR-based studies, but also to guide further tool development. However, they only provide a short-term solution as the time and computational resources required to run four tools may be impractical in certain applications.

Rapid analysis of metagenomic data using signature-based clustering.

BACKGROUND: Sequencing highly-variable 16S regions is a common and often effective approach to the study of microbial communities, and next-generation sequencing (NGS) technologies provide abundant quantities of data for analysis. However, the speed of existing analysis pipelines may limit our ability to work with these quantities of data. Furthermore, the limited coverage of existing 16S databases may hamper our ability to characterise these communities, particularly in the context of complex or poorly studied environments. RESULTS: In this article we present the SigClust algorithm, a novel clustering method involving the transformation of sequence reads into binary signatures. When compared to other published methods, SigClust yields superior cluster coherence and separation of metagenomic read data, while operating within substantially reduced timeframes. We demonstrate its utility on published Illumina datasets and on a large collection of labelled wound reads sourced from patients in a wound clinic. The temporal analysis is based on tracking the dominant clusters of wound samples over time. The analysis can identify markers of both healing and non-healing wounds in response to treatment. Prominent clusters are found, corresponding to bacterial species known to be associated with unfavourable healing outcomes, including a number of strains of Staphylococcus aureus. CONCLUSIONS: SigClust identifies clusters rapidly and supports an improved understanding of the wound microbiome without reliance on a reference database. The results indicate a promising use for a SigClust-based pipeline in wound analysis and prediction, and a possible novel method for wound management and treatment.

A mammalian circadian clock model incorporating daytime expression elements.

Models of the mammalian clock have traditionally been based around two feedback loops-the self-repression of Per/Cry by interfering with activation by BMAL/CLOCK, and the repression of Bmal/Clock by the REV-ERB proteins. Recent experimental evidence suggests that the D-box, a transcription factor binding site associated with daytime expression, plays a larger role in clock function than has previously been understood. We present a simplified clock model that highlights the role of the D-box and illustrate an approach for finding maximum-entropy ensembles of model parameters, given experimentally imposed constraints. Parameter variability can be mitigated using prior probability distributions derived from genome-wide studies of cellular kinetics. Our model reproduces predictions concerning the dual regulation of Cry1 by the D-box and Rev-ErbA/ROR response element (RRE) promoter elements and allows for ensemble-based predictions of phase response curves (PRCs). Nonphotic signals such as Neuropeptide Y (NPY) may act by promoting Cry1 expression, whereas photic signals likely act by stimulating expression from the E/E' box. Ensemble generation with parameter probability restraints reveals more about a model's behavior than a single optimal parameter set.

Predicting Carcass Weight of Grass-Fed Beef Cattle before Slaughter Using Statistical Modelling.

Gaining insights into the utilization of farm-level data for decision-making within the beef industry is vital for improving production and profitability. In this study, we present a statistical model to predict the carcass weight (CW) of grass-fed beef cattle at different stages before slaughter using historical cattle data. Models were developed using two approaches: boosted regression trees and multiple linear regression. A sample of 2995 grass-fed beef cattle from 3 major properties in Northern Australia was used in the modeling. Four timespans prior to the slaughter, i.e., 1 month, 3 months, 9-10 months, and at weaning, were considered in the predictive modelling. Seven predictors, i.e., weaning weight, weight gain since weaning to each stage before slaughter, time since weaning to each stage before slaughter, breed, sex, weaning season (wet and dry), and property, were used as the potential predictors of the CW. To assess the predictive performance in each scenario, a test set which was not used to train the models was utilized. The results showed that the CW of the cattle was strongly associated with the animal's body weight at each stage before slaughter. The results showed that the CW can be predicted with a mean absolute percentage error (MAPE) of 4% (~12-16 kg) at three months before slaughter. The predictive error increased gradually when moving away from the slaughter date, e.g., the prediction error at weaning was ~8% (~20-25 kg). The overall predictive performances of the two statistical approaches was approximately similar, and neither of the models substantially outperformed each other. Predicting the CW in advance of slaughter may allow farmers to adequately prepare for forthcoming needs at the farm level, such as changing husbandry practices, control inventory, and estimate price return, thus allowing them to maximize the profitability of the industry.

Rapid Whole-Genome Identification of High Quality CRISPR Guide RNAs with the Crackling Method.

The design of CRISPR-Cas9 guide RNAs is not trivial and is a computationally demanding task. Design tools need to identify target sequences that will maximize the likelihood of obtaining the desired cut, while minimizing off-target risk. There is a need for a tool that can meet both objectives while remaining practical to use on large genomes. In this study, we present Crackling, a new method that is more suitable for meeting these objectives. We test its performance on 12 genomes and on data from validation studies. Crackling maximizes guide efficiency by combining multiple scoring approaches. On experimental data, the guides it selects are better than those selected by others. It also incorporates Inverted Signature Slice Lists (ISSL) for faster off-target scoring. ISSL provides a gain of an order of magnitude in speed compared with other popular tools, such as Cas-OFFinder, Crisflash, and FlashFry, while preserving the same level of accuracy. Overall, this makes Crackling a faster and better method to design guide RNAs at scale. Crackling is available at https://github.com/bmds-lab/Crackling under the Berkeley Software Distribution (BSD) 3-Clause license.

Metagenomic Geolocation Using Read Signatures.

We present a novel approach to the Metagenomic Geolocation Challenge based on random projection of the sample reads from each location. This approach explores the direct use of k-mer composition to characterise samples so that we can avoid the computationally demanding step of aligning reads to available microbial reference sequences. Each variable-length read is converted into a fixed-length, k-mer-based read signature. Read signatures are then clustered into location signatures which provide a more compact characterisation of the reads at each location. Classification is then treated as a problem in ranked retrieval of locations, where signature similarity is used as a measure of similarity in microbial composition. We evaluate our approach using the CAMDA 2020 Challenge dataset and obtain promising results based on nearest neighbour classification. The main findings of this study are that k-mer representations carry sufficient information to reveal the origin of many of the CAMDA 2020 Challenge metagenomic samples, and that this reference-free approach can be achieved with much less computation than methods that need reads to be assigned to operational taxonomic units-advantages which become clear through comparison to previously published work on the CAMDA 2019 Challenge data.

Molecular and Serologic Diagnostic Technologies for SARS-CoV-2.

The COVID-19 pandemic has presented many challenges that have spurred biotechnological research to address specific problems. Diagnostics is one area where biotechnology has been critical. Diagnostic tests play a vital role in managing a viral threat by facilitating the detection of infected and/or recovered individuals. From the perspective of what information is provided, these tests fall into two major categories, molecular and serological. Molecular diagnostic techniques assay whether a virus is present in a biological sample, thus making it possible to identify individuals who are currently infected. Additionally, when the immune system is exposed to a virus, it responds by producing antibodies specific to the virus. Serological tests make it possible to identify individuals who have mounted an immune response to a virus of interest and therefore facilitate the identification of individuals who have previously encountered the virus. These two categories of tests provide different perspectives valuable to understanding the spread of SARS-CoV-2. Within these categories, different biotechnological approaches offer specific advantages and disadvantages. Here we review the categories of tests developed for the detection of the SARS-CoV-2 virus or antibodies against SARS-CoV-2 and discuss the role of diagnostics in the COVID-19 pandemic.

Complexity and high-end computing in biology and medicine.

Biomedical systems involve a large number of entities and intricate interactions between these. Their direct analysis is, therefore, difficult, and it is often necessary to rely on computational models. These models require significant resources and parallel computing solutions. These approaches are particularly suited, given parallel aspects in the nature of biomedical systems. Model hybridisation also permits the integration and simultaneous study of multiple aspects and scales of these systems, thus providing an efficient platform for multidisciplinary research.

Crowdsourcing digital health measures to predict Parkinson's disease severity: the Parkinson's Disease Digital Biomarker DREAM Challenge.

Consumer wearables and sensors are a rich source of data about patients' daily disease and symptom burden, particularly in the case of movement disorders like Parkinson's disease (PD). However, interpreting these complex data into so-called digital biomarkers requires complicated analytical approaches, and validating these biomarkers requires sufficient data and unbiased evaluation methods. Here we describe the use of crowdsourcing to specifically evaluate and benchmark features derived from accelerometer and gyroscope data in two different datasets to predict the presence of PD and severity of three PD symptoms: tremor, dyskinesia, and bradykinesia. Forty teams from around the world submitted features, and achieved drastically improved predictive performance for PD status (best AUROC = 0.87), as well as tremor- (best AUPR = 0.75), dyskinesia- (best AUPR = 0.48) and bradykinesia-severity (best AUPR = 0.95).

Cell type-dependent, infection-induced, aberrant DNA methylation in gastric cancer.

Epigenetic changes correspond to heritable modifications of the chromatin structure, which do not involve any alteration of the DNA sequence but nonetheless affect gene expression. These mechanisms play an important role in cell differentiation, but aberrant occurrences are also associated with a number of diseases, including cancer and neural development disorders. In particular, aberrant DNA methylation induced by H. Pylori has been found to be a significant risk factor in gastric cancer. To investigate the sensitivity of different genes and cell types to this infection, a computational model of methylation in gastric crypts is developed. In this article, we review existing results from physical experiments and outline their limitations, before presenting the computational model and investigating the influence of its parameters.

Recursive module extraction using Louvain and PageRank.

Biological networks are highly modular and contain a large number of clusters, which are often associated with a specific biological function or disease. Identifying these clusters, or modules, is therefore valuable, but it is not trivial. In this article we propose a recursive method based on the Louvain algorithm for community detection and the PageRank algorithm for authoritativeness weighting in networks. PageRank is used to initialise the weights of nodes in the biological network; the Louvain algorithm with the Newman-Girvan criterion for modularity is then applied to the network to identify modules. Any identified module with more than k nodes is further processed by recursively applying PageRank and Louvain, until no module contains more than k nodes (where k is a parameter of the method, no greater than 100). This method is evaluated on a heterogeneous set of six biological networks from the Disease Module Identification DREAM Challenge. Empirical findings suggest that the method is effective in identifying a large number of significant modules, although with substantial variability across restarts of the method.

Promises and Challenges in the Use of Consumer-Grade Devices for Sleep Monitoring.

The market for smartphones, smartwatches, and wearable devices is booming. In recent years, individuals and researchers have used these devices as additional tools to monitor and track sleep, physical activity, and behavior. Their use in sleep research and clinical applications could address the difficulties in scaling up studies that rely on polysomnography, the gold-standard. However, the use of commercial devices for large-scale sleep studies is not without challenges. With this in mind, this paper presents an extensive review of sleep monitoring systems and the techniques used in their development. We also discuss their performance in terms of reliability and validity, and consider the needs and expectations of users, whether they are experts, patients, or the general public. Through this review, we highlight a number of challenges with current studies: a lack of standard evaluation methods for consumer-grade devices (e.g., reliability and validity assessment); limitations in the populations studied; consumer expectations of monitoring devices; constraints on the resources of consumer-grade devices (e.g., power consumption).