A corpus of GA4GH Phenopackets: case-level phenotyping for genomic diagnostics and discovery.

The Global Alliance for Genomics and Health (GA4GH) Phenopacket Schema was released in 2022 and approved by ISO as a standard for sharing clinical and genomic information about an individual, including phenotypic descriptions, numerical measurements, genetic information, diagnoses, and treatments. A phenopacket can be used as an input file for software that supports phenotype-driven genomic diagnostics and for algorithms that facilitate patient classification and stratification for identifying new diseases and treatments. There has been a great need for a collection of phenopackets to test software pipelines and algorithms. Here, we present phenopacket-store. Version 0.1.12 of phenopacket-store includes 4916 phenopackets representing 277 Mendelian and chromosomal diseases associated with 236 genes, and 2872 unique pathogenic alleles curated from 605 different publications. This represents the first large-scale collection of case-level, standardized phenotypic information derived from case reports in the literature with detailed descriptions of the clinical data and will be useful for many purposes, including the development and testing of software for prioritizing genes and diseases in diagnostic genomics, machine learning analysis of clinical phenotype data, patient stratification, and genotype-phenotype correlations. This corpus also provides best-practice examples for curating literature-derived data using the GA4GH Phenopacket Schema.

Measuring emergency physician productivity and work patterns.

I propose a methodology to compare individual emergency physician (EP) work patterns. This is intended to generate discussion within the specialty. A work pattern graph shows individual EP productivity and, assuming the EPs case selection is similar, can be used to compare group activity. Using a simple mathematical model, an averaged calculation can be made of the number of patients needed to be seen by each treating clinician during a standard shift.

Array-based dynamic allele specific hybridization (Array-DASH): Optimization-free microarray processing for multiple simultaneous genomic assays.

We report proof-of-principle experiments regarding a dynamic microarray protocol enabling accurate and semi-quantitative DNA analysis for re-sequencing, fingerprinting and genotyping. Single-stranded target molecules hybridise to surface-bound probes during initial gradual cooling with high-fidelity. Real-time tracking of target denaturation (via fluorescence) during a 'dynamic' gradual heating phase permits 'melt-curve' analysis. The probe most closely matching the target sequence is identified based on the highest melting temperature. We demonstrated a >99% re-sequencing accuracy and a potential detection rate of 1% for SNPs. Experiments employing Hypericum ribosomal ITS regions and HIV genomes illustrated a reliable detection level of 5% plus simultaneous re-sequencing and genotyping. Such performance suggests a range of potential real-world applications involving rapid sequence interrogation, for example, in the Covid-19 pandemic. Guidance is offered towards the development of a commercial platform and dedicated software required to bring this technique into mainstream science.

Verifying nomenclature of DNA variants in submitted manuscripts: Guidance for journals.

Documenting variation in our genomes is important for research and clinical care. Accuracy in the description of DNA variants is therefore essential. To address this issue, the Human Variome Project convened a committee to evaluate the feasibility of requiring authors to verify that all variants submitted for publication complied with a widely accepted standard for description. After a pilot study of two journals, the committee agreed that requiring authors to verify that variants complied with Human Genome Variation Society nomenclature is a reasonable step toward standardizing the worldwide inventory of human variation.

Using interactive digital notebooks for bioscience and informatics education.

Interactive digital notebooks provide an opportunity for researchers and educators to carry out data analysis and report the results in a single digital format. Further to just being digital, the format allows for rich content to be created in order to interact with the code and data contained in such a notebook to form an educational narrative. This primer introduces some of the fundamental aspects involved in using Jupyter notebooks in an educational setting for teaching in the bio/health informatics disciplines. We also provide 2 case studies that detail how we used Jupyter notebooks to teach non-coders programming skills on a blended Master's degree module for a Health Informatics programme and a fully online distance learning unit on Programming for a postgraduate certificate (PG Cert) in Clinical Bioinformatics with a more technical audience.

The first reported case of the rare mitochondrial haplotype H4a1 in ancient Egypt.

Takabuti, was a female who lived in ancient Egypt during the 25th Dynasty, c.660 BCE. Her mummified remains were brought to Belfast, Northern Ireland, in 1834 and are currently displayed in the Ulster Museum. To gain insight into Takabuti's ancestry, we used deep sampling of vertebral bone, under X-ray control, to obtain non-contaminated bone tissue from which we extracted ancient DNA (aDNA) using established protocols. We targeted the maternally inherited mitochondrial DNA (mtDNA), known to be highly informative for human ancestry, and identified 38 single nucleotide variants using next generation sequencing. The specific combination of these SNVs suggests that Takabuti belonged to mitochondrial haplogroup H4a1. Neither H4 nor H4a1 have been reported in ancient Egyptian samples, prior to this study. The modern distribution of H4a1 is rare and sporadic and has been identified in areas including the Canary Islands, southern Iberia and the Lebanon. H4a1 has also been reported in ancient samples from Bell Beaker and Unetice contexts in Germany, as well as Bronze Age Bulgaria. We believe that this is an important finding because first, it adds to the depth of knowledge about the distribution of the H4a1 haplogroup in existing mtDNA, thus creating a baseline for future occurrences of this haplogroup in ancient Egyptian remains. Second, it is of great importance for archaeological sciences, since a predominantly European haplogroup has been identified in an Egyptian individual in Southern Egypt, prior to the Roman and Greek influx (332BCE).

Embedding best transfusion practice and blood management in neonatal intensive care.

BACKGROUND: Transfusion is a common procedure for neonates receiving intensive care management. Recognising a paucity of patient blood management (PBM) programmes in neonates, we aimed to embed blood management and best transfusion principles in the neonatal intensive care unit (NICU) by aligning local policies, providing targeted education and partnering with parents. METHODS: Practice-based evidence for clinical practice improvement (PBE-CPI) methodology was used. Previous hospital accreditation audits were reviewed and a neonate-specific transfusion audit was developed. Audit was performed at baseline and repeated following the intervention period. NICU clinicians received targeted education in obtaining informed consent, prescription and safe administration of blood components during a 'Blood Month' awareness period. A neonate-specific parent handout about transfusion was developed in partnership with parents. A pilot video demonstrating a shared consent discussion was also developed to assist in the consent process. Parents' knowledge, concerns and feedback regarding transfusion practice was sought at baseline (survey) and on project completion (experience trackers). RESULTS: Neonate-specific baseline transfusion audit showed inconsistent consent, monitoring and documentation processes in neonatal transfusions. Post-targeted education audit showed improvement in these parameters. The targeted PBM and transfusion-related education delivered during 'Blood Month' was well-received by staff. Parents' feedback about the NICU transfusion consenting process was consistently positive. NICU medical and nursing clinicians (n=25) surveyed agreed that the parent handout was well set out, easy to understand and recommended that it be used to complement practice. CONCLUSION: PBE-CPI tools aligned with Australian PBM guidelines for clinicians and parents were well-accepted by clinical stakeholders and were associated with practice improvement in PBM awareness and transfusion consent processes. This PBE-CPI project developed NICU-specific consent information, not previously available, by partnering with parents to ensure quality of care in transfusion practice. Adoption of this also helps to meet accreditation for Australian Blood Management Standards. These strategies and tools translate readily into other NICUs to embed and support best PBM and transfusion practice.

Combining novel research and community-engaged learning in an undergraduate physiology laboratory course.

To better prepare physiology students for 21st century careers, we incorporated classroom-based undergraduate research experiences and service learning/community-engaged learning (SLCE) into a college-level physiology laboratory course. The interventions were incorporated over 4 yr and assessed using validated surveys of student-reported learning gains related to attitudes toward science, the scientific process, and career paths. Students reported the greatest learning gains in those years when students did novel research oriented around a common theme of water quality. The gains were greater than those of a matched cohort that participated in an apprentice-style summer undergraduate research experience. With respect to the SLCE related to youth science literacy, students provided evidence of learning related to academics, personal growth, and civic mindedness. For example, many expressed discomfort about being in a new situation, often describing the differences between themselves and the youth with whom they interacted. However, students also grew in confidence about collaborating with people who were different from them and in their role as the "scientist." Limitations of the study include the quasi-experimental design and the incorporation of multiple interventions at the same time. Future studies should examine improvement in content acquisition and competency-based learning skills. Nonetheless, these results suggest that both novel research and SLCE increase student learning in the context of an undergraduate physiology laboratory course. Many of the learning gains observed with the SLCE are particularly important for physiology students, many of whom aspire to careers in health sciences, where they will be regularly working with nonscientists.

Practical steps to digital organism models, from laboratory model species to 'Crops in silico.

A recent initiative named 'Crops in silico' proposes that multi-scale models 'have the potential to fill in missing mechanistic details and generate new hypotheses to prioritize directed engineering efforts' in plant science, particularly directed to crop species. To that end, the group called for 'a paradigm shift in plant modelling, from largely isolated efforts to a connected community'. 'Wet' (experimental) research has been especially productive in plant science, since the adoption of Arabidopsis thaliana as a laboratory model species allowed the emergence of an Arabidopsis research community. Parts of this community invested in 'dry' (theoretical) research, under the rubric of Systems Biology. Our past research combined concepts from Systems Biology and crop modelling. Here we outline the approaches that seem most relevant to connected, 'digital organism' initiatives. We illustrate the scale of experimental research required, by collecting the kinetic parameter values that are required for a quantitative, dynamic model of a gene regulatory network. By comparison with the Systems Biology Markup Language (SBML) community, we note computational resources and community structures that will help to realize the potential for plant Systems Biology to connect with a broader crop science community.

hgvs: A Python package for manipulating sequence variants using HGVS nomenclature: 2018 Update.

The Human Genome Variation Society (HGVS) nomenclature guidelines encourage the accurate and standard description of DNA, RNA, and protein sequence variants in public variant databases and the scientific literature. Inconsistent application of the HGVS guidelines can lead to misinterpretation of variants in clinical settings. Reliable software tools are essential to ensure consistent application of the HGVS guidelines when reporting and interpreting variants. We present the hgvs Python package, a comprehensive tool for manipulating sequence variants according to the HGVS nomenclature guidelines. Distinguishing features of the hgvs package include: (1) parsing, formatting, validating, and normalizing variants on genome, transcript, and protein sequences; (2) projecting variants between aligned sequences, including those with gapped alignments; (3) flexible installation using remote or local data (fully local installations eliminate network dependencies); (4) extensive automated tests; and (5) open source development by a community from eight organizations worldwide. This report summarizes recent and significant updates to the hgvs package since its original release in 2014, and presents results of extensive validation using clinical relevant variants from ClinVar and HGMD.

SMYD2 promoter DNA methylation is associated with abdominal aortic aneurysm (AAA) and SMYD2 expression in vascular smooth muscle cells.

Background: Abdominal aortic aneurysm (AAA) is a deadly cardiovascular disease characterised by the gradual, irreversible dilation of the abdominal aorta. AAA is a complex genetic disease but little is known about the role of epigenetics. Our objective was to determine if global DNA methylation and CpG-specific methylation at known AAA risk loci is associated with AAA, and the functional effects of methylation changes. Results: We assessed global methylation in peripheral blood mononuclear cell DNA from 92 individuals with AAA and 93 controls using enzyme-linked immunosorbent assays, identifying hyper-methylation in those with large AAA and a positive linear association with AAA diameter (P < 0.0001, R2 = 0.3175).We then determined CpG methylation status of regulatory regions in genes located at AAA risk loci identified in genome-wide association studies, using bisulphite next-generation sequencing (NGS) in vascular smooth muscle cells (VSMCs) taken from aortic tissues of 44 individuals (24 AAAs and 20 controls). In IL6R, 2 CpGs were hyper-methylated (P = 0.0145); in ERG, 13 CpGs were hyper-methylated (P = 0.0005); in SERPINB9, 6 CpGs were hypo-methylated (P = 0.0037) and 1 CpG was hyper-methylated (P = 0.0098); and in SMYD2, 4 CpGs were hypo-methylated (P = 0.0012).RT-qPCR was performed for each differentially methylated gene on mRNA from the same VSMCs and compared with methylation. This analysis revealed downregulation of SMYD2 and SERPINB9 in AAA, and a direct linear relationship between SMYD2 promoter methylation and SMYD2 expression (P = 0.038). Furthermore, downregulation of SMYD2 at the site of aneurysm in the aortic wall was further corroborated in 6 of the same samples used for methylation and gene expression analysis with immunohistochemistry. Conclusions: This study is the first to assess DNA methylation in VSMCs from individuals with AAA using NGS, and provides further evidence there is an epigenetic basis to AAA. Our study shows that methylation status of the SMYD2 promoter may be linked with decreased SMYD2 expression in disease pathobiology. In support of our work, downregulated SMYD2 has previously been associated with adverse cardiovascular physiology and inflammation, which are both hallmarks of AAA. The identification of such adverse epigenetic modifications could potentially contribute towards the development of epigenetic treatment strategies in the future.

VariantValidator: Accurate validation, mapping, and formatting of sequence variation descriptions.

The Human Genome Variation Society (HGVS) variant nomenclature is widely used to describe sequence variants in scientific publications, clinical reports, and databases. However, the HGVS recommendations are complex and this often results in inaccurate variant descriptions being reported. The open-source hgvs Python package (https://github.com/biocommons/hgvs) provides a programmatic interface for parsing, manipulating, formatting, and validating of variants according to the HGVS recommendations, but does not provide a user-friendly Web interface. We have developed a Web-based variant validation tool, VariantValidator (https://variantvalidator.org/), which utilizes the hgvs Python package and provides additional functionality to assist users who wish to accurately describe and report sequence-level variations that are compliant with the HGVS recommendations. VariantValidator was designed to ensure that users are guided through the intricacies of the HGVS nomenclature, for example, if the user makes a mistake, VariantValidator automatically corrects the mistake if it can, or provides helpful guidance if it cannot. In addition, VariantValidator has the facility to interconvert genomic variant descriptions in HGVS and Variant Call Format with a degree of accuracy that surpasses most competing solutions.

Testing hypotheses about individual variation in plasma corticosterone in free-living salamanders.

In vertebrates, many responses to stress as well as homeostatic maintenance of basal metabolism are regulated by plasma glucocorticoid hormones (GCs). Despite having crucial functions, levels of GCs are typically variable among individuals. We examined the contribution of several physiological factors to individual variation in plasma corticosterone (CORT) and the number of corticotropin-releasing hormone (CRH) neurons in the magnocellular preoptic area of the brain in free-living Allegheny Mountain dusky salamanders. We addressed three hypotheses: the current-condition hypothesis, the facilitation hypothesis and the trade-off hypothesis. Differential white blood cell count was identified as a strong contributor to individual variation in baseline CORT, stress-induced CORT and the number of CRH neurons. In contrast, we found no relationship between CORT (or CRH) and body condition, energy stores or reproductive investment, providing no support for the current-condition hypothesis or the trade-off hypothesis involving reproduction. Because of the difficulties of interpreting the functional consequences of variation in differential white blood cell counts, we were unable to distinguish between the facilitation hypothesis or the trade-off hypothesis related to immune function. However, the strong association between differential white blood cell count and hypothalamic-pituitary-adrenal/interrenal (HPA/I) activation suggests that a more thorough examination of immune profiles is critical to understanding variation in HPA/I activation.

New technologies for DNA analysis--a review of the READNA Project.

The REvolutionary Approaches and Devices for Nucleic Acid analysis (READNA) project received funding from the European Commission for 41/2 years. The objectives of the project revolved around technological developments in nucleic acid analysis. The project partners have discovered, created and developed a huge body of insights into nucleic acid analysis, ranging from improvements and implementation of current technologies to the most promising sequencing technologies that constitute a 3(rd) and 4(th) generation of sequencing methods with nanopores and in situ sequencing, respectively.

Development of a productive research culture in emergency medicine: Report of the outcomes of a research forum.

In recent years, the Australasian College for Emergency Medicine (ACEM) has increasingly focused on the need for high-quality research in emergency medicine (EM). One important initiative was the establishment of the ACEM Foundation, which among other responsibilities, is required to support clinical research through the provision of research funding and other measures. In February 2015, the Foundation held a Research Forum that was attended by the leading EM researchers from Australasia. The Forum aimed to determine how a productive research culture could be developed within the ACEM. Nine key objectives were determined including that research should be a core business of the ACEM and a core activity of the EM workforce, and that EM research should be sustainable and adequately supported. This report describes the background and conduct of the Forum, its recommendations and the way in which they could be implemented.

Environmental acidification is not associated with altered plasma corticosterone levels in the stream-side salamander, Desmognathus ochrophaeus.

As environments become increasingly altered due to anthropogenic factors, interest is growing in how endocrine systems respond to pollution and environmental degradation. Glucocorticoid hormones (GCs) are a type of stress hormones that are released upon activation of the hypothalamic-pituitary-adrenal axis and have widespread effects throughout the body. We tested the hypothesis that exposure to environmental acidification is associated with altered levels of plasma GCs in adult, stream-side Allegheny Mountain dusky salamanders (Desmognathus ochrophaeus). We compared plasma corticosterone (CORT) in salamanders living in 9 streams that differed in pH. Although capture and handling induced a robust increase in plasma CORT in all populations of salamanders, we discerned no significant effect of environmental pH on baseline CORT or handling-induced CORT levels. In a laboratory study, low pH decreased salamander locomotory activity compared to acid-neutral controls, but there was no effect of pH on plasma CORT. Decreased locomotory activity is a common amphibian response to stress, indicating that low pH has adverse effects on Allegheny Mountain dusky salamanders. Overall, we conclude that the effects of environmental pH on salamander behavior and other potential responses are not mediated by changes in plasma CORT levels. We discuss alternative explanations for our results and describe difficulties involved in searching for relationships between plasma GCs and environmental degradation.

A mechanistic basis for amplification differences between samples and between genome regions.

BACKGROUND: For many analytical methods the efficiency of DNA amplification varies across the genome and between samples. The most affected genome regions tend to correlate with high C + G content, however this relationship is complex and does not explain why the direction and magnitude of effects varies considerably between samples. RESULTS: Here, we provide evidence that sequence elements that are particularly high in C + G content can remain annealed even when aggressive melting conditions are applied. In turn, this behavior creates broader 'Thermodynamically Ultra-Fastened' (TUF) regions characterized by incomplete denaturation of the two DNA strands, so reducing amplification efficiency throughout these domains. CONCLUSIONS: This model provides a mechanistic explanation for why some genome regions are particularly difficult to amplify and assay in many procedures, and importantly it also explains inter-sample variability of this behavior. That is, DNA samples of varying quality will carry more or fewer nicks and breaks, and hence their intact TUF regions will have different lengths and so be differentially affected by this amplification suppression mechanism - with 'higher' quality DNAs being the most vulnerable. A major practical consequence of this is that inter-region and inter-sample variability can be largely overcome by employing routine fragmentation methods (e.g. sonication or restriction enzyme digestion) prior to sample amplification.