Biology in balance: human diploid genome integrity, gene dosage, and genomic medicine.

The path to completion of the functional annotation of the haploid human genome reference build, exploration of the clan genomics hypothesis, understanding human gene and genome functional biology, and gene genome and organismal evolution, is in reach.

Clinical-Genomic Analysis of 1261 Patients with Ehlers-Danlos Syndrome Outlines an Articulo-Autonomic Gene Network (Entome).

Systematic evaluation of 80 history and 40 history findings diagnosed 1261 patients with Ehlers-Danlos syndrome (EDS) by direct or online interaction, and 60 key findings were selected for their relation to clinical mechanisms and/or management. Genomic testing results in 566 of these patients supported EDS relevance by their differences from those in 82 developmental disability patients and by their association with general rather than type-specific EDS findings. The 437 nuclear and 79 mitochondrial DNA changes included 71 impacting joint matrix (49 COL5), 39 bone (30 COL1/2/9/11), 22 vessel (12 COL3/8VWF), 43 vessel-heart (17FBN1/11TGFB/BR), 59 muscle (28 COL6/12), 56 neural (16 SCN9A/10A/11A), and 74 autonomic (13 POLG/25porphyria related). These genes were distributed over all chromosomes but the Y, a network analogized to an 'entome' where DNA change disrupts truncal mechanisms (skin constraint, neuromuscular support, joint vessel flexibility) and produces a mirroring cascade of articular and autonomic symptoms. The implied sequences of genes from nodal proteins to hypermobility to branching tissue laxity or dysautonomia symptoms would be ideal for large language/artificial intelligence analyses.

The Need for a Human Pangenome Reference Sequence.

The reference human genome sequence is inarguably the most important and widely used resource in the fields of human genetics and genomics. It has transformed the conduct of biomedical sciences and brought invaluable benefits to the understanding and improvement of human health. However, the commonly used reference sequence has profound limitations, because across much of its span, it represents the sequence of just one human haplotype. This single, monoploid reference structure presents a critical barrier to representing the broad genomic diversity in the human population. In this review, we discuss the modernization of the reference human genome sequence to a more complete reference of human genomic diversity, known as a human pangenome.

An integrated method for targeted Oxford Nanopore sequencing and automated bioinformatics for the simultaneous detection of bacteria, fungi, and ARG.

AIMS: The use of metagenomics for pathogen identification in clinical practice has been limited. Here we describe a workflow to encourage the clinical utility and potential of NGS for the screening of bacteria, fungi, and antimicrobial resistance genes (ARGs). METHODS AND RESULTS: The method includes target enrichment, long-read sequencing, and automated bioinformatics. Evaluation of several tools and databases was undertaken across standard organisms (n = 12), clinical isolates (n = 114), and blood samples from patients with suspected bloodstream infections (n = 33). The strategy used could offset the presence of host background DNA, error rates of long-read sequencing, and provide accurate and reproducible detection of pathogens. Eleven targets could be successfully tested in a single assay. Organisms could be confidently identified considering ≥60% of best hits of a BLAST-based threshold of e-value 0.001 and a percent identity of >80%. For ARGs, reads with percent identity of >90% and >60% overlap of the complete gene could be confidently annotated. A kappa of 0.83 was observed compared to standard diagnostic methods. Thus, a workflow for the direct-from-sample, on-site sequencing combined with automated genomics was demonstrated to be reproducible. CONCLUSION: NGS-based technologies overcome several limitations of current day diagnostics. Highly sensitive and comprehensive methods of pathogen screening are the need of the hour. We developed a framework for reliable, on-site, screening of pathogens.

Clinical interpretation of whole-genome and whole-transcriptome sequencing for precision oncology.

Whole-genome sequencing either alone or in combination with whole-transcriptome sequencing has started to be used to analyze clinical tumor samples to improve diagnosis, provide risk stratification, and select patient-specific therapies. Compared with current genomic testing strategies, largely focused on small number of genes tested individually or targeted panels, whole-genome and transcriptome sequencing (WGTS) provides novel opportunities to identify and report a potentially much larger number of actionable alterations with diagnostic, prognostic, and/or predictive impact. Such alterations include point mutations, indels, copy- number aberrations and structural variants, but also germline variants, fusion genes, noncoding alterations and mutational signatures. Nevertheless, these comprehensive tests are accompanied by many challenges ranging from the extent and diversity of sequence alterations detected by these methods to the complexity and limited existing standardization in interpreting them. We describe the challenges of WGTS interpretation and the opportunities with comprehensive genomic testing.

Investigations of sequencing data and sample type on HLA class Ia typing with different computational tools.

Human leukocyte antigen (HLA) can encode the human major histocompatibility complex (MHC) proteins and play a key role in adaptive and innate immunity. Emerging clinical evidences suggest that the presentation of tumor neoantigens and neoantigen-specific T cell response associated with MHC class I molecules are of key importance to activate the adaptive immune systemin cancer immunotherapy. Therefore, accurate HLA typing is very essential for the clinical application of immunotherapy. In this study, we conducted performance evaluations of 4 widely used HLA typing tools (OptiType, Phlat, Polysolver and seq2hla) for predicting HLA class Ia genes from WES and RNA-seq data of 28 cancer patients. HLA genotyping data using PCR-SBT method was firstly obtained as the golden standard and was subsequently compared with HLA typing data by using NGS techniques. For both WES data and RNA-seq data, OptiType showed the highest accuracy for HLA-Ia typing than the other 3 programs at 2-digit and 4-digit resolution. Additionally, HLA typing accuracy from WES data was higher than from RNA-seq data (99.11% for WES data versus 96.42% for RNA-seq data). The accuracy of HLA-Ia typing by OptiType can reach 100% with the average depth of HLA gene regions >20x. Besides, the accuracy of 2-digit and 4-digit HLA-Ia typing based on control samples was higher than tumor tissues. In conclusion, OptiType by using WES data from control samples with the high average depth (>20x) of HLA gene regions can present a probably superior performance for HLA-Ia typing, enabling its application in cancer immunotherapy.

Evaluation of an automated genome interpretation model for rare disease routinely used in a clinical genetic laboratory.

PURPOSE: The analysis of exome and genome sequencing data for the diagnosis of rare diseases is challenging and time-consuming. In this study, we evaluated an artificial intelligence model, based on machine learning for automating variant prioritization for diagnosing rare genetic diseases in the Baylor Genetics clinical laboratory. METHODS: The automated analysis model was developed using a supervised learning approach based on thousands of manually curated variants. The model was evaluated on 2 cohorts. The model accuracy was determined using a retrospective cohort comprising 180 randomly selected exome cases (57 singletons, 123 trios); all of which were previously diagnosed and solved through manual interpretation. Diagnostic yield with the modified workflow was estimated using a prospective "production" cohort of 334 consecutive clinical cases. RESULTS: The model accurately pinpointed all manually reported variants as candidates. The reported variants were ranked in top 10 candidate variants in 98.4% (121/123) of trio cases, in 93.0% (53/57) of single proband cases, and 96.7% (174/180) of all cases. The accuracy of the model was reduced in some cases because of incomplete variant calling (eg, copy number variants) or incomplete phenotypic description. CONCLUSION: The automated model for case analysis assists clinical genetic laboratories in prioritizing candidate variants effectively. The use of such technology may facilitate the interpretation of genomic data for a large number of patients in the era of precision medicine.

Standardizing variation: Scaling up clinical genomics in Australia.

PURPOSE: Clinical genomics demands close interaction of physicians, laboratory scientists, and genetic professionals. Taking genomics to scale requires an understanding of the underlying processes from the perspective of nongenetic physicians who are new to the field. We identified components of the processes amenable to adaptation when scaling up clinical genomics. METHODS: Semistructured interviews informed by the Theoretical Domains Framework with nongenetic physicians, who were using clinical genomics in practice, were guided by an annotated process map with 7 steps following the patient's journey. Findings from the individual maps were synthesized into an overview process map and a series of individual maps by common location and specialty. Interviews were analyzed using the Theoretical Domains Framework. RESULTS: In total, 16 nongenetic physicians (eg, nephrologists, immunologists) participated, generating 1 overview and 10 individual process maps. Sixteen common steps were identified across clinical specialties and locations, with variations over 9 steps. We report the potential for standardization across these 9 steps. CONCLUSION: When scaling up complex interventions, it is essential to identify steps where variation can be accommodated. With these results we show how process mapping can be used to identify steps where variation is acceptable during scale up to accommodate adaptation to local context, allowing for the inevitable evolution of factors influencing ongoing implementation and sustainability.

Identification of copy number variants with genome sequencing: Clinical experiences from the NYCKidSeq program.

Copy number variations (CNVs) play a significant role in human disease. While chromosomal microarray has traditionally been the first-tier test for CNV detection, use of genome sequencing (GS) is increasing. We report the frequency of CNVs detected with GS in a diverse pediatric cohort from the NYCKidSeq program and highlight specific examples of its clinical impact. A total of 1052 children (0-21 years) with neurodevelopmental, cardiac, and/or immunodeficiency phenotypes received GS. Phenotype-driven analysis was used, resulting in 183 (17.4%) participants with a diagnostic result. CNVs accounted for 20.2% of participants with a diagnostic result (37/183) and ranged from 0.5 kb to 16 Mb. Of participants with a diagnostic result (n = 183) and phenotypes in more than one category, 5/17 (29.4%) were solved by a CNV finding, suggesting a high prevalence of diagnostic CNVs in participants with complex phenotypes. Thirteen participants with a diagnostic CNV (35.1%) had previously uninformative genetic testing, of which nine included a chromosomal microarray. This study demonstrates the benefits of GS for reliable detection of CNVs in a pediatric cohort with variable phenotypes.

Beacon v2 and Beacon networks: A "lingua franca" for federated data discovery in biomedical genomics, and beyond.

Beacon is a basic data discovery protocol issued by the Global Alliance for Genomics and Health (GA4GH). The main goal addressed by version 1 of the Beacon protocol was to test the feasibility of broadly sharing human genomic data, through providing simple "yes" or "no" responses to queries about the presence of a given variant in datasets hosted by Beacon providers. The popularity of this concept has fostered the design of a version 2, that better serves real-world requirements and addresses the needs of clinical genomics research and healthcare, as assessed by several contributing projects and organizations. Particularly, rare disease genetics and cancer research will benefit from new case level and genomic variant level requests and the enabling of richer phenotype and clinical queries as well as support for fuzzy searches. Beacon is designed as a "lingua franca" to bridge data collections hosted in software solutions with different and rich interfaces. Beacon version 2 works alongside popular standards like Phenopackets, OMOP, or FHIR, allowing implementing consortia to return matches in beacon responses and provide a handover to their preferred data exchange format. The protocol is being explored by other research domains and is being tested in several international projects.

Lessons Learned from Large-Scale, First-Tier Clinical Exome Sequencing in a Highly Consanguineous Population.

We report the results of clinical exome sequencing (CES) on >2,200 previously unpublished Saudi families as a first-tier test. The predominance of autosomal-recessive causes allowed us to make several key observations. We highlight 155 genes that we propose to be recessive, disease-related candidates. We report additional mutational events in 64 previously reported candidates (40 recessive), and these events support their candidacy. We report recessive forms of genes that were previously associated only with dominant disorders and that have phenotypes ranging from consistent with to conspicuously distinct from the known dominant phenotypes. We also report homozygous loss-of-function events that can inform the genetics of complex diseases. We were also able to deduce the likely causal variant in most couples who presented after the loss of one or more children, but we lack samples from those children. Although a similar pattern of mostly recessive causes was observed in the prenatal setting, the higher proportion of loss-of-function events in these cases was notable. The allelic series presented by the wealth of recessive variants greatly expanded the phenotypic expression of the respective genes. We also make important observations about dominant disorders; these observations include the pattern of de novo variants, the identification of 74 candidate dominant, disease-related genes, and the potential confirmation of 21 previously reported candidates. Finally, we describe the influence of a predominantly autosomal-recessive landscape on the clinical utility of rapid sequencing (Flash Exome). Our cohort's genotypic and phenotypic data represent a unique resource that can contribute to improved variant interpretation through data sharing.

Re-analysis of genomic data: An overview of the mechanisms and complexities of clinical adoption.

Re-analyzing genomic information from a patient suspected of having an underlying genetic condition can improve the diagnostic yield of sequencing tests, potentially providing significant benefits to the patient and to the health care system. Although a significant number of studies have shown the clinical potential of re-analysis, less work has been performed to characterize the mechanisms responsible for driving the increases in diagnostic yield. Complexities surrounding re-analysis have also emerged. The terminology itself represents a challenge because "re-analysis" can refer to a range of different concepts. Other challenges include the increased workload that re-analysis demands of curators, adequate reimbursement pathways for clinical and diagnostic services, and the development of systems to handle large volumes of data. Re-analysis also raises ethical implications for patients and families, most notably when re-classification of a variant alters diagnosis, treatment, and prognosis. This review highlights the possibilities and complexities associated with the re-analysis of existing clinical genomic data. We propose a terminology that builds on the foundation presented in a recent statement from the American College of Medical Genetics and Genomics and describes each re-analysis process. We identify mechanisms for increasing diagnostic yield and provide perspectives on the range of challenges that must be addressed by health care systems and individual patients.

The difficulties of broad data sharing in genomic medicine: Empirical evidence from diverse participants in prenatal and pediatric clinical genomics research.

PURPOSE: This study aimed to understand broad data sharing decisions among predominantly underserved families participating in genomic research. METHODS: Drawing on clinic observations, semistructured interviews, and survey data from prenatal and pediatric families enrolled in a genomic medicine study focused on historically underserved and underrepresented populations, this paper expands empirical evidence regarding genomic data sharing communication and decision-making. RESULTS: One-third of parents declined to share family data, and pediatric participants were significantly more likely to decline than prenatal participants. The pediatric population was significantly more socioeconomically disadvantaged and more likely to require interpreters. Opt-in was tied to altruism and participants' perception that data sharing was inherent to research participation. Opt-out was associated with privacy concerns and influenced by clinical staff's presentation of data handling procedures. The ability of participants to make informed choices during enrollment about data sharing was weakened by suboptimal circumstances, which was revealed by poor understanding of data sharing in follow-up interviews as well as discrepancies between expressed participant desires and official recorded choices. CONCLUSION: These empirical data suggest that the context within which informed consent process is conducted in clinical genomics may be inadequate for respecting participants' values and preferences and does not support informed decision-making processes.

Recommendations for next generation sequencing data reanalysis of unsolved cases with suspected Mendelian disorders: A systematic review and meta-analysis.

PURPOSE: The study aimed to determine the diagnostic yield, optimal timing, and methodology of next generation sequencing data reanalysis in suspected Mendelian disorders. METHODS: We conducted a systematic review and meta-analysis of studies that conducted data reanalysis in patients with suspected Mendelian disorders. Random effects model was used to pool the estimated outcome with subgroup analysis stratified by timing, sequencing methodology, sample size, segregation, use of research validation, and artificial intelligence (AI) variant curation tools. RESULTS: A search of PubMed, Embase, Scopus, and Web of Science between 2007 and 2021 yielded 9327 articles, of which 29 were selected. Significant heterogeneity was noted between studies. Reanalysis had an overall diagnostic yield of 0.10 (95% CI = 0.06-0.13). Literature updates accounted for most new diagnoses. Diagnostic yield was higher after 24 months, although this was not statistically significant. Increased diagnoses were obtained with research validation and data sharing. AI-based tools did not adversely affect reanalysis diagnostic rate. CONCLUSION: Next generation sequencing data reanalysis can improve diagnostic yield. Owing to the heterogeneity of the studies, the optimal time to reanalysis and the impact of AI-based tools could not be determined with confidence. We propose standardized guidelines for future studies to reduce heterogeneity and improve the quality of the conclusions.

Phenotype based prediction of exome sequencing outcome using machine learning for neurodevelopmental disorders.

PURPOSE: Although the introduction of exome sequencing (ES) has led to the diagnosis of a significant portion of patients with neurodevelopmental disorders (NDDs), the diagnostic yield in actual clinical practice has remained stable at approximately 30%. We hypothesized that improving the selection of patients to test on the basis of their phenotypic presentation will increase diagnostic yield and therefore reduce unnecessary genetic testing. METHODS: We tested 4 machine learning methods and developed PredWES from these: a statistical model predicting the probability of a positive ES result solely on the basis of the phenotype of the patient. RESULTS: We first trained the tool on 1663 patients with NDDs and subsequently showed that diagnostic ES on the top 10% of patients with the highest probability of a positive ES result would provide a diagnostic yield of 56%, leading to a notable 114% increase. Inspection of our model revealed that for patients with NDDs, comorbid abnormal (lower) muscle tone and microcephaly positively correlated with a conclusive ES diagnosis, whereas autism was negatively associated with a molecular diagnosis. CONCLUSION: In conclusion, PredWES allows prioritizing patients with NDDs eligible for diagnostic ES on the basis of their phenotypic presentation to increase the diagnostic yield, making a more efficient use of health care resources.

Galaxy Is a Suitable Bioinformatics Platform for the Molecular Diagnosis of Human Genetic Disorders Using High-Throughput Sequencing Data Analysis: Five Years of Experience in a Clinical Laboratory.

BACKGROUND: To date, the usage of Galaxy, an open-source bioinformatics platform, has been reported primarily in research. We report 5 years' experience (2015 to 2020) with Galaxy in our hospital, as part of the "Assistance Publique-Hôpitaux de Paris" (AP-HP), to demonstrate its suitability for high-throughput sequencing (HTS) data analysis in a clinical laboratory setting. METHODS: Our Galaxy instance has been running since July 2015 and is used daily to study inherited diseases, cancer, and microbiology. For the molecular diagnosis of hereditary diseases, 6970 patients were analyzed with Galaxy (corresponding to a total of 7029 analyses). RESULTS: Using Galaxy, the time to process a batch of 23 samples-equivalent to a targeted DNA sequencing MiSeq run-from raw data to an annotated variant call file was generally less than 2 h for panels between 1 and 500 kb. Over 5 years, we only restarted the server twice for hardware maintenance and did not experience any significant troubles, demonstrating the robustness of our Galaxy installation in conjunction with HTCondor as a job scheduler and a PostgreSQL database. The quality of our targeted exome sequencing method was externally evaluated annually by the European Molecular Genetics Quality Network (EMQN). Sensitivity was mean (SD)% 99 (2)% for single nucleotide variants and 93 (9)% for small insertion-deletions. CONCLUSION: Our experience with Galaxy demonstrates it to be a suitable platform for HTS data analysis with vast potential to benefit patient care in a clinical laboratory setting.

AnFiSA: An open-source computational platform for the analysis of sequencing data for rare genetic disease.

Despite genomic sequencing rapidly transforming from being a bench-side tool to a routine procedure in a hospital, there is a noticeable lack of genomic analysis software that supports both clinical and research workflows as well as crowdsourcing. Furthermore, most existing software packages are not forward-compatible in regards to supporting ever-changing diagnostic rules adopted by the genetics community. Regular updates of genomics databases pose challenges for reproducible and traceable automated genetic diagnostics tools. Lastly, most of the software tools score low on explainability amongst clinicians. We have created a fully open-source variant curation tool, AnFiSA, with the intention to invite and accept contributions from clinicians, researchers, and professional software developers. The design of AnFiSA addresses the aforementioned issues via the following architectural principles: using a multidimensional database management system (DBMS) for genomic data to address reproducibility, curated decision trees adaptable to changing clinical rules, and a crowdsourcing-friendly interface to address difficult-to-diagnose cases. We discuss how we have chosen our technology stack and describe the design and implementation of the software. Finally, we show in detail how selected workflows can be implemented using the current version of AnFiSA by a medical geneticist.

vcf2fhir: a utility to convert VCF files into HL7 FHIR format for genomics-EHR integration.

BACKGROUND: VCF formatted files are the lingua franca of next-generation sequencing, whereas HL7 FHIR is emerging as a standard language for electronic health record interoperability. A growing number of FHIR-based clinical genomics applications are emerging. Here, we describe an open source utility for converting variants from VCF format into HL7 FHIR format. RESULTS: vcf2fhir converts VCF variants into a FHIR Genomics Diagnostic Report. Conversion translates each VCF row into a corresponding FHIR-formatted variant in the generated report. In scope are simple variants (SNVs, MNVs, Indels), along with zygosity and phase relationships, for autosomes, sex chromosomes, and mitochondrial DNA. Input parameters include VCF file and genome build ('GRCh37' or 'GRCh38'); and optionally a conversion region that indicates the region(s) to convert, a studied region that lists genomic regions studied by the lab, and a non-callable region that lists studied regions deemed uncallable by the lab. Conversion can be limited to a subset of VCF by supplying genomic coordinates of the conversion region(s). If studied and non-callable regions are also supplied, the output FHIR report will include 'region-studied' observations that detail which portions of the conversion region were studied, and of those studied regions, which portions were deemed uncallable. We illustrate the vcf2fhir utility via two case studies. The first, 'SMART Cancer Navigator', is a web application that offers clinical decision support by linking patient EHR information to cancerous gene variants. The second, 'Precision Genomics Integration Platform', intersects a patient's FHIR-formatted clinical and genomic data with knowledge bases in order to provide on-demand delivery of contextually relevant genomic findings and recommendations to the EHR. CONCLUSIONS: Experience to date shows that the vcf2fhir utility can be effectively woven into clinically useful genomic-EHR integration pipelines. Additional testing will be a critical step towards the clinical validation of this utility, enabling it to be integrated in a variety of real world data flow scenarios. For now, we propose the use of this utility primarily to accelerate FHIR Genomics understanding and to facilitate experimentation with further integration of genomics data into the EHR.

VIP-HL: Semi-automated ACMG/AMP variant interpretation platform for genetic hearing loss.

The American College of Medical Genetics and Genomics, and the Association for Molecular Pathology (ACMG/AMP) have proposed a set of evidence-based guidelines to support sequence variant interpretation. The ClinGen hearing loss expert panel (HL-EP) introduced further specifications into the ACMG/AMP framework for genetic hearing loss. This study developed a tool named Variant Interpretation Platform for genetic Hearing Loss (VIP-HL), aiming to semi-automate the HL ACMG/AMP rules. VIP-HL aggregates information from external databases to automate 13 out of 24 ACMG/AMP rules specified by HL-EP, namely PVS1, PS1, PM1, PM2, PM4, PM5, PP3, BA1, BS1, BS2, BP3, BP4, and BP7. We benchmarked VIP-HL using 50 variants in which 82 rules were activated by the ClinGen HL-EP. VIP-HL concordantly activated 93% (76/82) rules, significantly higher than that of by InterVar (48%; 39/82). VIP-HL is an integrated online tool for reliable automated variant classification in hearing loss genes. It assists curators in variant interpretation and provides a platform for users to share classifications with each other. VIP-HL is available with a user-friendly web interface at http://hearing.genetics.bgi.com/.

A novel statistical method for interpreting the pathogenicity of rare variants.

PURPOSE: To achieve the ultimate goal of personalized treatment of patients, accurate molecular diagnosis and precise interpretation of the impact of genetic variants on gene function is essential. With sequencing cost becoming increasingly affordable, the accurate distinguishing of benign from pathogenic variants becomes the major bottleneck. Although large normal population sequence databases have become a key resource in filtering benign variants, they are not effective at filtering extremely rare variants. METHODS: To address this challenge, we developed a novel statistical test by combining sequencing data from a patient cohort with a normal control population database. By comparing the expected and observed allele frequency in the patient cohort, variants that are likely benign can be identified. RESULTS: The performance of this new method is evaluated on both simulated and real data sets coupled with experimental validation. As a result, we demonstrate this new test is well powered to identify benign variants, and is particularly effective for variants with low frequency in the normal population. CONCLUSION: Overall, as a general test that can be applied to any type of variants in the context of all Mendelian diseases, our work provides a general framework for filtering benign variants with very low population allele frequency.