Parallel RNA and DNA analysis after deep sequencing (PRDD-seq) reveals cell type-specific lineage patterns in human brain.

Elucidating the lineage relationships among different cell types is key to understanding human brain development. Here we developed parallel RNA and DNA analysis after deep sequencing (PRDD-seq), which combines RNA analysis of neuronal cell types with analysis of nested spontaneous DNA somatic mutations as cell lineage markers, identified from joint analysis of single-cell and bulk DNA sequencing by single-cell MosaicHunter (scMH). PRDD-seq enables simultaneous reconstruction of neuronal cell type, cell lineage, and sequential neuronal formation ("birthdate") in postmortem human cerebral cortex. Analysis of two human brains showed remarkable quantitative details that relate mutation mosaic frequency to clonal patterns, confirming an early divergence of precursors for excitatory and inhibitory neurons, and an "inside-out" layer formation of excitatory neurons as seen in other species. In addition our analysis allows an estimate of excitatory neuron-restricted precursors (about 10) that generate the excitatory neurons within a cortical column. Inhibitory neurons showed complex, subtype-specific patterns of neurogenesis, including some patterns of development conserved relative to mouse, but also some aspects of primate cortical interneuron development not seen in mouse. PRDD-seq can be broadly applied to characterize cell identity and lineage from diverse archival samples with single-cell resolution and in potentially any developmental or disease condition.

Integrated genome and transcriptome sequencing identifies a noncoding mutation in the genome replication factor DONSON as the cause of microcephaly-micromelia syndrome.

While next-generation sequencing has accelerated the discovery of human disease genes, progress has been largely limited to the "low hanging fruit" of mutations with obvious exonic coding or canonical splice site impact. In contrast, the lack of high-throughput, unbiased approaches for functional assessment of most noncoding variants has bottlenecked gene discovery. We report the integration of transcriptome sequencing (RNA-seq), which surveys all mRNAs to reveal functional impacts of variants at the transcription level, into the gene discovery framework for a unique human disease, microcephaly-micromelia syndrome (MMS). MMS is an autosomal recessive condition described thus far in only a single First Nations population and causes intrauterine growth restriction, severe microcephaly, craniofacial anomalies, skeletal dysplasia, and neonatal lethality. Linkage analysis of affected families, including a very large pedigree, identified a single locus on Chromosome 21 linked to the disease (LOD > 9). Comprehensive genome sequencing did not reveal any pathogenic coding or canonical splicing mutations within the linkage region but identified several nonconserved noncoding variants. RNA-seq analysis detected aberrant splicing in DONSON due to one of these noncoding variants, showing a causative role for DONSON disruption in MMS. We show that DONSON is expressed in progenitor cells of embryonic human brain and other proliferating tissues, is co-expressed with components of the DNA replication machinery, and that Donson is essential for early embryonic development in mice as well, suggesting an essential conserved role for DONSON in the cell cycle. Our results demonstrate the utility of integrating transcriptomics into the study of human genetic disease when DNA sequencing alone is not sufficient to reveal the underlying pathogenic mutation.

Risk of sexual transmission of human immunodeficiency virus with antiretroviral therapy, suppressed viral load and condom use: a systematic review.

BACKGROUND: The Public Health Agency of Canada reviewed sexual transmission of HIV between serodiscordant partners to support examination of the criminal justice system response to HIV nondisclosure by the Department of Justice of Canada. We sought to determine HIV transmission risk when an HIV-positive partner takes antiretroviral therapy, has a suppressed viral load or uses condoms. METHODS: We conducted an overview and systematic review update by searching MEDLINE and other databases (Jan. 1, 2007, to Mar. 13, 2017; and Nov. 1, 2012, to Apr. 27, 2017, respectively). We considered reviews and studies about absolute risk of sexual transmission of HIV between serodiscordant partners to be eligible for inclusion. We used A Measurement Tool to Assess Systematic Reviews (AMSTAR) for review quality, Quality in Prognosis Studies (QUIPS) instrument for study risk of bias and then the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) approach to assess the quality of evidence across studies. We calculated HIV incidence per 100 person-years with 95% confidence intervals (CIs). We assigned risk categories according to potential for and evidence of HIV transmission. RESULTS: We identified 12 reviews. We selected 1 review to estimate risk of HIV transmission for condom use without antiretroviral therapy (1.14 transmissions/100 person-years, 95% CI 0.56-2.04; low risk). We identified 11 studies with 23 transmissions over 10 511 person-years with antiretroviral therapy (0.22 transmissions/ 100 person-years, 95% CI 0.14-0.33; low risk). We found no transmissions with antiretroviral therapy and a viral load of less than 200 copies/mL across consecutive measurements 4 to 6 months apart (0.00 transmissions/100 person-years, 95% CI 0.00-0.28; negligible risk regardless of condom use). INTERPRETATION: Based on high-quality evidence, there is a negligible risk of sexual transmission of HIV when an HIV-positive sex partner adheres to antiretroviral therapy and maintains a suppressed viral load of less than 200 copies/mL measured every 4 to 6 months. Sexual transmissions of HIV have occurred when viral load was more than 200 copies/mL with antiretroviral therapy or condoms alone were used, although the risk remains low. These findings will help to support patient and clinician decision-making, affect public health case management and contact tracing, and inform justice system responses to HIV nondisclosure.

Preferred Reporting Items for a Systematic Review and Meta-analysis of Diagnostic Test Accuracy Studies: The PRISMA-DTA Statement.

Importance: Systematic reviews of diagnostic test accuracy synthesize data from primary diagnostic studies that have evaluated the accuracy of 1 or more index tests against a reference standard, provide estimates of test performance, allow comparisons of the accuracy of different tests, and facilitate the identification of sources of variability in test accuracy. Objective: To develop the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) diagnostic test accuracy guideline as a stand-alone extension of the PRISMA statement. Modifications to the PRISMA statement reflect the specific requirements for reporting of systematic reviews and meta-analyses of diagnostic test accuracy studies and the abstracts for these reviews. Design: Established standards from the Enhancing the Quality and Transparency of Health Research (EQUATOR) Network were followed for the development of the guideline. The original PRISMA statement was used as a framework on which to modify and add items. A group of 24 multidisciplinary experts used a systematic review of articles on existing reporting guidelines and methods, a 3-round Delphi process, a consensus meeting, pilot testing, and iterative refinement to develop the PRISMA diagnostic test accuracy guideline. The final version of the PRISMA diagnostic test accuracy guideline checklist was approved by the group. Findings: The systematic review (produced 64 items) and the Delphi process (provided feedback on 7 proposed items; 1 item was later split into 2 items) identified 71 potentially relevant items for consideration. The Delphi process reduced these to 60 items that were discussed at the consensus meeting. Following the meeting, pilot testing and iterative feedback were used to generate the 27-item PRISMA diagnostic test accuracy checklist. To reflect specific or optimal contemporary systematic review methods for diagnostic test accuracy, 8 of the 27 original PRISMA items were left unchanged, 17 were modified, 2 were added, and 2 were omitted. Conclusions and Relevance: The 27-item PRISMA diagnostic test accuracy checklist provides specific guidance for reporting of systematic reviews. The PRISMA diagnostic test accuracy guideline can facilitate the transparent reporting of reviews, and may assist in the evaluation of validity and applicability, enhance replicability of reviews, and make the results from systematic reviews of diagnostic test accuracy studies more useful.

Canada-United States-Mexico Trilateral Cooperation on Childhood Obesity Initiative.

Childhood obesity is an important public health problem that affects countries in the Americas. In 2014, Pan American Health Organization (PAHO) Member States agreed on a Plan of Action for the Prevention of Obesity in Children and Adolescents in an effort to address the impact of this disorder in the Americas region. The interventions laid out in this regional plan are multi-faceted and require multi-sectoral partnerships. Building on a strong history of successful trilateral collaboration, Canada, Mexico, and the United States formed a partnership to address the growing epidemic of childhood obesity in the North American region. This collaborative effort, known as the Trilateral Cooperation on Childhood Obesity Initiative, is the first initiative in the region to address chronic noncommunicable diseases by bringing together technical and policy experts, with strong leadership and support from the secretaries and ministers of health. The Initiative's goals include increasing levels of physical activity and reducing sedentary behavior through 1) increased social mobilization and citizen engagement, 2) community- based outreach, and 3) changes to the built (man-made) environment. This article describes the background and development process of the Initiative; specific goals, activities, and actions achieved to date; and opportunities and next steps. This information may be useful for those forming other partnerships designed to address childhood obesity or other complex public health challenges in the region.

L'obésité de l'enfant est un problème de santé publique important dans les pays des Amériques. En 2014, les États Membres de l'Organisation Panaméricaine de la Santé (OPS) ont approuvé un Plan d'action pour la prévention de l'obésité chez les enfants et les adolescents pour combattre la prévalence de ce trouble dans la Région des Amériques. Les interventions énoncées dans ce plan régional sont multidimensionnelles et nécessitent des partenariats multisectoriels. S'appuyant sur une riche histoire de collaboration trilatérale fructueuse, le Canada, les États-Unis et le Mexique ont établi un partenariat pour combattre l'épidémie croissante d'obésité chez les enfants dans la région nord-américaine. Cet effort de collaboration, connu sous le nom d'Initiative de Coopération Trilatérale pour Réduire l'Obésité de l'Enfant, est la première initiative de la Région pour lutter contre les maladies chroniques non transmissibles en réunissant des experts techniques et stratégiques, sous le solide leadership et avec le soutien des secrétaires et des ministres de la Santé. Les objectifs de l'initiative visent à accroître les niveaux d'activité physique et réduire les comportements sédentaires grâce à 1) une augmentation de la mobilisation sociale et de la participation des citoyens, 2) une sensibilisation au niveau de la communauté et 3) des modifications de l'environnement bâti (par l'homme). Cet article décrit le contexte et le processus de conception et d'élaboration de l'initiative, les objectifs spécifiques atteints à ce jour, ainsi que les activités et les actions réalisées, les perspectives et les étapes à venir. Cette information peut être utile à ceux qui établissent des partenariats en vue de combattre l'obésité chez les enfants ou de relever d'autres défis difficiles de santé publique dans la Région.

La obesidad infantil es un problema de salud pública importante que afecta a los países de las Américas. En el 2014, los Estados Miembros de la Organización Panamericana de la Salud (OPS) acordaron un Plan de acción para la prevención de la obesidad en la niñez y la adolescencia con el fin de hacer frente a las repercusiones de este trastorno en la Región de las Américas. Las intervenciones que componen este plan regional son multifacéticas y exigen la formación de alianzas multisectoriales. Aprovechando las bases sentadas por una larga tradición de colaboración trilateral, el Canadá, los Estados Unidos y México formaron una alianza para controlar la epidemia creciente de obesidad infantil en la subregión norteamericana. Esta iniciativa colaborativa, que se conoce por Iniciativa de Cooperación Trilateral contra la Obesidad Infantil, es la primera iniciativa en la Región en tratar de hacer frente al problema de las enfermedades crónicas no transmisibles convocando a un grupo de expertos técnicos y de expertos en asuntos normativos, con el liderazgo y respaldo de los secretarios y ministros de salud de los países implicados. La Iniciativa tiene por objetivos aumentar los niveles de actividad física y reducir el sedentarismo mediante a) una mayor movilización social y participación ciudadana, b) medidas de extensión comunitaria y c) modificaciones de las zonas edificadas (construidas por el hombre). En el presente artículo se describen los antecedentes de la Iniciativa y su creación; las metas, actividades y medidas específicas que ha habido hasta ahora; y las oportunidades y los pasos que hay que dar en lo sucesivo. Esta información podría resultar útil para quienes estén formando otras alianzas encaminadas a controlar la obesidad infantil u otros problemas de salud pública complejos en la Región.

Soluble biomarkers for Neuromyelitis Optica Spectrum Disorders: a mini review.

The Neuromyelitis Optica Spectrum Disorders (NMOSD) constitute a spectrum of rare autoimmune diseases of the central nervous system characterized by episodes of transverse myelitis, optic neuritis, and other demyelinating attacks. Previously thought to be a subtype of multiple sclerosis, NMOSD is now known to be a distinct disease with unique pathophysiology, clinical course, and treatment options. Although there have been significant recent advances in the diagnosis and treatment of NMOSD, the field still lacks clinically validated biomarkers that can be used to stratify disease severity, monitor disease activity, and inform treatment decisions. Here we review many emerging NMOSD biomarkers including markers of cellular damage, neutrophil-to-lymphocyte ratio, complement, and cytokines, with a focus on how each biomarker can potentially be used for initial diagnosis, relapse surveillance, disability prediction, and treatment monitoring.

A case of immunotherapy-responsive autoimmune hemichorea.

INTRODUCTION: Subacute adult-acquired hemichorea is a striking presentation with a broad differential, including ischemic, metabolic, and inflammatory causes. CASE: We encountered a 74-year-old woman with rapid onset of hemichorea and associated encephalopathy. Following a thorough workup without identification of clear imaging or laboratory abnormalities, we empirically treated with IVIg. Her hemichorea dramatically improved. Due to relapses of hemichorea, she required repeat immunotherapy with IVIg or high dose steroids followed by maintenance mycophenolate. DISCUSSION: This case of seronegative autoimmune hemichorea highlights the importance of a high index of suspicion for an inflammatory etiology of chorea when other causes are ruled out and performing an immunotherapy trial.

Dissemination and implementation of clinical practice guidelines: a longitudinal, mixed-methods evaluation of the Canadian Task Force on Preventive Health Care's knowledge translation efforts.

BACKGROUND: The Canadian Task Force on Preventive Health Care (task force) develops evidence-based preventive health care guidelines and knowledge translation (KT) tools to facilitate guideline dissemination and implementation. We aimed to determine practitioners' awareness of task force guidelines and KT tools and explore barriers and facilitators to their use. METHODS: The task force's KT team completed annual evaluations using surveys and interviews with primary care providers in Canada from 2014 to 2020, to assess practitioners' awareness and determinants of use of task force guidelines and tools. We transcribed interviews verbatim and double-coded them using a framework analysis approach. RESULTS: A total of 1284 primary care practitioners completed surveys and 183 participated in interviews. On average, 79.9% of participants were aware of the task force's 7 cancer screening guidelines, 36.2% were aware of the other 6 screening guidelines and 18.6% were aware of the 3 lifestyle or prevention guidelines. Participants identified 13 barriers and 7 facilitators to guideline and KT tool implementation; these were consistent over time. Participants identified strategies at the public and patient, provider and health systems levels to improve uptake of guidelines. INTERPRETATION: Canadian primary care practitioners were more aware of task force cancer screening guidelines than its other preventive health guidelines. Over the 6-year period, participants consistently reported barriers to guideline uptake, including misalignment with patient preferences and other provincial or specialty guideline organizations. Further evaluations will assess tailored strategies to address the barriers identified.

Population surveillance approach to detect and respond to new clusters of COVID-19.

BACKGROUND: To maintain control of the coronavirus disease 2019 (COVID-19) epidemic as lockdowns are lifted, it will be crucial to enhance alternative public health measures. For surveillance, it will be necessary to detect a high proportion of any new cases quickly so that they can be isolated, and people who have been exposed to them traced and quarantined. Here we introduce a mathematical approach that can be used to determine how many samples need to be collected per unit area and unit time to detect new clusters of COVID-19 cases at a stage early enough to control an outbreak. METHODS: We present a sample size determination method that uses a relative weighted approach. Given the contribution of COVID-19 test results from sub-populations to detect the disease at a threshold prevalence level to control the outbreak to 1) determine if the expected number of weekly samples provided from current healthcare-based surveillance for respiratory virus infections may provide a sample size that is already adequate to detect new clusters of COVID-19 and, if not, 2) to determine how many additional weekly samples were needed from volunteer sampling. RESULTS: In a demonstration of our method at the weekly and Canadian provincial and territorial (P/T) levels, we found that only the more populous P/T have sufficient testing numbers from healthcare visits for respiratory illness to detect COVID-19 at our target prevalence level-assumed to be high enough to identify and control new clusters. Furthermore, detection of COVID-19 is most efficient (fewer samples required) when surveillance focuses on healthcare symptomatic testing demand. In the volunteer populations: the higher the contact rates; the higher the expected prevalence level; and the fewer the samples were needed to detect COVID-19 at a predetermined threshold level. CONCLUSION: This study introduces a targeted surveillance strategy, combining both passive and active surveillance samples, to determine how many samples to collect per unit area and unit time to detect new clusters of COVID-19 cases. The goal of this strategy is to allow for early enough detection to control an outbreak.

The impact of vaccination status on importation of COVID-19 among international travellers.

Governments worldwide are looking for ways to safely enable international travel while mitigating the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the associated coronavirus disease 2019 (COVID-19). However, few data describe the impact of vaccination on importation of COVID-19. We took advantage of the sequential introduction of two government policies in Canada to evaluate the real-world evidence of vaccine effectiveness among 30,361 international travellers arriving by air in Alberta, Canada. The proportion of COVID-19-positive results for travellers who were either fully vaccinated or partially vaccinated was 0.02% (95% CI: 0.00-0.10) (i.e. one positive case among 5,817 travellers). In contrast, 1.42% (95% CI: 1.27-1.58) of unvaccinated travellers tested positive for SARS-CoV-2 (341 cases among 24,034 travellers). These findings suggest that COVID-19 vaccinations approved in Canada, substantially reduced the risk of travel-related importation of COVID-19 when combined with other public health measures. The low absolute rate of infection among fully vaccinated or partially vaccinated international travellers may inform quarantine requirements in this population.

Large mosaic copy number variations confer autism risk.

Although germline de novo copy number variants (CNVs) are known causes of autism spectrum disorder (ASD), the contribution of mosaic (early-developmental) copy number variants (mCNVs) has not been explored. In this study, we assessed the contribution of mCNVs to ASD by ascertaining mCNVs in genotype array intensity data from 12,077 probands with ASD and 5,500 unaffected siblings. We detected 46 mCNVs in probands and 19 mCNVs in siblings, affecting 2.8-73.8% of cells. Probands carried a significant burden of large (>4-Mb) mCNVs, which were detected in 25 probands but only one sibling (odds ratio = 11.4, 95% confidence interval = 1.5-84.2, P = 7.4 × 10-4). Event size positively correlated with severity of ASD symptoms (P = 0.016). Surprisingly, we did not observe mosaic analogues of the short de novo CNVs recurrently observed in ASD (eg, 16p11.2). We further experimentally validated two mCNVs in postmortem brain tissue from 59 additional probands. These results indicate that mCNVs contribute a previously unexplained component of ASD risk.

MIPP-Seq: ultra-sensitive rapid detection and validation of low-frequency mosaic mutations.

BACKGROUND: Mosaic mutations contribute to numerous human disorders. As such, the identification and precise quantification of mosaic mutations is essential for a wide range of research applications, clinical diagnoses, and early detection of cancers. Currently, the low-throughput nature of single allele assays (e.g., allele-specific ddPCR) commonly used for genotyping known mutations at very low alternate allelic fractions (AAFs) have limited the integration of low-level mosaic analyses into clinical and research applications. The growing importance of mosaic mutations requires a more rapid, low-cost solution for mutation detection and validation. METHODS: To overcome these limitations, we developed Multiple Independent Primer PCR Sequencing (MIPP-Seq) which combines the power of ultra-deep sequencing and truly independent assays. The accuracy of MIPP-seq to quantifiable detect and measure extremely low allelic fractions was assessed using a combination of SNVs, insertions, and deletions at known allelic fractions in blood and brain derived DNA samples. RESULTS: The Independent amplicon analyses of MIPP-Seq markedly reduce the impact of allelic dropout, amplification bias, PCR-induced, and sequencing artifacts. Using low DNA inputs of either 25 ng or 50 ng of DNA, MIPP-Seq provides sensitive and quantitative assessments of AAFs as low as 0.025% for SNVs, insertion, and deletions. CONCLUSIONS: MIPP-Seq provides an ultra-sensitive, low-cost approach for detecting and validating known and novel mutations in a highly scalable system with broad utility spanning both research and clinical diagnostic testing applications. The scalability of MIPP-Seq allows for multiplexing mutations and samples, which dramatically reduce costs of variant validation when compared to methods like ddPCR. By leveraging the power of individual analyses of multiple unique and independent reactions, MIPP-Seq can validate and precisely quantitate extremely low AAFs across multiple tissues and mutational categories including both indels and SNVs. Furthermore, using Illumina sequencing technology, MIPP-seq provides a robust method for accurate detection of novel mutations at an extremely low AAF.

The landscape of somatic mutation in cerebral cortex of autistic and neurotypical individuals revealed by ultra-deep whole-genome sequencing.

We characterize the landscape of somatic mutations-mutations occurring after fertilization-in the human brain using ultra-deep (~250×) whole-genome sequencing of prefrontal cortex from 59 donors with autism spectrum disorder (ASD) and 15 control donors. We observe a mean of 26 somatic single-nucleotide variants per brain present in ≥4% of cells, with enrichment of mutations in coding and putative regulatory regions. Our analysis reveals that the first cell division after fertilization produces ~3.4 mutations, followed by 2-3 mutations in subsequent generations. This suggests that a typical individual possesses ~80 somatic single-nucleotide variants present in ≥2% of cells-comparable to the number of de novo germline mutations per generation-with about half of individuals having at least one potentially function-altering somatic mutation somewhere in the cortex. ASD brains show an excess of somatic mutations in neural enhancer sequences compared with controls, suggesting that mosaic enhancer mutations may contribute to ASD risk.

COVID-19 infection among international travellers: a prospective analysis.

OBJECTIVES: This report estimates the risk of COVID-19 importation and secondary transmission associated with a modified quarantine programme in Canada. DESIGN AND PARTICIPANTS: Prospective analysis of international asymptomatic travellers entering Alberta, Canada. INTERVENTIONS: All participants were required to receive a PCR COVID-19 test on arrival. If negative, participants could leave quarantine but were required to have a second test 6 or 7 days after arrival. If the arrival test was positive, participants were required to remain in quarantine for 14 days. MAIN OUTCOME MEASURES: Proportion and rate of participants testing positive for COVID-19; number of cases of secondary transmission. RESULTS: The analysis included 9535 international travellers entering Alberta by air (N=8398) or land (N=1137) that voluntarily enrolled in the Alberta Border Testing Pilot Programme (a subset of all travellers); most (83.1%) were Canadian citizens. Among the 9310 participants who received at least one test, 200 (21.5 per 1000, 95% CI 18.6 to 24.6) tested positive. Sixty-nine per cent (138/200) of positive tests were detected on arrival (14.8 per 1000 travellers, 95% CI 12.5 to 17.5). 62 cases (6.7 per 1000 travellers, 95% CI 5.1 to 8.5; 31.0% of positive cases) were identified among participants that had been released from quarantine following a negative test result on arrival. Of 192 participants who developed symptoms, 51 (26.6%) tested positive after arrival. Among participants with positive tests, four (2.0%) were hospitalised for COVID-19; none required critical care or died. Contact tracing among participants who tested positive identified 200 contacts; of 88 contacts tested, 22 were cases of secondary transmission (14 from those testing positive on arrival and 8 from those testing positive thereafter). SARS-CoV-2 B.1.1.7 lineage was not detected in any of the 200 positive cases. CONCLUSIONS: 21.5 per 1000 international travellers tested positive for COVID-19. Most (69%) tested positive on arrival and 31% tested positive during follow-up. These findings suggest the need for ongoing vigilance in travellers testing negative on arrival and highlight the value of follow-up testing and contact tracing to monitor and limit secondary transmission where possible.

Developing trustworthy recommendations as part of an urgent response (1-2 weeks): a GRADE concept paper.

OBJECTIVES: The aim of this study is to propose an approach for developing trustworthy recommendations as part of urgent responses (1-2 week) in the clinical, public health, and health systems fields. STUDY DESIGN AND SETTING: We conducted a review of the literature, outlined a draft approach, refined the concept through iterative discussions, a workshop by the Grading of Recommendations Assessment, Development and Evaluation Rapid Guidelines project group, and obtained feedback from the larger Grading of Recommendations Assessment, Development and Evaluation working group. RESULTS: A request for developing recommendations within 2 week is the usual trigger for an urgent response. Although the approach builds on the general principles of trustworthy guideline development, we highlight the following steps: (1) assess the level of urgency; (2) assess feasibility; (3) set up the organizational logistics; (4) specify the question(s); (5) collect the information needed; (6) assess the adequacy of identified information; (7) develop the recommendations using one of the 4 potential approaches: adopt existing recommendations, adapt existing recommendations, develop new recommendations using existing adequate systematic review, or develop new recommendations using expert panel input; and (8) consider an updating plan. CONCLUSION: An urgent response for developing recommendations requires building a cohesive, skilled, and highly motivated multidisciplinary team with the necessary clinical, scientific, and methodological expertise; adapting to shifting needs; complying with the principles of transparency; and properly managing conflicts of interest.

Comprehensive identification of somatic nucleotide variants in human brain tissue.

BACKGROUND: Post-zygotic mutations incurred during DNA replication, DNA repair, and other cellular processes lead to somatic mosaicism. Somatic mosaicism is an established cause of various diseases, including cancers. However, detecting mosaic variants in DNA from non-cancerous somatic tissues poses significant challenges, particularly if the variants only are present in a small fraction of cells. RESULTS: Here, the Brain Somatic Mosaicism Network conducts a coordinated, multi-institutional study to examine the ability of existing methods to detect simulated somatic single-nucleotide variants (SNVs) in DNA mixing experiments, generate multiple replicates of whole-genome sequencing data from the dorsolateral prefrontal cortex, other brain regions, dura mater, and dural fibroblasts of a single neurotypical individual, devise strategies to discover somatic SNVs, and apply various approaches to validate somatic SNVs. These efforts lead to the identification of 43 bona fide somatic SNVs that range in variant allele fractions from ~ 0.005 to ~ 0.28. Guided by these results, we devise best practices for calling mosaic SNVs from 250× whole-genome sequencing data in the accessible portion of the human genome that achieve 90% specificity and sensitivity. Finally, we demonstrate that analysis of multiple bulk DNA samples from a single individual allows the reconstruction of early developmental cell lineage trees. CONCLUSIONS: This study provides a unified set of best practices to detect somatic SNVs in non-cancerous tissues. The data and methods are freely available to the scientific community and should serve as a guide to assess the contributions of somatic SNVs to neuropsychiatric diseases.