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Microsatellites are highly mutable sequences that can serve as markers for relationships among individuals or cells within a population. The accuracy and resolution of reconstructing these relationships depends on the fidelity of microsatellite profiling and the number of microsatellites profiled. However, current methods for targeted profiling of microsatellites incur significant "stutter" artifacts that interfere with accurate genotyping, and sequencing costs preclude whole-genome microsatellite profiling of a large number of samples. We developed a novel method for accurate and cost-effective targeted profiling of a panel of more than 150,000 microsatellites per sample, along with a computational tool for designing large-scale microsatellite panels. Our method addresses the greatest challenge for microsatellite profiling-"stutter" artifacts-with a low-temperature hybridization capture that significantly reduces these artifacts. We also developed a computational tool for accurate genotyping of the resulting microsatellite sequencing data that uses an ensemble approach integrating three microsatellite genotyping tools, which we optimize by analysis of de novo microsatellite mutations in human trios. Altogether, our suite of experimental and computational tools enables high-fidelity, large-scale profiling of microsatellites, which may find utility in diverse applications such as lineage tracing, population genetics, ecology, and forensics., (© 2024 Loh et al.; Published by Cold Spring Harbor Laboratory Press.)

Mutations accumulate in the genome of every cell of the body throughout life, causing cancer and other diseases 1,2 . Most mutations begin as nucleotide mismatches or damage in one of the two strands of the DNA before becoming double-strand mutations if unrepaired or misrepaired 3,4 . However, current DNA-sequencing technologies cannot accurately resolve these initial single-strand events. Here we develop a single-molecule, long-read sequencing method (Hairpin Duplex Enhanced Fidelity sequencing (HiDEF-seq)) that achieves single-molecule fidelity for base substitutions when present in either one or both DNA strands. HiDEF-seq also detects cytosine deamination-a common type of DNA damage-with single-molecule fidelity. We profiled 134 samples from diverse tissues, including from individuals with cancer predisposition syndromes, and derive from them single-strand mismatch and damage signatures. We find correspondences between these single-strand signatures and known double-strand mutational signatures, which resolves the identity of the initiating lesions. Tumours deficient in both mismatch repair and replicative polymerase proofreading show distinct single-strand mismatch patterns compared to samples that are deficient in only polymerase proofreading. We also define a single-strand damage signature for APOBEC3A. In the mitochondrial genome, our findings support a mutagenic mechanism occurring primarily during replication. As double-strand DNA mutations are only the end point of the mutation process, our approach to detect the initiating single-strand events at single-molecule resolution will enable studies of how mutations arise in a variety of contexts, especially in cancer and ageing., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

The diagnosis and treatment of tumors often depends on molecular-genetic data. However, rapid and iterative access to molecular data is not currently feasible during surgery, complicating intraoperative diagnosis and precluding measurement of tumor cell burdens at surgical margins to guide resections. To address this gap, we developed Ultra-Rapid droplet digital PCR (UR-ddPCR), which can be completed in 15 minutes from tissue to result with an accuracy comparable to standard ddPCR. We demonstrate UR-ddPCR assays for the IDH1 R132H and BRAF V600E clonal mutations that are present in many low-grade gliomas and melanomas, respectively. We illustrate the clinical feasibility of UR-ddPCR by performing it intraoperatively for 13 glioma cases. We further combine UR-ddPCR measurements with UR-stimulated Raman histology intraoperatively to estimate tumor cell densities in addition to tumor cell percentages. We anticipate that UR-ddPCR, along with future refinements in assay instrumentation, will enable novel point-of-care diagnostics and the development of molecularly-guided surgeries that improve clinical outcomes., Competing Interests: New York University Grossman School of Medicine has filed a provisional patent on the technology, listing Z.R.M., D.A.O., and G.D.E. as inventors. D.A.O. is a medical advisor and shareholder of Invenio Imaging, Inc., a company developing and marketing stimulated Raman histology microscopes. D.A.O. is also a consultant to Servier, a company whose portfolio includes IDH inhibitors. M.S. is a scientific advisor and shareholder of Heidelberg Epignostix and Halo Dx, is a scientific advisor of Arima Genomics and InnoSIGN, and has received research funding from Lilly USA. Other authors declare no competing interests.

Detecting mutations from single DNA molecules is crucial in many fields but challenging. Next-generation sequencing (NGS) affords tremendous throughput but cannot directly sequence double-stranded DNA molecules ('single duplexes') to discern the true mutations on both strands. Here we present Concatenating Original Duplex for Error Correction (CODEC), which confers single duplex resolution to NGS. CODEC affords 1,000-fold higher accuracy than NGS, using up to 100-fold fewer reads than duplex sequencing. CODEC revealed mutation frequencies of 2.72 × 10 -8 in sperm of a 39-year-old individual, and somatic mutations acquired with age in blood cells. CODEC detected genome-wide, clonal hematopoiesis mutations from single DNA molecules, single mutated duplexes from tumor genomes and liquid biopsies, microsatellite instability with 10-fold greater sensitivity and mutational signatures, and specific tumor mutations with up to 100-fold fewer reads. CODEC enables more precise genetic testing and reveals biologically significant mutations, which are commonly obscured by NGS errors., (© 2023. The Author(s).)

Congenital myasthenic syndrome (CMS) is a heterogeneous condition associated with 34 different genes, including SLC5A7, which encodes the high-affinity choline transporter 1 (CHT1). CHT1 is expressed in presynaptic neurons of the neuromuscular junction where it uses the inward sodium gradient to reuptake choline. Biallelic CHT1 mutations often lead to neonatal lethality, and less commonly to non-lethal motor weakness and developmental delays. Here, we report detailed biochemical characterization of two novel mutations in CHT1, p.I294T and p.D349N, which we identified in an 11-year-old patient with a history of neonatal respiratory distress, and subsequent hypotonia and global developmental delay. Heterologous expression of each CHT1 mutant in human embryonic kidney cells showed two different mechanisms of reduced protein function. The p.I294T CHT1 mutant transporter function was detectable, but its abundance and half-life were significantly reduced. In contrast, the p.D349N CHT1 mutant was abundantly expressed at the cell membrane, but transporter function was absent. The residual function of the p.I294T CHT1 mutant may explain the non-lethal form of CMS in this patient, and the divergent mechanisms of reduced CHT1 function that we identified may guide future functional studies of the CHT1 myasthenic syndrome. Based on these in vitro studies that provided a diagnosis, treatment with cholinesterase inhibitor together with physical and occupational therapy significantly improved the patient's strength and quality of life., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Numerous applications in molecular biology and genomics require characterization of mutant DNA molecules present at low levels within a larger sample of non-mutant DNA. This is often achieved either by selectively amplifying mutant DNA, or by sequencing all the DNA followed by computational identification of the mutant DNA. However, selective amplification is challenging for insertions and deletions (indels). Additionally, sequencing all the DNA in a sample may not be cost effective when only the presence of a mutation needs to be ascertained rather than its allelic fraction. The MutS protein evolved to detect DNA heteroduplexes in which the two DNA strands are mismatched. Prior methods have utilized MutS to enrich mutant DNA by hybridizing mutant to non-mutant DNA to create heteroduplexes. However, the purity of heteroduplex DNA these methods achieve is limited because they can only feasibly perform one or two enrichment cycles. We developed a MutS-magnetic bead system that enables rapid serial enrichment cycles. With six cycles, we achieve complete purification of heteroduplex indel DNA originally present at a 5% fraction and over 40-fold enrichment of heteroduplex DNA originally present at a 1% fraction. This system may enable novel approaches for enriching mutant DNA for targeted sequencing., (© 2022 Wiley-VCH GmbH.)

Au-Kline syndrome (AKS) is a neurodevelopmental disorder associated with multiple malformations and a characteristic facial gestalt. The first individuals ascertained carried de novo loss-of-function (LoF) variants in HNRNPK. Here, we report 32 individuals with AKS (26 previously unpublished), including 13 with de novo missense variants. We propose new clinical diagnostic criteria for AKS that differentiate it from the clinically overlapping Kabuki syndrome and describe a significant phenotypic expansion to include individuals with missense variants who present with subtle facial features and few or no malformations. Many gene-specific DNA methylation (DNAm) signatures have been identified for neurodevelopmental syndromes. Because HNRNPK has roles in chromatin and epigenetic regulation, we hypothesized that pathogenic variants in HNRNPK may be associated with a specific DNAm signature. Here, we report a unique DNAm signature for AKS due to LoF HNRNPK variants, distinct from controls and Kabuki syndrome. This DNAm signature is also identified in some individuals with de novo HNRNPK missense variants, confirming their pathogenicity and the phenotypic expansion of AKS to include more subtle phenotypes. Furthermore, we report that some individuals with missense variants have an "intermediate" DNAm signature that parallels their milder clinical presentation, suggesting the presence of an epi-genotype phenotype correlation. In summary, the AKS DNAm signature may help elucidate the underlying pathophysiology of AKS. This DNAm signature also effectively supported clinical syndrome delineation and is a valuable aid for variant interpretation in individuals where a clinical diagnosis of AKS is unclear, particularly for mild presentations., Competing Interests: Declaration of interests H.T.B. is a consultant for Mahzi therapeutics., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Over the past decade, genomic analyses of single cells-the fundamental units of life-have become possible. Single-cell DNA sequencing has shed light on biological questions that were previously inaccessible across diverse fields of research, including somatic mutagenesis, organismal development, genome function, and microbiology. Single-cell DNA sequencing also promises significant future biomedical and clinical impact, spanning oncology, fertility, and beyond. While single-cell approaches that profile RNA and protein have greatly expanded our understanding of cellular diversity, many fundamental questions in biology and important biomedical applications require analysis of the DNA of single cells. Here, we review the applications and biological questions for which single-cell DNA sequencing is uniquely suited or required. We include a discussion of the fields that will be impacted by single-cell DNA sequencing as the technology continues to advance.

Background: Over half of children with rare genetic diseases remain undiagnosed despite maximal clinical evaluation and DNA-based genetic testing. As part of an Undiagnosed Diseases Program applying transcriptome (RNA) sequencing to identify the causes of these unsolved cases, we studied a child with severe infantile osteopetrosis leading to cranial nerve palsies, bone deformities, and bone marrow failure, for whom whole-genome sequencing was nondiagnostic., Methods: We performed transcriptome (RNA) sequencing of whole blood followed by analysis of aberrant transcript isoforms and osteoclast functional studies., Results: We identified a pathogenic deep intronic variant in CLCN7 creating an unexpected, frameshifting pseudoexon causing complete loss of function. Functional studies, including osteoclastogenesis and bone resorption assays, confirmed normal osteoclast differentiation but loss of osteoclast function., Conclusion: This is the first report of a pathogenic deep intronic variant in CLCN7, and our approach provides a model for systematic identification of noncoding variants causing osteopetrosis-a disease for which molecular-genetic diagnosis can be pivotal for potentially curative hematopoietic stem cell transplantation. Our work illustrates that cryptic splice variants may elude DNA-only sequencing and supports broad first-line use of transcriptome sequencing for children with undiagnosed diseases., (© 2020 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

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., (© 2017 Evrony et al.; Published by Cold Spring Harbor Laboratory Press.)

Competing Interests: Disclosures: The author has completed and submitted the ICMJE Form for the Disclosure of Potential Conflicts of Interest and none were reported.

Whether somatic mutations contribute functional diversity to brain cells is a long-standing question. Single-neuron genomics enables direct measurement of somatic mutation rates in human brain and promises to answer this question. A recent study (Upton et al., 2015) reported high rates of somatic LINE-1 element (L1) retrotransposition in the hippocampus and cerebral cortex that would have major implications for normal brain function, and suggested that these events preferentially impact genes important for neuronal function. We identify aspects of the single-cell sequencing approach, bioinformatic analysis, and validation methods that led to thousands of artifacts being interpreted as somatic mutation events. Our reanalysis supports a mutation frequency of approximately 0.2 events per cell, which is about fifty-fold lower than reported, confirming that L1 elements mobilize in some human neurons but indicating that L1 mosaicism is not ubiquitous. Through consideration of the challenges identified, we provide a foundation and framework for designing single-cell genomics studies.

Neurons live for decades in a postmitotic state, their genomes susceptible to DNA damage. Here we survey the landscape of somatic single-nucleotide variants (SNVs) in the human brain. We identified thousands of somatic SNVs by single-cell sequencing of 36 neurons from the cerebral cortex of three normal individuals. Unlike germline and cancer SNVs, which are often caused by errors in DNA replication, neuronal mutations appear to reflect damage during active transcription. Somatic mutations create nested lineage trees, allowing them to be dated relative to developmental landmarks and revealing a polyclonal architecture of the human cerebral cortex. Thus, somatic mutations in the brain represent a durable and ongoing record of neuronal life history, from development through postmitotic function., (Copyright © 2015, American Association for the Advancement of Science.)

Objective: To identify the genetic cause of pontocerebellar hypoplasia type III (PCH3)., Methods: We studied the original reported pedigree of PCH3 and performed genetic analysis including genome-wide single nucleotide polymorphism genotyping, linkage analysis, whole-exome sequencing, and Sanger sequencing. Human fetal brain RNA sequencing data were then analyzed for the identified candidate gene., Results: The affected individuals presented with severe global developmental delay and seizures starting in the first year of life. Brain MRI of an affected individual showed diffuse atrophy of the cerebrum, cerebellum, and brainstem. Genome-wide single nucleotide polymorphism analysis confirmed the linkage to chromosome 7q we previously reported, and showed no other genomic areas of linkage. Whole-exome sequencing of 2 affected individuals identified a shared homozygous, nonsense variant in the PCLO (piccolo) gene. This variant segregated with the disease phenotype in the pedigree was rare in the population and was predicted to eliminate the PDZ and C2 domains in the C-terminus of the protein. RNA sequencing data of human fetal brain showed that PCLO was moderately expressed in the developing cerebral cortex., Conclusions: Here, we show that a homozygous, nonsense PCLO mutation underlies the autosomal recessive neurodegenerative disorder, PCH3. PCLO is a component of the presynaptic cytoskeletal matrix, and is thought to be involved in regulation of presynaptic proteins and synaptic vesicles. Our findings suggest that PCLO is crucial for the development and survival of a wide range of neuronal types in the human brain., (© 2015 American Academy of Neurology.)

Somatic mutations occur during brain development and are increasingly implicated as a cause of neurogenetic disease. However, the patterns in which somatic mutations distribute in the human brain are unknown. We used high-coverage whole-genome sequencing of single neurons from a normal individual to identify spontaneous somatic mutations as clonal marks to track cell lineages in human brain. Somatic mutation analyses in >30 locations throughout the nervous system identified multiple lineages and sublineages of cells marked by different LINE-1 (L1) retrotransposition events and subsequent mutation of poly-A microsatellites within L1. One clone contained thousands of cells limited to the left middle frontal gyrus, whereas a second distinct clone contained millions of cells distributed over the entire left hemisphere. These patterns mirror known somatic mutation disorders of brain development and suggest that focally distributed mutations are also prevalent in normal brains. Single-cell analysis of somatic mutation enables tracing of cell lineage clones in human brain., (Copyright © 2015 Elsevier Inc. All rights reserved.)