Severus: accurate detection and characterization of somatic structural variation in tumor genomes using long reads.

Most current studies rely on short-read sequencing to detect somatic structural variation (SV) in cancer genomes. Long-read sequencing offers the advantage of better mappability and long-range phasing, which results in substantial improvements in germline SV detection. However, current long-read SV detection methods do not generalize well to the analysis of somatic SVs in tumor genomes with complex rearrangements, heterogeneity, and aneuploidy. Here, we present Severus: a method for the accurate detection of different types of somatic SVs using a phased breakpoint graph approach. To benchmark various short- and long-read SV detection methods, we sequenced five tumor/normal cell line pairs with Illumina, Nanopore, and PacBio sequencing platforms; on this benchmark Severus showed the highest F1 scores (harmonic mean of the precision and recall) as compared to long-read and short-read methods. We then applied Severus to three clinical cases of pediatric cancer, demonstrating concordance with known genetic findings as well as revealing clinically relevant cryptic rearrangements missed by standard genomic panels.

Osteocalcin of maternal and embryonic origins synergize to establish homeostasis in offspring.

Many physiological osteocalcin-regulated functions are affected in adult offspring of mothers experiencing unhealthy pregnancy. Furthermore, osteocalcin signaling during gestation influences cognition and adrenal steroidogenesis in adult mice. Together these observations suggest that osteocalcin may broadly function during pregnancy to determine organismal homeostasis in adult mammals. To test this hypothesis, we analyzed in unchallenged wildtype and Osteocalcin-deficient, newborn and adult mice of various genotypes and origin maintained on different genetic backgrounds, the functions of osteocalcin in the pancreas, liver and testes and their molecular underpinnings. This analysis revealed that providing mothers are Osteocalcin-deficient, Osteocalcin haploinsufficiency in embryos hampers insulin secretion, liver gluconeogenesis, glucose homeostasis, testes steroidogenesis in adult offspring; inhibits cell proliferation in developing pancreatic islets and testes; and disrupts distinct programs of gene expression in these organs and in the brain. This study indicates that osteocalcin exerts dominant functions in most organs it influences. Furthermore, through their synergistic regulation of multiple physiological functions, osteocalcin of maternal and embryonic origins contributes to the establishment and maintenance of organismal homeostasis in newborn and adult offspring.

Osteocalcin of maternal and embryonic origins synergize to establish homeostasis in offspring.

Many physiological functions regulated by osteocalcin are affected in adult offspring of mothers experiencing an unhealthy pregnancy. Furthermore, osteocalcin signaling during gestation influences cognition and adrenal steroidogenesis in adult mice. Together these observations suggest that osteocalcin functions during pregnancy may be a broader determinant of organismal homeostasis in adult mammals than previously thought. To test this hypothesis, we analyzed in unchallenged wildtype and Osteocalcin -deficient, newborn, and adult mice of various genotypes and origin, and that were maintained on different genetic backgrounds, the functions of osteocalcin in the pancreas, liver and testes and their molecular underpinnings. This analysis revealed that providing mothers are themselves Osteocalcin -deficient, Osteocalcin haploinsufficiency in embryos hampers insulin secretion, liver gluconeogenesis, glucose homeostasis, testes steroidogenesis in adult offspring; inhibits cell proliferation in developing pancreatic islets and testes; and disrupts distinct programs of gene expression in these organs and in the brain. This study indicates that through their synergistic regulation of multiple physiological functions, osteocalcin ofmaternal and embryonic origins contributes to the establishment and maintenance of organismal homeostasis in newborn and adult offspring.

Unexpected frequency of the pathogenic AR CAG repeat expansion in the general population.

CAG repeat expansions in exon 1 of the AR gene on the X chromosome cause spinal and bulbar muscular atrophy, a male-specific progressive neuromuscular disorder associated with a variety of extra-neurological symptoms. The disease has a reported male prevalence of approximately 1:30 000 or less, but the AR repeat expansion frequency is unknown. We established a pipeline, which combines the use of the ExpansionHunter tool and visual validation, to detect AR CAG expansion on whole-genome sequencing data, benchmarked it to fragment PCR sizing, and applied it to 74 277 unrelated individuals from four large cohorts. Our pipeline showed sensitivity of 100% [95% confidence interval (CI) 90.8-100%], specificity of 99% (95% CI 94.2-99.7%), and a positive predictive value of 97.4% (95% CI 84.4-99.6%). We found the mutation frequency to be 1:3182 (95% CI 1:2309-1:4386, n = 117 734) X chromosomes-10 times more frequent than the reported disease prevalence. Modelling using the novel mutation frequency led to estimate disease prevalence of 1:6887 males, more than four times more frequent than the reported disease prevalence. This discrepancy is possibly due to underdiagnosis of this neuromuscular condition, reduced penetrance, and/or pleomorphic clinical manifestations.

Integrative transcriptomic analysis of the amyotrophic lateral sclerosis spinal cord implicates glial activation and suggests new risk genes.

Amyotrophic lateral sclerosis (ALS) is a progressively fatal neurodegenerative disease affecting motor neurons in the brain and spinal cord. In this study, we investigated gene expression changes in ALS via RNA sequencing in 380 postmortem samples from cervical, thoracic and lumbar spinal cord segments from 154 individuals with ALS and 49 control individuals. We observed an increase in microglia and astrocyte gene expression, accompanied by a decrease in oligodendrocyte gene expression. By creating a gene co-expression network in the ALS samples, we identified several activated microglia modules that negatively correlate with retrospective disease duration. We mapped molecular quantitative trait loci and found several potential ALS risk loci that may act through gene expression or splicing in the spinal cord and assign putative cell types for FNBP1, ACSL5, SH3RF1 and NFASC. Finally, we outline how common genetic variants associated with splicing of C9orf72 act as proxies for the well-known repeat expansion, and we use the same mechanism to suggest ATXN3 as a putative risk gene.

Benchmarking challenging small variants with linked and long reads.

Genome in a Bottle benchmarks are widely used to help validate clinical sequencing pipelines and develop variant calling and sequencing methods. Here we use accurate linked and long reads to expand benchmarks in 7 samples to include difficult-to-map regions and segmental duplications that are challenging for short reads. These benchmarks add more than 300,000 SNVs and 50,000 insertions or deletions (indels) and include 16% more exonic variants, many in challenging, clinically relevant genes not covered previously, such as PMS2. For HG002, we include 92% of the autosomal GRCh38 assembly while excluding regions problematic for benchmarking small variants, such as copy number variants, that should not have been in the previous version, which included 85% of GRCh38. It identifies eight times more false negatives in a short read variant call set relative to our previous benchmark. We demonstrate that this benchmark reliably identifies false positives and false negatives across technologies, enabling ongoing methods development.

High-coverage whole-genome sequencing of the expanded 1000 Genomes Project cohort including 602 trios.

The 1000 Genomes Project (1kGP) is the largest fully open resource of whole-genome sequencing (WGS) data consented for public distribution without access or use restrictions. The final, phase 3 release of the 1kGP included 2,504 unrelated samples from 26 populations and was based primarily on low-coverage WGS. Here, we present a high-coverage 3,202-sample WGS 1kGP resource, which now includes 602 complete trios, sequenced to a depth of 30X using Illumina. We performed single-nucleotide variant (SNV) and short insertion and deletion (INDEL) discovery and generated a comprehensive set of structural variants (SVs) by integrating multiple analytic methods through a machine learning model. We show gains in sensitivity and precision of variant calls compared to phase 3, especially among rare SNVs as well as INDELs and SVs spanning frequency spectrum. We also generated an improved reference imputation panel, making variants discovered here accessible for association studies.

Curated variation benchmarks for challenging medically relevant autosomal genes.

The repetitive nature and complexity of some medically relevant genes poses a challenge for their accurate analysis in a clinical setting. The Genome in a Bottle Consortium has provided variant benchmark sets, but these exclude nearly 400 medically relevant genes due to their repetitiveness or polymorphic complexity. Here, we characterize 273 of these 395 challenging autosomal genes using a haplotype-resolved whole-genome assembly. This curated benchmark reports over 17,000 single-nucleotide variations, 3,600 insertions and deletions and 200 structural variations each for human genome reference GRCh37 and GRCh38 across HG002. We show that false duplications in either GRCh37 or GRCh38 result in reference-specific, missed variants for short- and long-read technologies in medically relevant genes, including CBS, CRYAA and KCNE1. When masking these false duplications, variant recall can improve from 8% to 100%. Forming benchmarks from a haplotype-resolved whole-genome assembly may become a prototype for future benchmarks covering the whole genome.

Performance assessment of DNA sequencing platforms in the ABRF Next-Generation Sequencing Study.

Assessing the reproducibility, accuracy and utility of massively parallel DNA sequencing platforms remains an ongoing challenge. Here the Association of Biomolecular Resource Facilities (ABRF) Next-Generation Sequencing Study benchmarks the performance of a set of sequencing instruments (HiSeq/NovaSeq/paired-end 2 × 250-bp chemistry, Ion S5/Proton, PacBio circular consensus sequencing (CCS), Oxford Nanopore Technologies PromethION/MinION, BGISEQ-500/MGISEQ-2000 and GS111) on human and bacterial reference DNA samples. Among short-read instruments, HiSeq 4000 and X10 provided the most consistent, highest genome coverage, while BGI/MGISEQ provided the lowest sequencing error rates. The long-read instrument PacBio CCS had the highest reference-based mapping rate and lowest non-mapping rate. The two long-read platforms PacBio CCS and PromethION/MinION showed the best sequence mapping in repeat-rich areas and across homopolymers. NovaSeq 6000 using 2 × 250-bp read chemistry was the most robust instrument for capturing known insertion/deletion events. This study serves as a benchmark for current genomics technologies, as well as a resource to inform experimental design and next-generation sequencing variant calling.

Coding and noncoding variants in EBF3 are involved in HADDS and simplex autism.

BACKGROUND: Previous research in autism and other neurodevelopmental disorders (NDDs) has indicated an important contribution of protein-coding (coding) de novo variants (DNVs) within specific genes. The role of de novo noncoding variation has been observable as a general increase in genetic burden but has yet to be resolved to individual functional elements. In this study, we assessed whole-genome sequencing data in 2671 families with autism (discovery cohort of 516 families, replication cohort of 2155 families). We focused on DNVs in enhancers with characterized in vivo activity in the brain and identified an excess of DNVs in an enhancer named hs737. RESULTS: We adapted the fitDNM statistical model to work in noncoding regions and tested enhancers for excess of DNVs in families with autism. We found only one enhancer (hs737) with nominal significance in the discovery (p = 0.0172), replication (p = 2.5 × 10-3), and combined dataset (p = 1.1 × 10-4). Each individual with a DNV in hs737 had shared phenotypes including being male, intact cognitive function, and hypotonia or motor delay. Our in vitro assessment of the DNVs showed they all reduce enhancer activity in a neuronal cell line. By epigenomic analyses, we found that hs737 is brain-specific and targets the transcription factor gene EBF3 in human fetal brain. EBF3 is genome-wide significant for coding DNVs in NDDs (missense p = 8.12 × 10-35, loss-of-function p = 2.26 × 10-13) and is widely expressed in the body. Through characterization of promoters bound by EBF3 in neuronal cells, we saw enrichment for binding to NDD genes (p = 7.43 × 10-6, OR = 1.87) involved in gene regulation. Individuals with coding DNVs have greater phenotypic severity (hypotonia, ataxia, and delayed development syndrome [HADDS]) in comparison to individuals with noncoding DNVs that have autism and hypotonia. CONCLUSIONS: In this study, we identify DNVs in the hs737 enhancer in individuals with autism. Through multiple approaches, we find hs737 targets the gene EBF3 that is genome-wide significant in NDDs. By assessment of noncoding variation and the genes they affect, we are beginning to understand their impact on gene regulatory networks in NDDs.

The genomic basis of evolutionary differentiation among honey bees.

In contrast to the western honey bee, Apis mellifera, other honey bee species have been largely neglected despite their importance and diversity. The genetic basis of the evolutionary diversification of honey bees remains largely unknown. Here, we provide a genome-wide comparison of three honey bee species, each representing one of the three subgenera of honey bees, namely the dwarf (Apis florea), giant (A. dorsata), and cavity-nesting (A. mellifera) honey bees with bumblebees as an outgroup. Our analyses resolve the phylogeny of honey bees with the dwarf honey bees diverging first. We find that evolution of increased eusocial complexity in Apis proceeds via increases in the complexity of gene regulation, which is in agreement with previous studies. However, this process seems to be related to pathways other than transcriptional control. Positive selection patterns across Apis reveal a trade-off between maintaining genome stability and generating genetic diversity, with a rapidly evolving piRNA pathway leading to genomes depleted of transposable elements, and a rapidly evolving DNA repair pathway associated with high recombination rates in all Apis species. Diversification within Apis is accompanied by positive selection in several genes whose putative functions present candidate mechanisms for lineage-specific adaptations, such as migration, immunity, and nesting behavior.

Feather Gene Expression Elucidates the Developmental Basis of Plumage Iridescence in African Starlings.

Iridescence is widespread in the living world, occurring in organisms as diverse as bacteria, plants, and animals. Yet, compared to pigment-based forms of coloration, we know surprisingly little about the developmental and molecular bases of the structural colors that give rise to iridescence. Birds display a rich diversity of iridescent structural colors that are produced in feathers by the arrangement of melanin-containing organelles called melanosomes into nanoscale configurations, but how these often unusually shaped melanosomes form, or how they are arranged into highly organized nanostructures, remains largely unknown. Here, we use functional genomics to explore the developmental basis of iridescent plumage using superb starlings (Lamprotornis superbus), which produce both iridescent blue and non-iridescent red feathers. Through morphological and chemical analyses, we confirm that hollow, flattened melanosomes in iridescent feathers are eumelanin-based, whereas melanosomes in non-iridescent feathers are solid and amorphous, suggesting that high pheomelanin content underlies red coloration. Intriguingly, the nanoscale arrangement of melanosomes within the barbules was surprisingly similar between feather types. After creating a new genome assembly, we use transcriptomics to show that non-iridescent feather development is associated with genes related to pigmentation, metabolism, and mitochondrial function, suggesting non-iridescent feathers are more energetically expensive to produce than iridescent feathers. However, iridescent feather development is associated with genes related to structural and cellular organization, suggesting that, while nanostructures themselves may passively assemble, barbules and melanosomes may require active organization to give them their shape. Together, our analyses suggest that iridescent feathers form through a combination of passive self-assembly and active processes.

Somatic variant analysis of linked-reads sequencing data with Lancet.

SUMMARY: We present a new version of the popular somatic variant caller, Lancet, that supports the analysis of linked-reads sequencing data. By seamlessly integrating barcodes and haplotype read assignments within the colored De Bruijn graph local-assembly framework, Lancet computes a barcode-aware coverage and identifies variants that disagree with the local haplotype structure. AVAILABILITY AND IMPLEMENTATION: Lancet is implemented in C++ and available for academic and non-commercial research purposes as an open-source package at https://github.com/nygenome/lancet. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

A crowdsourced set of curated structural variants for the human genome.

A high quality benchmark for small variants encompassing 88 to 90% of the reference genome has been developed for seven Genome in a Bottle (GIAB) reference samples. However a reliable benchmark for large indels and structural variants (SVs) is more challenging. In this study, we manually curated 1235 SVs, which can ultimately be used to evaluate SV callers or train machine learning models. We developed a crowdsourcing app-SVCurator-to help GIAB curators manually review large indels and SVs within the human genome, and report their genotype and size accuracy. SVCurator displays images from short, long, and linked read sequencing data from the GIAB Ashkenazi Jewish Trio son [NIST RM 8391/HG002]. We asked curators to assign labels describing SV type (deletion or insertion), size accuracy, and genotype for 1235 putative insertions and deletions sampled from different size bins between 20 and 892,149 bp. 'Expert' curators were 93% concordant with each other, and 37 of the 61 curators had at least 78% concordance with a set of 'expert' curators. The curators were least concordant for complex SVs and SVs that had inaccurate breakpoints or size predictions. After filtering events with low concordance among curators, we produced high confidence labels for 935 events. The SVCurator crowdsourced labels were 94.5% concordant with the heuristic-based draft benchmark SV callset from GIAB. We found that curators can successfully evaluate putative SVs when given evidence from multiple sequencing technologies.

ExpansionHunter Denovo: a computational method for locating known and novel repeat expansions in short-read sequencing data.

Repeat expansions are responsible for over 40 monogenic disorders, and undoubtedly more pathogenic repeat expansions remain to be discovered. Existing methods for detecting repeat expansions in short-read sequencing data require predefined repeat catalogs. Recent discoveries emphasize the need for methods that do not require pre-specified candidate repeats. To address this need, we introduce ExpansionHunter Denovo, an efficient catalog-free method for genome-wide repeat expansion detection. Analysis of real and simulated data shows that our method can identify large expansions of 41 out of 44 pathogenic repeats, including nine recently reported non-reference repeat expansions not discoverable via existing methods.

ExpansionHunter: a sequence-graph-based tool to analyze variation in short tandem repeat regions.

SUMMARY: We describe a novel computational method for genotyping repeats using sequence graphs. This method addresses the long-standing need to accurately genotype medically important loci containing repeats adjacent to other variants or imperfect DNA repeats such as polyalanine repeats. Here we introduce a new version of our repeat genotyping software, ExpansionHunter, that uses this method to perform targeted genotyping of a broad class of such loci. AVAILABILITY AND IMPLEMENTATION: ExpansionHunter is implemented in C++ and is available under the Apache License Version 2.0. The source code, documentation, and Linux/macOS binaries are available at https://github.com/Illumina/ExpansionHunter/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

A strategy for building and using a human reference pangenome.

In March 2019, 45 scientists and software engineers from around the world converged at the University of California, Santa Cruz for the first pangenomics codeathon. The purpose of the meeting was to propose technical specifications and standards for a usable human pangenome as well as to build relevant tools for genome graph infrastructures. During the meeting, the group held several intense and productive discussions covering a diverse set of topics, including advantages of graph genomes over a linear reference representation, design of new methods that can leverage graph-based data structures, and novel visualization and annotation approaches for pangenomes. Additionally, the participants self-organized themselves into teams that worked intensely over a three-day period to build a set of pipelines and tools for specific pangenomic applications. A summary of the questions raised and the tools developed are reported in this manuscript.

Genome-wide somatic variant calling using localized colored de Bruijn graphs.

Reliable detection of somatic variations is of critical importance in cancer research. Here we present Lancet, an accurate and sensitive somatic variant caller, which detects SNVs and indels by jointly analyzing reads from tumor and matched normal samples using colored de Bruijn graphs. We demonstrate, through extensive experimental comparison on synthetic and real whole-genome sequencing datasets, that Lancet has better accuracy, especially for indel detection, than widely used somatic callers, such as MuTect, MuTect2, LoFreq, Strelka, and Strelka2. Lancet features a reliable variant scoring system, which is essential for variant prioritization, and detects low-frequency mutations without sacrificing the sensitivity to call longer insertions and deletions empowered by the local-assembly engine. In addition to genome-wide analysis, Lancet allows inspection of somatic variants in graph space, which augments the traditional read alignment visualization to help confirm a variant of interest. Lancet is available as an open-source program at https://github.com/nygenome/lancet.

YES1 amplification is a mechanism of acquired resistance to EGFR inhibitors identified by transposon mutagenesis and clinical genomics.

In ∼30% of patients with EGFR-mutant lung adenocarcinomas whose disease progresses on EGFR inhibitors, the basis for acquired resistance remains unclear. We have integrated transposon mutagenesis screening in an EGFR-mutant cell line and clinical genomic sequencing in cases of acquired resistance to identify mechanisms of resistance to EGFR inhibitors. The most prominent candidate genes identified by insertions in or near the genes during the screen were MET, a gene whose amplification is known to mediate resistance to EGFR inhibitors, and the gene encoding the Src family kinase YES1. Cell clones with transposon insertions that activated expression of YES1 exhibited resistance to all three generations of EGFR inhibitors and sensitivity to pharmacologic and siRNA-mediated inhibition of YES1 Analysis of clinical genomic sequencing data from cases of acquired resistance to EGFR inhibitors revealed amplification of YES1 in five cases, four of which lacked any other known mechanisms of resistance. Preinhibitor samples, available for two of the five patients, lacked YES1 amplification. None of 136 postinhibitor samples had detectable amplification of other Src family kinases (SRC and FYN). YES1 amplification was also found in 2 of 17 samples from ALK fusion-positive lung cancer patients who had progressed on ALK TKIs. Taken together, our findings identify acquired amplification of YES1 as a recurrent and targetable mechanism of resistance to EGFR inhibition in EGFR-mutant lung cancers and demonstrate the utility of transposon mutagenesis in discovering clinically relevant mechanisms of drug resistance.