Recurrent inversion polymorphisms in humans associate with genetic instability and genomic disorders.

Unlike copy number variants (CNVs), inversions remain an underexplored genetic variation class. By integrating multiple genomic technologies, we discover 729 inversions in 41 human genomes. Approximately 85% of inversions <2 kbp form by twin-priming during L1 retrotransposition; 80% of the larger inversions are balanced and affect twice as many nucleotides as CNVs. Balanced inversions show an excess of common variants, and 72% are flanked by segmental duplications (SDs) or retrotransposons. Since flanking repeats promote non-allelic homologous recombination, we developed complementary approaches to identify recurrent inversion formation. We describe 40 recurrent inversions encompassing 0.6% of the genome, showing inversion rates up to 2.7 × 10-4 per locus per generation. Recurrent inversions exhibit a sex-chromosomal bias and co-localize with genomic disorder critical regions. We propose that inversion recurrence results in an elevated number of heterozygous carriers and structural SD diversity, which increases mutability in the population and predisposes specific haplotypes to disease-causing CNVs.

Gaps and complex structurally variant loci in phased genome assemblies.

There has been tremendous progress in phased genome assembly production by combining long-read data with parental information or linked-read data. Nevertheless, a typical phased genome assembly generated by trio-hifiasm still generates more than 140 gaps. We perform a detailed analysis of gaps, assembly breaks, and misorientations from 182 haploid assemblies obtained from a diversity panel of 77 unique human samples. Although trio-based approaches using HiFi are the current gold standard, chromosome-wide phasing accuracy is comparable when using Strand-seq instead of parental data. Importantly, the majority of assembly gaps cluster near the largest and most identical repeats (including segmental duplications [35.4%], satellite DNA [22.3%], or regions enriched in GA/AT-rich DNA [27.4%]). Consequently, 1513 protein-coding genes overlap assembly gaps in at least one haplotype, and 231 are recurrently disrupted or missing from five or more haplotypes. Furthermore, we estimate that 6-7 Mbp of DNA are misorientated per haplotype irrespective of whether trio-free or trio-based approaches are used. Of these misorientations, 81% correspond to bona fide large inversion polymorphisms in the human species, most of which are flanked by large segmental duplications. We also identify large-scale alignment discontinuities consistent with 11.9 Mbp of deletions and 161.4 Mbp of insertions per haploid genome. Although 99% of this variation corresponds to satellite DNA, we identify 230 regions of euchromatic DNA with frequent expansions and contractions, nearly half of which overlap with 197 protein-coding genes. Such variable and incompletely assembled regions are important targets for future algorithmic development and pangenome representation.

Familial long-read sequencing increases yield of de novo mutations.

Studies of de novo mutation (DNM) have typically excluded some of the most repetitive and complex regions of the genome because these regions cannot be unambiguously mapped with short-read sequencing data. To better understand the genome-wide pattern of DNM, we generated long-read sequence data from an autism parent-child quad with an affected female where no pathogenic variant had been discovered in short-read Illumina sequence data. We deeply sequenced all four individuals by using three sequencing platforms (Illumina, Oxford Nanopore, and Pacific Biosciences) and three complementary technologies (Strand-seq, optical mapping, and 10X Genomics). Using long-read sequencing, we initially discovered and validated 171 DNMs across two children-a 20% increase in the number of de novo single-nucleotide variants (SNVs) and indels when compared to short-read callsets. The number of DNMs further increased by 5% when considering a more complete human reference (T2T-CHM13) because of the recovery of events in regions absent from GRCh38 (e.g., three DNMs in heterochromatic satellites). In total, we validated 195 de novo germline mutations and 23 potential post-zygotic mosaic mutations across both children; the overall true substitution rate based on this integrated callset is at least 1.41 × 10-8 substitutions per nucleotide per generation. We also identified six de novo insertions and deletions in tandem repeats, two of which represent structural variants. We demonstrate that long-read sequencing and assembly, especially when combined with a more complete reference genome, increases the number of DNMs by >25% compared to previous studies, providing a more complete catalog of DNM compared to short-read data alone.

A high-resolution map of small-scale inversions in the gibbon genome.

Gibbons are the most speciose family of living apes, characterized by a diverse chromosome number and rapid rate of large-scale rearrangements. Here we performed single-cell template strand sequencing (Strand-seq), molecular cytogenetics, and deep in silico analysis of a southern white-cheeked gibbon genome, providing the first comprehensive map of 238 previously hidden small-scale inversions. We determined that more than half are gibbon specific, at least fivefold higher than shown for other primate lineage-specific inversions, with a significantly high number of small heterozygous inversions, suggesting that accelerated evolution of inversions may have played a role in the high sympatric diversity of gibbons. Although the precise mechanisms underlying these inversions are not yet understood, it is clear that segmental duplication-mediated NAHR only accounts for a small fraction of events. Several genomic features, including gene density and repeat (e.g., LINE-1) content, might render these regions more break-prone and susceptible to inversion formation. In the attempt to characterize interspecific variation between southern and northern white-cheeked gibbons, we identify several large assembly errors in the current GGSC Nleu3.0/nomLeu3 reference genome comprising more than 49 megabases of DNA. Finally, we provide a list of 182 candidate genes potentially involved in gibbon diversification and speciation.

Age-related differences in resting-state functional connectivity from childhood to adolescence.

The human brain is active at rest, and spontaneous fluctuations in functional MRI BOLD signals reveal an intrinsic functional architecture. During childhood and adolescence, functional networks undergo varying patterns of maturation, and measures of functional connectivity within and between networks differ as a function of age. However, many aspects of these developmental patterns (e.g. trajectory shape and directionality) remain unresolved. In the present study, we characterised age-related differences in within- and between-network resting-state functional connectivity (rsFC) and integration (i.e. participation coefficient, PC) in a large cross-sectional sample of children and adolescents (n = 628) aged 8-21 years from the Lifespan Human Connectome Project in Development. We found evidence for both linear and non-linear differences in cortical, subcortical, and cerebellar rsFC, as well as integration, that varied by age. Additionally, we found that sex moderated the relationship between age and putamen integration where males displayed significant age-related increases in putamen PC compared with females. Taken together, these results provide evidence for complex, non-linear differences in some brain systems during development.

Graded Variation in T1w/T2w Ratio during Adolescence: Measurement, Caveats, and Implications for Development of Cortical Myelin.

Adolescence is characterized by the maturation of cortical microstructure and connectivity supporting complex cognition and behavior. Axonal myelination influences brain connectivity during development by enhancing neural signaling speed and inhibiting plasticity. However, the maturational timing of cortical myelination during human adolescence remains poorly understood. Here, we take advantage of recent advances in high-resolution cortical T1w/T2w mapping methods, including principled correction of B1+ transmit field effects, using data from the Human Connectome Project in Development (HCP-D; N = 628, ages 8-21). We characterize microstructural changes relevant to myelination by estimating age-related differences in T1w/T2w throughout the cerebral neocortex from childhood to early adulthood. We apply Bayesian spline models and clustering analysis to demonstrate graded variation in age-dependent cortical T1w/T2w differences that are correlated with the sensorimotor-association (S-A) axis of cortical organization reported by others. In sensorimotor areas, T1w/T2w ratio measures start at high levels at early ages, increase at a fast pace, and decelerate at later ages (18-21). In intermediate multimodal areas along the S-A axis, T1w/T2w starts at intermediate levels and increases linearly at an intermediate pace. In transmodal/paralimbic association areas, T1w/T2w starts at low levels and increases linearly at the slowest pace. These data provide evidence for graded variation of the T1w/T2w ratio along the S-A axis that may reflect cortical myelination changes during adolescence underlying the development of complex information processing and psychological functioning. We discuss the implications of these results as well as caveats in interpreting magnetic resonance imaging (MRI)-based estimates of myelination.SIGNIFICANCE STATEMENT Myelin is a lipid membrane that is essential to healthy brain function. Myelin wraps axons to increase neural signaling speed, enabling complex neuronal functioning underlying learning and cognition. Here, we characterize the developmental timing of myelination across the cerebral cortex during adolescence using a noninvasive proxy measure, T1w/T2w mapping. Our results provide new evidence demonstrating graded variation across the cortex in the timing of T1w/T2w changes during adolescence, with rapid T1w/T2w increases in lower-order sensory areas and gradual T1w/T2w increases in higher-order association areas. This spatial pattern of microstructural brain development closely parallels the sensorimotor-to-association axis of cortical organization and plasticity during ontogeny.

Single-cell strand sequencing of a macaque genome reveals multiple nested inversions and breakpoint reuse during primate evolution.

Rhesus macaque is an Old World monkey that shared a common ancestor with human ∼25 Myr ago and is an important animal model for human disease studies. A deep understanding of its genetics is therefore required for both biomedical and evolutionary studies. Among structural variants, inversions represent a driving force in speciation and play an important role in disease predisposition. Here we generated a genome-wide map of inversions between human and macaque, combining single-cell strand sequencing with cytogenetics. We identified 375 total inversions between 859 bp and 92 Mbp, increasing by eightfold the number of previously reported inversions. Among these, 19 inversions flanked by segmental duplications overlap with recurrent copy number variants associated with neurocognitive disorders. Evolutionary analyses show that in 17 out of 19 cases, the Hominidae orientation of these disease-associated regions is always derived. This suggests that duplicated sequences likely played a fundamental role in generating inversions in humans and great apes, creating architectures that nowadays predispose these regions to disease-associated genetic instability. Finally, we identified 861 genes mapping at 156 inversions breakpoints, with some showing evidence of differential expression in human and macaque cell lines, thus highlighting candidates that might have contributed to the evolution of species-specific features. This study depicts the most accurate fine-scale map of inversions between human and macaque using a two-pronged integrative approach, such as single-cell strand sequencing and cytogenetics, and represents a valuable resource toward understanding of the biology and evolution of primate species.

ASHLEYS: automated quality control for single-cell Strand-seq data.

SUMMARY: Single-cell DNA template strand sequencing (Strand-seq) enables chromosome length haplotype phasing, construction of phased assemblies, mapping sister-chromatid exchange events and structural variant discovery. The initial quality control of potentially thousands of single-cell libraries is still done manually by domain experts. ASHLEYS automates this tedious task, delivers near-expert performance and labels even large datasets in seconds. AVAILABILITY AND IMPLEMENTATION: github.com/friendsofstrandseq/ashleys-qc, MIT license. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

White matter alterations associated with lifetime and current depression in adolescents: Evidence for cingulum disruptions.

INTRODUCTION: Compared to research on adults with depression, relatively little work has examined white matter microstructure differences in depression arising earlier in life. Here we tested hypotheses about disruptions to white matter structure in adolescents with current and past depression, with an a priori focus on the cingulum bundles, uncinate fasciculi, corpus collosum, and superior longitudinal fasciculus. METHODS: One hundred thirty-one children from the Preschool Depression Study were assessed using a Human Connectome Project style diffusion imaging sequence which was processed with HCP pipelines and TRACULA to generate estimates of fractional anisotropy (FA), mean diffusivity (MD), axial diffusivity (AD) and radial diffusivity (RD). RESULTS: We found that reduced FA, reduced AD, and increased RD in the dorsal cingulum bundle were associated with a lifetime diagnosis of major depression and greater cumulative and current depression severity. Reduced FA, reduced AD, and increased RD in the ventral cingulum were associated with greater cumulative depression severity. CONCLUSION: These findings support the emergence of white matter differences detected in adolescence associated with earlier life and concurrent depression. They also highlight the importance of connections of the cingulate to other brain regions in association with depression, potentially relevant to understanding emotion dysregulation and functional connectivity differences in depression.

Genomic inversions and GOLGA core duplicons underlie disease instability at the 15q25 locus.

Human chromosome 15q25 is involved in several disease-associated structural rearrangements, including microdeletions and chromosomal markers with inverted duplications. Using comparative fluorescence in situ hybridization, strand-sequencing, single-molecule, real-time sequencing and Bionano optical mapping analyses, we investigated the organization of the 15q25 region in human and nonhuman primates. We found that two independent inversions occurred in this region after the fission event that gave rise to phylogenetic chromosomes XIV and XV in humans and great apes. One of these inversions is still polymorphic in the human population today and may confer differential susceptibility to 15q25 microdeletions and inverted duplications. The inversion breakpoints map within segmental duplications containing core duplicons of the GOLGA gene family and correspond to the site of an ancestral centromere, which became inactivated about 25 million years ago. The inactivation of this centromere likely released segmental duplications from recombination repression typical of centromeric regions. We hypothesize that this increased the frequency of ectopic recombination creating a hotspot of hominid inversions where dispersed GOLGA core elements now predispose this region to recurrent genomic rearrangements associated with disease.

VISOR: a versatile haplotype-aware structural variant simulator for short- and long-read sequencing.

SUMMARY: VISOR is a tool for haplotype-specific simulations of simple and complex structural variants (SVs). The method is applicable to haploid, diploid or higher ploidy simulations for bulk or single-cell sequencing data. SVs are implanted into FASTA haplotypes at single-basepair resolution, optionally with nearby single-nucleotide variants. Short or long reads are drawn at random from these haplotypes using standard error profiles. Double- or single-stranded data can be simulated and VISOR supports the generation of haplotype-tagged BAM files. The tool further includes methods to interactively visualize simulated variants in single-stranded data. The versatility of VISOR is unmet by comparable tools and it lays the foundation to simulate haplotype-resolved cancer heterogeneity data in bulk or at single-cell resolution. AVAILABILITY AND IMPLEMENTATION: VISOR is implemented in python 3.6, open-source and freely available at https://github.com/davidebolo1993/VISOR. Documentation is available at https://davidebolo1993.github.io/visordoc/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

breakpointR: an R/Bioconductor package to localize strand state changes in Strand-seq data.

MOTIVATION: Strand-seq is a specialized single-cell DNA sequencing technique centered around the directionality of single-stranded DNA. Computational tools for Strand-seq analyses must capture the strand-specific information embedded in these data. RESULTS: Here we introduce breakpointR, an R/Bioconductor package specifically tailored to process and interpret single-cell strand-specific sequencing data obtained from Strand-seq. We developed breakpointR to detect local changes in strand directionality of aligned Strand-seq data, to enable fine-mapping of sister chromatid exchanges, germline inversion and to support global haplotype assembly. Given the broad spectrum of Strand-seq applications we expect breakpointR to be an important addition to currently available tools and extend the accessibility of this novel sequencing technique. AVAILABILITY AND IMPLEMENTATION: R/Bioconductor package https://bioconductor.org/packages/breakpointR. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

A feasibility trial of parent HPV vaccine reminders and phone-based motivational interviewing.

BACKGROUND: We assessed the feasibility and acceptability of a sequential approach of parent-targeted HPV vaccine reminders and phone-based Motivation Interviewing (MI). METHODS: In 2016, we selected all 11- to 12-year-old boys and girls seen in one clinic whose vaccine records did not include the HPV vaccine (n=286). By gender, we individually randomized parents of adolescents to an interactive text message (74 girls and 45 boys), postcard reminder (46 boys and no girls because of previously demonstrated efficacy), or standard care group (75 girls and 46 boys). Reminders were sent with medical director permission and a HIPAA waiver. Two months after reminders, among the adolescents whose vaccine records still did not include the HPV vaccine, we selected a gender-stratified random sample of 20 parents for phone-based MI. We assessed the percentage of deliverable messages, the percentage of parents' responding to the interactive text message, parent acceptability of receiving a text message, and MI parent responsiveness and interviewer competence (MI Treatment Integrity Coding system). RESULTS: Nearly all messages were deliverable (98% of postcards and 74% of text messages). Six of the 88 parents (7%) receiving text messages scheduled an appointment through our interactive system. The acceptability survey response rate was 37% (38/102). Respondents were favorable toward vaccine reminders for all parents (82%). Among 20 sampled parents, 17 were reached by phone of whom 7 completed MI, 4 had or were getting the HPV vaccine for their child, and 5 expressed disinterest. Across the 7 MI calls, the interviewer was rated 100% MI adherent and scored an average 4.19 rating for Global Spirit. CONCLUSION: Without providing explicit consent to receive vaccine-related messages, parents nonetheless found postcards and interactive text messages acceptable. Centralizing MI to phone calls with trained staff was acceptable to parents and resulted in highly MI-adherent interviews.

Construction of Whole Genomes from Scaffolds Using Single Cell Strand-Seq Data.

Accurate reference genome sequences provide the foundation for modern molecular biology and genomics as the interpretation of sequence data to study evolution, gene expression, and epigenetics depends heavily on the quality of the genome assembly used for its alignment. Correctly organising sequenced fragments such as contigs and scaffolds in relation to each other is a critical and often challenging step in the construction of robust genome references. We previously identified misoriented regions in the mouse and human reference assemblies using Strand-seq, a single cell sequencing technique that preserves DNA directionality Here we demonstrate the ability of Strand-seq to build and correct full-length chromosomes by identifying which scaffolds belong to the same chromosome and determining their correct order and orientation, without the need for overlapping sequences. We demonstrate that Strand-seq exquisitely maps assembly fragments into large related groups and chromosome-sized clusters without using new assembly data. Using template strand inheritance as a bi-allelic marker, we employ genetic mapping principles to cluster scaffolds that are derived from the same chromosome and order them within the chromosome based solely on directionality of DNA strand inheritance. We prove the utility of our approach by generating improved genome assemblies for several model organisms including the ferret, pig, Xenopus, zebrafish, Tasmanian devil and the Guinea pig.

Improved assembly and variant detection of a haploid human genome using single-molecule, high-fidelity long reads.

The sequence and assembly of human genomes using long-read sequencing technologies has revolutionized our understanding of structural variation and genome organization. We compared the accuracy, continuity, and gene annotation of genome assemblies generated from either high-fidelity (HiFi) or continuous long-read (CLR) datasets from the same complete hydatidiform mole human genome. We find that the HiFi sequence data assemble an additional 10% of duplicated regions and more accurately represent the structure of tandem repeats, as validated with orthogonal analyses. As a result, an additional 5 Mbp of pericentromeric sequences are recovered in the HiFi assembly, resulting in a 2.5-fold increase in the NG50 within 1 Mbp of the centromere (HiFi 480.6 kbp, CLR 191.5 kbp). Additionally, the HiFi genome assembly was generated in significantly less time with fewer computational resources than the CLR assembly. Although the HiFi assembly has significantly improved continuity and accuracy in many complex regions of the genome, it still falls short of the assembly of centromeric DNA and the largest regions of segmental duplication using existing assemblers. Despite these shortcomings, our results suggest that HiFi may be the most effective standalone technology for de novo assembly of human genomes.

Characterizing polymorphic inversions in human genomes by single-cell sequencing.

Identifying genomic features that differ between individuals and cells can help uncover the functional variants that drive phenotypes and disease susceptibilities. For this, single-cell studies are paramount, as it becomes increasingly clear that the contribution of rare but functional cellular subpopulations is important for disease prognosis, management, and progression. Until now, studying these associations has been challenged by our inability to map structural rearrangements accurately and comprehensively. To overcome this, we coupled single-cell sequencing of DNA template strands (Strand-seq) with custom analysis software to rapidly discover, map, and genotype genomic rearrangements at high resolution. This allowed us to explore the distribution and frequency of inversions in a heterogeneous cell population, identify several polymorphic domains in complex regions of the genome, and locate rare alleles in the reference assembly. We then mapped the entire genomic complement of inversions within two unrelated individuals to characterize their distinct inversion profiles and built a nonredundant global reference of structural rearrangements in the human genome. The work described here provides a powerful new framework to study structural variation and genomic heterogeneity in single-cell samples, whether from individuals for population studies or tissue types for biomarker discovery.

Direct chromosome-length haplotyping by single-cell sequencing.

Haplotypes are fundamental to fully characterize the diploid genome of an individual, yet methods to directly chart the unique genetic makeup of each parental chromosome are lacking. Here we introduce single-cell DNA template strand sequencing (Strand-seq) as a novel approach to phasing diploid genomes along the entire length of all chromosomes. We demonstrate this by building a complete haplotype for a HapMap individual (NA12878) at high accuracy (concordance 99.3%), without using generational information or statistical inference. By use of this approach, we mapped all meiotic recombination events in a family trio with high resolution (median range ∼14 kb) and phased larger structural variants like deletions, indels, and balanced rearrangements like inversions. Lastly, the single-cell resolution of Strand-seq allowed us to observe loss of heterozygosity regions in a small number of cells, a significant advantage for studies of heterogeneous cell populations, such as cancer cells. We conclude that Strand-seq is a unique and powerful approach to completely phase individual genomes and map inheritance patterns in families, while preserving haplotype differences between single cells.

Strand-seq enables reliable separation of long reads by chromosome via expectation maximization.

Motivation: Current sequencing technologies are able to produce reads orders of magnitude longer than ever possible before. Such long reads have sparked a new interest in de novo genome assembly, which removes reference biases inherent to re-sequencing approaches and allows for a direct characterization of complex genomic variants. However, even with latest algorithmic advances, assembling a mammalian genome from long error-prone reads incurs a significant computational burden and does not preclude occasional misassemblies. Both problems could potentially be mitigated if assembly could commence for each chromosome separately. Results: To address this, we show how single-cell template strand sequencing (Strand-seq) data can be leveraged for this purpose. We introduce a novel latent variable model and a corresponding Expectation Maximization algorithm, termed SaaRclust, and demonstrates its ability to reliably cluster long reads by chromosome. For each long read, this approach produces a posterior probability distribution over all chromosomes of origin and read directionalities. In this way, it allows to assess the amount of uncertainty inherent to sparse Strand-seq data on the level of individual reads. Among the reads that our algorithm confidently assigns to a chromosome, we observed more than 99% correct assignments on a subset of Pacific Bioscience reads with 30.1× coverage. To our knowledge, SaaRclust is the first approach for the in silico separation of long reads by chromosome prior to assembly. Availability and implementation: https://github.com/daewoooo/SaaRclust.

DNA template strand sequencing of single-cells maps genomic rearrangements at high resolution.

DNA rearrangements such as sister chromatid exchanges (SCEs) are sensitive indicators of genomic stress and instability, but they are typically masked by single-cell sequencing techniques. We developed Strand-seq to independently sequence parental DNA template strands from single cells, making it possible to map SCEs at orders-of-magnitude greater resolution than was previously possible. On average, murine embryonic stem (mES) cells exhibit eight SCEs, which are detected at a resolution of up to 23 bp. Strikingly, Strand-seq of 62 single mES cells predicts that the mm 9 mouse reference genome assembly contains at least 17 incorrectly oriented segments totaling nearly 1% of the genome. These misoriented contigs and fragments have persisted through several iterations of the mouse reference genome and have been difficult to detect using conventional sequencing techniques. The ability to map SCE events at high resolution and fine-tune reference genomes by Strand-seq dramatically expands the scope of single-cell sequencing.

Cell-type-specific consequences of mosaic structural variants in hematopoietic stem and progenitor cells.

The functional impact and cellular context of mosaic structural variants (mSVs) in normal tissues is understudied. Utilizing Strand-seq, we sequenced 1,133 single-cell genomes from 19 human donors of increasing age, and discovered the heterogeneous mSV landscapes of hematopoietic stem and progenitor cells. While mSVs are continuously acquired throughout life, expanded subclones in our cohort are confined to individuals >60. Cells already harboring mSVs are more likely to acquire additional somatic structural variants, including megabase-scale segmental aneuploidies. Capitalizing on comprehensive single-cell micrococcal nuclease digestion with sequencing reference data, we conducted high-resolution cell-typing for eight hematopoietic stem and progenitor cells. Clonally expanded mSVs disrupt normal cellular function by dysregulating diverse cellular pathways, and enriching for myeloid progenitors. Our findings underscore the contribution of mSVs to the cellular and molecular phenotypes associated with the aging hematopoietic system, and establish a foundation for deciphering the molecular links between mSVs, aging and disease susceptibility in normal tissues.