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: R/Bioconductor package https://bioconductor.org/packages/breakpointR

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

Abstract 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

Dense and accurate whole-chromosome haplotyping of individual genomes

The diploid nature of the human genome is neglected in many analyses done today, where a genome is perceived as a set of unphased variants with respect to a reference genome. This lack of haplotype-level analyses can be explained by a lack of methods that can produce dense and accurate chromosome-length haplotypes at reasonable costs. Here we introduce an integrative phasing strategy that combines global, but sparse haplotypes obtained from strand-specific single-cell sequencing (Strand-seq) with dense, yet local, haplotype information available through long-read or linked-read sequencing. We provide comprehensive guidance on the required sequencing depths and reliably assign more than 95% of alleles (NA12878) to their parental haplotypes using as few as 10 Strand-seq libraries in combination with 10-fold coverage PacBio data or, alternatively, 10X Genomics linked-read sequencing data. We conclude that the combination of Strand-seq with different technologies represents an attractive solution to chart the genetic variation of diploid genomes

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

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

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

Aptamer-based multiplexed proteomic technology for biomarker discovery

Interrogation of the human proteome in a highly multiplexed and efficient manner remains a coveted and challenging goal in biology. We present a new aptamer-based proteomic technology for biomarker discovery capable of simultaneously measuring thousands of proteins from small sample volumes (15 [mu]L of serum or plasma). Our current assay allows us to measure ~800 proteins with very low limits of detection (1 pM average), 7 logs of overall dynamic range, and 5% average coefficient of variation. This technology is enabled by a new generation of aptamers that contain chemically modified nucleotides, which greatly expand the physicochemical diversity of the large randomized nucleic acid libraries from which the aptamers are selected. Proteins in complex matrices such as plasma are measured with a process that transforms a signature of protein concentrations into a corresponding DNA aptamer concentration signature, which is then quantified with a DNA microarray. In essence, our assay takes advantage of the dual nature of aptamers as both folded binding entities with defined shapes and unique sequences recognizable by specific hybridization probes. To demonstrate the utility of our proteomics biomarker discovery technology, we applied it to a clinical study of chronic kidney disease (CKD). We identified two well known CKD biomarkers as well as an additional 58 potential CKD biomarkers. These results demonstrate the potential utility of our technology to discover unique protein signatures characteristic of various disease states. More generally, we describe a versatile and powerful tool that allows large-scale comparison of proteome profiles among discrete populations. This unbiased and highly multiplexed search engine will enable the discovery of novel biomarkers in a manner that is unencumbered by our incomplete knowledge of biology, thereby helping to advance the next generation of evidence-based medicine

Functional analysis of structural variants in single cells using Strand-seq

Somatic structural variants (SVs) are widespread in cancer, but their impact on disease evolution is understudied due to a lack of methods to directly characterize their functional consequences. We present a computational method, scNOVA, which uses Strand-seq to perform haplotype-aware integration of SV discovery and molecular phenotyping in single cells by using nucleosome occupancy to infer gene expression as a readout. Application to leukemias and cell lines identifies local effects of copy-balanced rearrangements on gene deregulation, and consequences of SVs on aberrant signaling pathways in subclones. We discovered distinct SV subclones with dysregulated Wnt signaling in a chronic lymphocytic leukemia patient. We further uncovered the consequences of subclonal chromothripsis in T cell acute lymphoblastic leukemia, which revealed c-Myb activation, enrichment of a primitive cell state and informed successful targeting of the subclone in cell culture, using a Notch inhibitor. By directly linking SVs to their functional effects, scNOVA enables systematic single-cell multiomic studies of structural variation in heterogeneous cell populations

Search for gravitational waves from binary inspirals in S3 and S4 LIGO data

We report on a search for gravitational waves from the coalescence of compact binaries during the third and fourth LIGO science runs. The search focused on gravitational waves generated during the inspiral phase of the binary evolution. In our analysis, we considered three categories of compact binary systems, ordered by mass: (i) primordial black hole binaries with masses in the range 0.35 M(sun) < m1, m2 < 1.0 M(sun), (ii) binary neutron stars with masses in the range 1.0 M(sun) < m1, m2 < 3.0 M(sun), and (iii) binary black holes with masses in the range 3.0 M(sun)< m1, m2 < m_(max) with the additional constraint m1+ m2 < m_(max), where m_(max) was set to 40.0 M(sun) and 80.0 M(sun) in the third and fourth science runs, respectively. Although the detectors could probe to distances as far as tens of Mpc, no gravitational-wave signals were identified in the 1364 hours of data we analyzed. Assuming a binary population with a Gaussian distribution around 0.75-0.75 M(sun), 1.4-1.4 M(sun), and 5.0-5.0 M(sun), we derived 90%-confidence upper limit rates of 4.9 yr^(-1) L10^(-1) for primordial black hole binaries, 1.2 yr^(-1) L10^(-1) for binary neutron stars, and 0.5 yr^(-1) L10^(-1) for stellar mass binary black holes, where L10 is 10^(10) times the blue light luminosity of the Sun.Comment: 12 pages, 11 figure

Searching for gravitational waves from known pulsars

We present upper limits on the amplitude of gravitational waves from 28 isolated pulsars using data from the second science run of LIGO. The results are also expressed as a constraint on the pulsars' equatorial ellipticities. We discuss a new way of presenting such ellipticity upper limits that takes account of the uncertainties of the pulsar moment of inertia. We also extend our previous method to search for known pulsars in binary systems, of which there are about 80 in the sensitive frequency range of LIGO and GEO 600.Comment: Accepted by CQG for the proceeding of GWDAW9, 7 pages, 2 figure