In this video Aaron Wenger describes the new structural variant detection application, known as pbsv, available in SMRT Link v5.0. This application identifies large (default: =50 bp) insertions and deletions…
Explore human genetic variation and learn how SMRT Sequencing uncovers the full spectrum of structural variation to advance understanding of genetic disease and broaden our knowledge of human diversity.
Most of the basepairs that differ between two human genomes are in intermediate-sized structural variants (50 bp to 5 kb), which are too small to detect with array CGH but…
In this ASHG 2017 presentation, Jonas Korlach, the CSO of PacBio shared updates on three applications featuring SMRT Sequencing on the Sequel System, highlighting structural variant detection, targeted sequencing and…
Howard Jacob, Chief Genomics Officer at the HudsonAlpha Institute for Biotechnology, explored the role of genomics in diagnosing rare diseases. In this podcast he shared his views on the economics…
In this video, Aaron Wenger, a research scientist at PacBio, describes the use of long-read SMRT Sequencing to detect structural variants in the human genome. He shares that structural variations…
Structural variants (SVs, differences >50 base pairs) account for most of the base pairs that differ between two human genomes, and are known to cause over 1,000 genetic disorders including…
The lack of diversity in genomic data has been an issue of growing concern. It threatens to limit the benefits from the massive investment that has been made to date…
In this presentation Fritz Sedlazeck describes his latest work to obtain comprehensive genomes leveraging long-read sequencing and linked reads.
In this PacBio User Group Meeting presentation, Jonas Korlach and Roberto Lleras share the latest updates to the structural variation application and analysis tools.
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. © 2019 John Wiley & Sons Ltd/University College London.
New technologies and analysis methods are enabling genomic structural variants (SVs) to be detected with ever-increasing accuracy, resolution, and comprehensiveness. Translating these methods to routine research and clinical practice requires robust benchmark sets. We developed the first benchmark set for identification of both false negative and false positive germline SVs, which complements recent efforts emphasizing increasingly comprehensive characterization of SVs. To create this benchmark for a broadly consented son in a Personal Genome Project trio with broadly available cells and DNA, the Genome in a Bottle (GIAB) Consortium integrated 19 sequence-resolved variant calling methods, both alignment- and de novo assembly-based, from short-, linked-, and long-read sequencing, as well as optical and electronic mapping. The final benchmark set contains 12745 isolated, sequence-resolved insertion and deletion calls =50 base pairs (bp) discovered by at least 2 technologies or 5 callsets, genotyped as heterozygous or homozygous variants by long reads. The Tier 1 benchmark regions, for which any extra calls are putative false positives, cover 2.66 Gbp and 9641 SVs supported by at least one diploid assembly. Support for SVs was assessed using svviz with short-, linked-, and long-read sequence data. In general, there was strong support from multiple technologies for the benchmark SVs, with 90 % of the Tier 1 SVs having support in reads from more than one technology. The Mendelian genotype error rate was 0.3 %, and genotype concordance with manual curation was >98.7 %. We demonstrate the utility of the benchmark set by showing it reliably identifies both false negatives and false positives in high-quality SV callsets from short-, linked-, and long-read sequencing and optical mapping.
After nearly two decades of improvements, the current human reference genome (GRCh38) is the most accurate and complete vertebrate genome ever produced. However, no one chromosome has been finished end to end, and hundreds of unresolved gaps persist. The remaining gaps include ribosomal rDNA arrays, large near-identical segmental duplications, and satellite DNA arrays. These regions harbor largely unexplored variation of unknown consequence, and their absence from the current reference genome can lead to experimental artifacts and hide true variants when re-sequencing additional human genomes. Here we present a de novo human genome assembly that surpasses the continuity of GRCh38, along with the first gapless, telomere-to-telomere assembly of a human chromosome. This was enabled by high-coverage, ultra-long-read nanopore sequencing of the complete hydatidiform mole CHM13 genome, combined with complementary technologies for quality improvement and validation. Focusing our efforts on the human X chromosome 3, we reconstructed the ~2.8 megabase centromeric satellite DNA array and closed all 29 remaining gaps in the current reference, including new sequence from the human pseudoautosomal regions and cancer-testis ampliconic gene families (CT-X and GAGE). This complete chromosome X, combined with the ultra-long nanopore data, also allowed us to map methylation patterns across complex tandem repeats and satellite arrays for the first time. These results demonstrate that finishing the human genome is now within reach and will enable ongoing efforts to complete the remaining human chromosomes.
Accurate detection and genotyping of structural variations (SVs) from short-read data is a long-standing area of development in genomics research and clinical sequencing pipelines. We introduce Paragraph, a fast and accurate genotyper that models SVs using sequence graphs and SV annotations produced by a range of methods and technologies. We demonstrate the accuracy of Paragraph on whole genome sequence data from a control sample with both short and long read sequencing data available, and then apply it at scale to a cohort of 100 samples of diverse ancestry sequenced with short-reads. Comparative analyses indicate that Paragraph has better accuracy than other existing genotypers. The Paragraph software is open-source and available at ?https://github.com/Illumina/paragraph
The activity of the retrotransposon LINE-1 has created a substantial portion of the human genome. Most of this sequence comprises fractured and debilitated LINE-1s. An accurate approximation of the number, location, and sequence of the LINE-1 elements present in any single genome has proven elusive due to the difficulty of assembling and phasing the repetitive and polymorphic regions of the human genome. Through an in-depth analysis of publicly-available, deep, long-read assemblies of nearly homozygous human genomes, we defined the location and sequence of all intact LINE-1s in these assemblies. We found 148 and 142 intact LINE-1s in two nearly homozygous assemblies. A combination of these assemblies suggests a diploid human genome contains at least 50% more intact LINE-1s than previous estimates textendash in this case, 290 intact LINE-1s at 194 loci. We think this is the best approximation, to date, of the number of intact LINE-1s in a single diploid human genome. In addition to counting intact LINE-1 elements, we resolved the sequence of each element, including some LINE-1 elements in unassembled, presumably centromeric regions of the genome. A comparison of the intact LINE-1s in each assembly shows the specific pattern of variation between these genomes, including LINE-1s that remain intact in only one genome, allelic variation in shared intact LINE-1s, and LINE-1s that are unique (presumably young) insertions in only one genome. We found that many old elements (> 6 million years old) remain intact, and comparison of the young and intact LINE-1s across assemblies reinforces the notion that only a small portion of all LINE-1 sequences that may be intact in the genomes of the human population has been uncovered. This dataset provides the first nearly comprehensive estimate of LINE-1 diversity within an individual, an important dataset in the quest to understand the functional consequences of sequence variation in LINE-1 and the complete set of LINE-1s in the human population.
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