Structural variation accounts for much of the variation among human genomes. Structural variants of all types are known to cause Mendelian disease and contribute to complex disease. Learn how long-read sequencing is enabling detection of the full spectrum of structural variants to advance the study of human disease, evolution and genetic diversity.
During the past decade, the search for pathogenic mutations in rare human genetic diseases has involved huge efforts to sequence coding regions, or the entire genome, using massively parallel short-read sequencers. However, the approximate current diagnostic rate is
Achieving high intraspecific genetic diversity is a critical goal in ecological restoration as it increases the adaptive potential and long-term resilience of populations. Thus, we investigated genetic diversity within and between pristine sites in a fossil floodplain and compared it to sites restored by hay-transfer between 1997 and 2014. RAD-seq genotyping revealed that the stenoecious flood-plain species Arabis nemorensis is co-occurring with individuals that, based on ploidy, ITS-sequencing and morphology, probably belong to the close relative Arabis sagittata, which has a documented preference for dry calcareous grasslands but has not been reported in floodplain meadows. We show that hay-transfer maintains…
Phenotypic change is a hallmark of bacterial adaptation during chronic infection. In the case of chronic Pseudomonas aeruginosa lung infection in patients with cystic fibrosis, well-characterized phenotypic variants include mucoid and small colony variants (SCVs). It has previously been shown that SCVs can be reproducibly isolated from the murine lung following the establishment of chronic infection with mucoid P. aeruginosa strain NH57388A. Using a combination of single-molecule real-time (PacBio) and Illumina sequencing we identify a large genomic inversion in the SCV through recombination between homologous regions of two rRNA operons and an associated truncation of one of the 16S rRNA…
Gloeostereum incarnatum is a precious edible mushroom that is widely grown in Asia and known for its useful medicinal properties. Here, we present a high-quality genome of G. incarnatum using the single-molecule real-time (SMRT) sequencing platform. The G. incarnatum genome, which is the first complete genome to be sequenced in the family Cyphellaceae, was 38.67 Mbp, with an N50 of 3.5 Mbp, encoding 15,251 proteins. Based on our phylogenetic analysis, the Cyphellaceae diverged ~174 million years ago. Several genes and gene clusters associated with lignocellulose degradation, secondary metabolites, and polysaccharide biosynthesis were identified in G. incarnatum, and compared with other…
Tandem repeat (TR) expansions have been implicated in dozens of genetic diseases, including Huntington’s Disease, Fragile X Syndrome, and hereditary ataxias. Furthermore, TRs have recently been implicated in a range of complex traits, including gene expression and cancer risk. While the human genome harbors hundreds of thousands of TRs, analysis of TR expansions has been mainly limited to known pathogenic loci. A major challenge is that expanded repeats are beyond the read length of most next-generation sequencing (NGS) datasets and are not profiled by existing genome-wide tools. We present GangSTR, a novel algorithm for genome-wide genotyping of both short and…
To date, clinical sequencing has focused on genomic DNA using targeted panels and exome sequencing. Sequencing of a large hypertrophic cardiomyopathy (HCM) cohort revealed that positive identification of a disease-associated variant was returned in only 32% of patients, with an additional 15% receiving inconclusive results. When genome sequencing fails to reveal causative variants, the transcriptome may provide additional diagnostic clarity. A recent study examining patients with genetically undiagnosed muscle disorders found that RNA sequencing, when used as a complement to exome and whole genome sequencing, had an overall diagnosis rate of 35%.
Downy mildews are obligate biotrophic oomycete pathogens that cause devastating plant diseases on economically important crops. Plasmopara viticola is the causal agent of grapevine downy mildew, a major disease in vineyards worldwide. We sequenced the genome of Pl. viticola with PacBio long reads and obtained a new 92.94?Mb assembly with high contiguity (359 scaffolds for a N50 of 706.5?kb) due to a better resolution of repeat regions. This assembly presented a high level of gene completeness, recovering 1,592 genes encoding secreted proteins involved in plant-pathogen interactions. Plasmopara viticola had a two-speed genome architecture, with secreted protein-encoding genes preferentially located in…
In the past several years, single-molecule sequencing platforms, such as those by Pacific Biosciences and Oxford Nanopore Technologies, have become available to researchers and are currently being tested for clinical applications. They offer exceptionally long reads that permit direct sequencing through regions of the genome inaccessible or difficult to analyze by short-read platforms. This includes disease-causing long repetitive elements, extreme GC content regions, and complex gene loci. Similarly, these platforms enable structural variation characterization at previously unparalleled resolution and direct detection of epigenetic marks in native DNA. Here, we review how these technologies are opening up new clinical avenues that…
The discovery of mutations associated with human genetic dis- ease is an exercise in comparative genomics (see Glossary). Although there are many different strategies and approaches, the central premise is that affected persons harbor a significant excess of pathogenic DNA variants as com- pared with a group of unaffected persons (controls) that is either clinically defined1 or established by surveying large swaths of the general population.2 The more exclu- sive the variant is to the disease, the greater its penetrance, the larger its effect size, and the more relevant it becomes to both disease diagnosis and future therapeutic investigation. The…
Benchmark small variant calls are required for developing, optimizing and assessing the performance of sequencing and bioinformatics methods. Here, as part of the Genome in a Bottle (GIAB) Consortium, we apply a reproducible, cloud-based pipeline to integrate multiple short- and linked-read sequencing datasets and provide benchmark calls for human genomes. We generate benchmark calls for one previously analyzed GIAB sample, as well as six genomes from the Personal Genome Project. These new genomes have broad, open consent, making this a ‘first of its kind’ resource that is available to the community for multiple downstream applications. We produce 17% more benchmark…
We report a family with progressive myoclonic epilepsy who underwent whole-exome sequencing but was negative for pathogenic variants. Similar clinical courses of a devastating neurodegenerative phenotype of two affected siblings were highly suggestive of a genetic etiology, which indicates that the survey of genetic variation by whole-exome sequencing was not comprehensive. To investigate the presence of a variant that remained unrecognized by standard genetic testing, PacBio long-read sequencing was performed. Structural variant (SV) detection using low-coverage (6×) whole-genome sequencing called 17,165 SVs (7,216 deletions and 9,949 insertions). Our SV selection narrowed down potential candidates to only five SVs (two deletions…
The locus for familial cortical myoclonic tremor with epilepsy (FCMTE) has long been mapped to 8q24 in linkage studies, but the causative mutations remain unclear. Recently, expansions of intronic TTTCA and TTTTA repeat motifs within SAMD12 were found to be involved in the pathogenesis of FCMTE in Japanese pedigrees. We aim to identify the causative mutations of FCMTE in Chinese pedigrees.We performed genetic linkage analysis by microsatellite markers in a five-generation Chinese pedigree with 55 members. We also used array-comparative genomic hybridisation (CGH) and next-generation sequencing (NGS) technologies (whole-exome sequencing, capture region deep sequencing and whole-genome sequencing) to identify the…
Combining high-throughput sequencing with targeted sequence capture has become an attractive tool to study specific genomic regions of interest. Most studies have so far focused on the exome using short-read technology. These approaches are not designed to capture intergenic regions needed to reconstruct genomic organization, including regulatory regions and gene synteny. Here, we demonstrate the power of combining targeted sequence capture with long-read sequencing technology for comparative genomic analyses of the haemoglobin (Hb) gene clusters across eight species separated by up to 70 million years. Guided by the reference genome assembly of the Atlantic cod (Gadus morhua) together with genome…
Neuronal intranuclear inclusion disease (NIID) is a progressive neurodegenerative disease that is characterized by eosinophilic hyaline intranuclear inclusions in neuronal and somatic cells. The wide range of clinical manifestations in NIID makes ante-mortem diagnosis difficult1-8, but skin biopsy enables its ante-mortem diagnosis9-12. The average onset age is 59.7 years among approximately 140 NIID cases consisting of mostly sporadic and several familial cases. By linkage mapping of a large NIID family with several affected members (Family 1), we identified a 58.1 Mb linked region at 1p22.1-q21.3 with a maximum logarithm of the odds score of 4.21. By long-read sequencing, we identified…