scholarly journals Long-read genome sequencing for the diagnosis of neurodevelopmental disorders

2020 ◽  
Author(s):  
Susan M. Hiatt ◽  
James M.J. Lawlor ◽  
Lori H. Handley ◽  
Ryne C. Ramaker ◽  
Brianne B. Rogers ◽  
...  
Keyword(s):  
Genome Sequencing ◽  
Neurodevelopmental Disorders ◽  
De Novo ◽  
Genomic Analysis ◽  
Data Sets ◽  
Short Read ◽  
Long Read ◽  
Variant Detection ◽  
Circular Consensus Sequencing

AbstractPurposeExome and genome sequencing have proven to be effective tools for the diagnosis of neurodevelopmental disorders (NDDs), but large fractions of NDDs cannot be attributed to currently detectable genetic variation. This is likely, at least in part, a result of the fact that many genetic variants are difficult or impossible to detect through typical short-read sequencing approaches.MethodsHere, we describe a genomic analysis using Pacific Biosciences circular consensus sequencing (CCS) reads, which are both long (>10 kb) and accurate (>99% bp accuracy). We used CCS on six proband-parent trios with NDDs that were unexplained despite extensive testing, including genome sequencing with short reads.ResultsWe identified variants and created de novo assemblies in each trio, with global metrics indicating these data sets are more accurate and comprehensive than those provided by short-read data. In one proband, we identified a likely pathogenic (LP), de novo L1-mediated insertion in CDKL5 that results in duplication of exon 3, leading to a frameshift. In a second proband, we identified multiple large de novo structural variants, including insertion-translocations affecting DGKB and MLLT3, which we show disrupt MLLT3 transcript levels. We consider this extensive structural variation likely pathogenic.ConclusionThe breadth and quality of variant detection, coupled to finding variants of clinical and research interest in two of six probands with unexplained NDDs strongly support the value of long-read genome sequencing for understanding rare disease.

scholarly journals Highly-accurate long-read sequencing improves variant detection and assembly of a human genome

2019 ◽  
Author(s):  
Aaron M. Wenger ◽  
Paul Peluso ◽  
William J. Rowell ◽  
Pi-Chuan Chang ◽  
Richard J. Hall ◽  
...  
Keyword(s):  
Single Molecule ◽  
De Novo ◽  
Structural Variants ◽  
Short Reads ◽  
Sequencing Technologies ◽  
Long Reads ◽  
Long Read ◽  
Variant Detection ◽  
High Quality Genome ◽  
Circular Consensus Sequencing

AbstractThe major DNA sequencing technologies in use today produce either highly-accurate short reads or noisy long reads. We developed a protocol based on single-molecule, circular consensus sequencing (CCS) to generate highly-accurate (99.8%) long reads averaging 13.5 kb and applied it to sequence the well-characterized human HG002/NA24385. We optimized existing tools to comprehensively detect variants, achieving precision and recall above 99.91% for SNVs, 95.98% for indels, and 95.99% for structural variants. We estimate that 2,434 discordances are correctable mistakes in the high-quality Genome in a Bottle benchmark. Nearly all (99.64%) variants are phased into haplotypes, which further improves variant detection. De novo assembly produces a highly contiguous and accurate genome with contig N50 above 15 Mb and concordance of 99.998%. CCS reads match short reads for small variant detection, while enabling structural variant detection and de novo assembly at similar contiguity and markedly higher concordance than noisy long reads.

scholarly journals Complex Structural Variants Resolved by Short-Read and Long-Read Whole Genome Sequencing in Mendelian Disorders

2018 ◽  
Author(s):  
Alba Sanchis-Juan ◽  
Jonathan Stephens ◽  
Courtney E French ◽  
Nicholas Gleadall ◽  
Karyn Mégy ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
De Novo ◽  
Genomic Variation ◽  
Mendelian Disease ◽  
Whole Genome ◽  
Structural Variants ◽  
Short Read ◽  
Long Read ◽  
Complex Structural

AbstractComplex structural variants (cxSVs) are genomic rearrangements comprising multiple structural variants, typically involving three or more breakpoint junctions. They contribute to human genomic variation and can cause Mendelian disease, however they are not typically considered during genetic testing. Here, we investigate the role of cxSVs in Mendelian disease using short-read whole genome sequencing (WGS) data from 1,324 individuals with neurodevelopmental or retinal disorders from the NIHR BioResource project. We present four cases of individuals with a cxSV affecting Mendelian disease-associated genes. Three of the cxSVs are pathogenic: a de novo duplication-inversion-inversion-deletion affecting ARID1B in an individual with Coffin-Siris syndrome, a deletion-inversion-duplication affecting HNRNPU in an individual with intellectual disability and seizures, and a homozygous deletion-inversion-deletion affecting CEP78 in an individual with cone-rod dystrophy. Additionally, we identified a de novo duplication-inversion-duplication overlapping CDKL5 in an individual with neonatal hypoxic-ischaemic encephalopathy. Long-read sequencing technology used to resolve the breakpoints demonstrated the presence of both a disrupted and an intact copy of CDKL5 on the same allele; therefore, it was classified as a variant of uncertain significance. Analysis of sequence flanking all breakpoint junctions in all the cxSVs revealed both microhomology and longer repetitive sequences, suggesting both replication and homology based processes. Accurate resolution of cxSVs is essential for clinical interpretation, and here we demonstrate that long-read WGS is a powerful technology by which to achieve this. Our results show cxSVs are an important although rare cause of Mendelian disease, and we therefore recommend their consideration during research and clinical investigations.

scholarly journals Long-read genome sequencing for the molecular diagnosis of neurodevelopmental disorders

2021 ◽  
Vol 2 (2) ◽  
pp. 100023
Author(s):  
Susan M. Hiatt ◽  
James M.J. Lawlor ◽  
Lori H. Handley ◽  
Ryne C. Ramaker ◽  
Brianne B. Rogers ◽  
...  
Keyword(s):  
Genome Sequencing ◽  
Molecular Diagnosis ◽  
Neurodevelopmental Disorders ◽  
Long Read

scholarly journals Genotyping structural variants in pangenome graphs using the vg toolkit

2019 ◽  
Author(s):  
Glenn Hickey ◽  
David Heller ◽  
Jean Monlong ◽  
Jonas A. Sibbesen ◽  
Jouni Sirén ◽  
...  
Keyword(s):  
De Novo ◽  
State Of The Art ◽  
Effective Means ◽  
Point Mutations ◽  
Structural Variants ◽  
Short Read ◽  
Yeast Strains ◽  
Sequencing Studies ◽  
Long Read

AbstractStructural variants (SVs) remain challenging to represent and study relative to point mutations despite their demonstrated importance. We show that variation graphs, as implemented in the vg toolkit, provide an effective means for leveraging SV catalogs for short-read SV genotyping experiments. We benchmarked vg against state-of-the-art SV genotypers using three sequence-resolved SV catalogs generated by recent long-read sequencing studies. In addition, we use assemblies from 12 yeast strains to show that graphs constructed directly from aligned de novo assemblies improve genotyping compared to graphs built from intermediate SV catalogs in the VCF format.

scholarly journals Mapping and phasing of structural variation in patient genomes using nanopore sequencing

2017 ◽  
Author(s):  
Mircea Cretu Stancu ◽  
Markus J. van Roosmalen ◽  
Ivo Renkens ◽  
Marleen Nieboer ◽  
Sjors Middelkamp ◽  
...  
Keyword(s):  
Single Molecule ◽  
De Novo ◽  
Structural Variants ◽  
Human Genetic Disease ◽  
Structural Genomic ◽  
Short Read ◽  
Sequencing Technologies ◽  
Genome Wide ◽  
Long Read ◽  
Complex Structural

AbstractStructural genomic variants form a common type of genetic alteration underlying human genetic disease and phenotypic variation. Despite major improvements in genome sequencing technology and data analysis, the detection of structural variants still poses challenges, particularly when variants are of high complexity. Emerging long-read single-molecule sequencing technologies provide new opportunities for detection of structural variants. Here, we demonstrate sequencing of the genomes of two patients with congenital abnormalities using the ONT MinION at 11x and 16x mean coverage, respectively. We developed a bioinformatic pipeline - NanoSV - to efficiently map genomic structural variants (SVs) from the long-read data. We demonstrate that the nanopore data are superior to corresponding short-read data with regard to detection of de novo rearrangements originating from complex chromothripsis events in the patients. Additionally, genome-wide surveillance of SVs, revealed 3,253 (33%) novel variants that were missed in short-read data of the same sample, the majority of which are duplications < 200bp in size. Long sequencing reads enabled efficient phasing of genetic variations, allowing the construction of genome-wide maps of phased SVs and SNVs. We employed read-based phasing to show that all de novo chromothripsis breakpoints occurred on paternal chromosomes and we resolved the long-range structure of the chromothripsis. This work demonstrates the value of long-read sequencing for screening whole genomes of patients for complex structural variants.

scholarly journals Complete Genome Resequencing of Thermus thermophilus Strain TMY by Hybrid Assembly of Long- and Short-Read Sequencing Technologies

2021 ◽  
Vol 10 (46) ◽  
Author(s):  
Kentaro Miyazaki ◽  
Natsuko Tokito
Keyword(s):  
Complete Genome ◽  
Thermus Thermophilus ◽  
Genomic Analysis ◽  
Comparative Genomic ◽  
Hybrid Assembly ◽  
Genome Resequencing ◽  
Short Read ◽  
Content Type ◽  
Sequencing Technologies ◽  
Long Read

Complete genome resequencing was conducted for Thermus thermophilus strain TMY by hybrid assembly of Oxford Nanopore Technologies long-read and MGI short-read data. Errors in the previously reported genome sequence determined by PacBio technology alone were corrected, allowing for high-quality comparative genomic analysis of closely related T. thermophilus genomes.

scholarly journals 4478 Not just GLUT1: genome sequencing reveals genetic heterogeneity in Doose syndrome

2020 ◽  
Vol 4 (s1) ◽  
pp. 13-13
Author(s):  
Jeffrey Dennis Calhoun ◽  
Jonathan Gunti ◽  
Katie Angione ◽  
Elizabeth Geiger ◽  
Krista Eschbach ◽  
...  
Keyword(s):  
Genome Sequencing ◽  
Genetic Heterogeneity ◽  
Subject Matter ◽  
De Novo ◽  
Structural Abnormality ◽  
Short Read ◽  
Coding Regions ◽  
Initial Analysis ◽  
Pathogenic Variants ◽  
Unrelated Subject

OBJECTIVES/GOALS: Epilepsy with myoclonic-atonic seizures (EMAS) is a childhood onset epilepsy disorder characterized by seizures with sudden loss of posture, or drop seizures. Our objective was to use short-read genome sequencing in 40 EMAS trios to better understand variants contributing to the development of EMAS. METHODS/STUDY POPULATION: Eligibility for the cohort included a potential diagnosis of EMAS by child neurology faculty at Children’s Hospital Colorado. Exclusion criteria included lack of drop seizures upon chart review or structural abnormality on MRI. Some individuals had prior genetic testing and priority for genome sequencing was given to individuals without clear genetic diagnosis based on previous testing. We analyzed single nucleotide variants (SNVs), small insertions and deletions (INDELs), and larger structural variants (SVs) from trio genomes and determined those that were likely contributory based on standardized American College of Medical Genetics (ACMG) criteria. RESULTS/ANTICIPATED RESULTS: Our initial analysis focused on variants in coding regions of known epilepsy-associated genes. We identified pathogenic or likely pathogenic variants in 6 different individuals involving 6 unique genes. Of these, 5 are de novo SNVs or INDELs and 1 is a de novo SV. One of these involve a de novo heterozygous variant in an X-linked gene (ARHGEF9) in a female individual. We hypothesize the skewed X-inactivation may result in primarily expression of the pathogenic variant. We anticipate identifying additional candidate variants in coding regions of genes previously not associated with EMAS or pediatric epilepsies as well as in noncoding regions of the genome. DISCUSSION/SIGNIFICANCE OF IMPACT: Despite the genetic heterogeneity of EMAS, our initial analysis identified de novo pathogenic or likely pathogenic variants in 15% (6/40) of our cohort. As the cost continues to decline, short read genome sequencing represents a promising diagnostic tool for EMAS and other pediatric onset epilepsy syndromes. CONFLICT OF INTEREST DESCRIPTION: The authors have no conflicts of interest to disclose. SD has consulted for Upsher-Smith, Biomarin and Neurogene on an unrelated subject matter. GLC holds a research collaborative grant with Stoke therapeutics on unrelated subject matter.

scholarly journals Improved structural variant interpretation for hereditary cancer susceptibility using long-read sequencing

2020 ◽  
Vol 22 (11) ◽  
pp. 1892-1897 ◽  
Author(s):  
My Linh Thibodeau ◽  
Kieran O’Neill ◽  
Katherine Dixon ◽  
Caralyn Reisle ◽  
Karen L. Mungall ◽  
...  
Keyword(s):  
Genome Sequencing ◽  
Hereditary Cancer ◽  
Cancer Susceptibility ◽  
Return Of Results ◽  
Advanced Cancer Patients ◽  
Short Read ◽  
Cancer Susceptibility Genes ◽  
Oxford Nanopore ◽  
Long Read ◽  
Tumor Genome

Abstract Purpose Structural variants (SVs) may be an underestimated cause of hereditary cancer syndromes given the current limitations of short-read next-generation sequencing. Here we investigated the utility of long-read sequencing in resolving germline SVs in cancer susceptibility genes detected through short-read genome sequencing. Methods Known or suspected deleterious germline SVs were identified using Illumina genome sequencing across a cohort of 669 advanced cancer patients with paired tumor genome and transcriptome sequencing. Candidate SVs were subsequently assessed by Oxford Nanopore long-read sequencing. Results Nanopore sequencing confirmed eight simple pathogenic or likely pathogenic SVs, resolving three additional variants whose impact could not be fully elucidated through short-read sequencing. A recurrent sequencing artifact on chromosome 16p13 and one complex rearrangement on chromosome 5q35 were subsequently classified as likely benign, obviating the need for further clinical assessment. Variant configuration was further resolved in one case with a complex pathogenic rearrangement affecting TSC2. Conclusion Our findings demonstrate that long-read sequencing can improve the validation, resolution, and classification of germline SVs. This has important implications for return of results, cascade carrier testing, cancer screening, and prophylactic interventions.

scholarly journals Whole-Genome Sequencing of a Human Clinical Isolate of the Novel Species Klebsiella quasivariicola sp. nov

2017 ◽  
Vol 5 (42) ◽  
Author(s):  
S. Wesley Long ◽  
Sarah E. Linson ◽  
Matthew Ojeda Saavedra ◽  
Concepcion Cantu ◽  
James J. Davis ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Clinical Isolate ◽  
Genome Sequencing ◽  
Novel Species ◽  
Whole Genome ◽  
The Novel ◽  
Short Read ◽  
Oxford Nanopore ◽  
Long Read

ABSTRACT In a study of 1,777 Klebsiella strains, we discovered KPN1705, which was distinct from all recognized Klebsiella spp. We closed the genome of strain KPN1705 using a hybrid of Illumina short-read and Oxford Nanopore long-read technologies. For this novel species, we propose the name Klebsiella quasivariicola sp. nov.

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