Linked-Read Whole Genome Sequencing Solves a Double DMD Gene Rearrangement

Next generation sequencing (NGS) has changed our approach to diagnosis of genetic disorders. Nowadays, the most comprehensive application of NGS is whole genome sequencing (WGS) that is able to detect virtually all DNA variations. However, even after accurate WGS, many genetic conditions remain unsolved. This may be due to the current NGS protocols, based on DNA fragmentation and short reads. To overcome these limitations, we applied a linked-read sequencing technology that combines single-molecule barcoding with short-read WGS. We were able to assemble haplotypes and distinguish between alleles along the genome. As an exemplary case, we studied the case of a female carrier of X-linked muscular dystrophy with an unsolved genetic status. A deletion of exons 16–29 in DMD gene was responsible for the disease in her family, but she showed a normal dosage of these exons by Multiplex Ligation-dependent Probe Amplification (MLPA) and array CGH. This situation is usually considered compatible with a “non-carrier” status. Unexpectedly, the girl also showed an increased dosage of flanking exons 1–15 and 30–34. Using linked-read WGS, we were able to distinguish between the two X chromosomes. In the first allele, we found the 16–29 deletion, while the second allele showed a 1–34 duplication: in both cases, linked-read WGS correctly mapped the borders at single-nucleotide resolution. This duplication in trans apparently restored the normal dosage of exons 16–29 seen by quantitative assays. This had a dramatic impact in genetic counselling, by converting a non-carrier into a double carrier status prediction. We conclude that linked-read WGS should be considered as a valuable option to improve our understanding of unsolved genetic conditions.

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Genetics of neuropsychiatric disease

Oxford Textbook of Neuropsychiatry ◽

10.1093/med/9780198757139.003.0012 ◽

2020 ◽

pp. 127-140

Author(s):

Stefania Bruno ◽

Nayana Lahiri

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Genetic Disorders ◽

Single Gene ◽

Diagnostic Techniques ◽

Genetic Influences ◽

Whole Genome ◽

Neuropsychiatric Disease ◽

Genomic Technologies ◽

Whole Exome

To better understand the intricacies of genetic influences on neuropsychiatric disease, it is important first to have a grounding in the models of human inheritance and current diagnostic techniques. This chapter covers the fundamentals of genetic disorders, giving insights into chromosomal, single-gene, and mitochondrial disorders. Moreover, it explores the changing applications of genomic technologies, such as whole exome and whole genome sequencing, through the lens of their implications for neuropsychiatry. Clinical examples are provided to give an idea of the genetic underpinnings of Alzheimer’s disease, Parkinson’s disease, and other familiar disorders.

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Best practices for the analytical validation of clinical whole-genome sequencing intended for the diagnosis of germline disease

npj Genomic Medicine ◽

10.1038/s41525-020-00154-9 ◽

2020 ◽

Vol 5 (1) ◽

Author(s):

Christian R. Marshall ◽

◽

Shimul Chowdhury ◽

Ryan J. Taft ◽

Mathew S. Lebo ◽

...

Keyword(s):

Best Practices ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Test Performance ◽

Genetic Disorders ◽

Chromosomal Microarray ◽

Chromosomal Microarray Analysis ◽

Whole Genome ◽

Analytical Validation ◽

Whole Exome

Abstract Whole-genome sequencing (WGS) has shown promise in becoming a first-tier diagnostic test for patients with rare genetic disorders; however, standards addressing the definition and deployment practice of a best-in-class test are lacking. To address these gaps, the Medical Genome Initiative, a consortium of leading healthcare and research organizations in the US and Canada, was formed to expand access to high-quality clinical WGS by publishing best practices. Here, we present consensus recommendations on clinical WGS analytical validation for the diagnosis of individuals with suspected germline disease with a focus on test development, upfront considerations for test design, test validation practices, and metrics to monitor test performance. This work also provides insight into the current state of WGS testing at each member institution, including the utilization of reference and other standards across sites. Importantly, members of this initiative strongly believe that clinical WGS is an appropriate first-tier test for patients with rare genetic disorders, and at minimum is ready to replace chromosomal microarray analysis and whole-exome sequencing. The recommendations presented here should reduce the burden on laboratories introducing WGS into clinical practice, and support safe and effective WGS testing for diagnosis of germline disease.

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Rapid Whole Genome Sequencing Decreases Morbidity and Healthcare Cost of Hospitalized Infants

10.1101/253534 ◽

2018 ◽

Cited By ~ 1

Author(s):

Lauge Farnaes ◽

Amber Hildreth ◽

Nathaly M. Sweeney ◽

Michelle M. Clark ◽

Shimul Chowdhury ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Clinical Utility ◽

Genetic Disorders ◽

Standard Of Care ◽

Inpatient Cost ◽

Whole Genome ◽

Improved Outcomes ◽

Historical Controls ◽

Etiologic Diagnosis

ABSTRACTBACKGROUNDGenetic disorders are a leading cause of morbidity and mortality in infants. Rapid Whole Genome Sequencing (rWGS) can diagnose genetic disorders in time to change acute medical or surgical management (clinical utility) and improve outcomes in acutely ill infants.METHODSRetrospective cohort study of acutely ill inpatient infants in a regional children’s hospital from July 2016–March 2017. Forty-two families received rWGS for etiologic diagnosis of genetic disorders. Probands received standard genetic testing as clinically indicated. Primary end-points were rate of diagnosis, clinical utility, and healthcare utilization. The latter was modelled in six infants by comparing actual utilization with matched historical controls and/or counterfactual utilization had rWGS been performed at different time points.FINDINGSThe diagnostic sensitivity was 43% (eighteen of 42 infants) for rWGS and 10% (four of 42 infants) for standard of care (P=.0005). The rate of clinical utility for rWGS (31%, thirteen of 42 infants) was significantly greater than for standard of care (2%, one of 42; P=.0015). Eleven (26%) infants with diagnostic rWGS avoided morbidity, one had 43% reduction in likelihood of mortality, and one started palliative care. In six of the eleven infants, the changes in management reduced inpatient cost by $800, 000 to $2,000,000.DISCUSSIONThese findings replicate a prior study of the clinical utility of rWGS in acutely ill inpatient infants, and demonstrate improved outcomes and net healthcare savings. rWGS merits consideration as a first tier test in this setting.

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Evaluation of Single-Molecule Sequencing Technologies for Structural Variant Detection in Two Swedish Human Genomes

Genes ◽

10.3390/genes11121444 ◽

2020 ◽

Vol 11 (12) ◽

pp. 1444

Author(s):

Nazeefa Fatima ◽

Anna Petri ◽

Ulf Gyllensten ◽

Lars Feuk ◽

Adam Ameur

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Single Molecule ◽

Large Scale ◽

Whole Genome Sequencing Data ◽

Whole Genome ◽

Sequencing Data ◽

Structural Variations ◽

Single Molecule Sequencing ◽

Human Samples

Long-read single molecule sequencing is increasingly used in human genomics research, as it allows to accurately detect large-scale DNA rearrangements such as structural variations (SVs) at high resolution. However, few studies have evaluated the performance of different single molecule sequencing platforms for SV detection in human samples. Here we performed Oxford Nanopore Technologies (ONT) whole-genome sequencing of two Swedish human samples (average 32× coverage) and compared the results to previously generated Pacific Biosciences (PacBio) data for the same individuals (average 66× coverage). Our analysis inferred an average of 17k and 23k SVs from the ONT and PacBio data, respectively, with a majority of them overlapping with an available multi-platform SV dataset. When comparing the SV calls in the two Swedish individuals, we find a higher concordance between ONT and PacBio SVs detected in the same individual as compared to SVs detected by the same technology in different individuals. Downsampling of PacBio reads, performed to obtain similar coverage levels for all datasets, resulted in 17k SVs per individual and improved overlap with the ONT SVs. Our results suggest that ONT and PacBio have a similar performance for SV detection in human whole genome sequencing data, and that both technologies are feasible for population-scale studies.

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Identification and Prioritization of Causal Variants of Human Genetic Disorders from Exome or Whole Genome Sequencing Data

10.21926/obm.genet.1802017 ◽

2018 ◽

Vol 2 (2) ◽

pp. 1-1

Author(s):

Nagarajan Paramasivam ◽

◽

Martin Granzow ◽

Christina Evers ◽

Katrin Hinderhofer ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Genetic Disorders ◽

Whole Genome Sequencing Data ◽

Whole Genome ◽

Sequencing Data ◽

Causal Variants ◽

Human Genetic Disorders

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Whole-Genome Sequencing in Diagnostics of Selected Slovenian Undiagnosed Patients with Rare Disorders

Life ◽

10.3390/life11030205 ◽

2021 ◽

Vol 11 (3) ◽

pp. 205

Author(s):

Gaber Bergant ◽

Aleš Maver ◽

Borut Peterlin

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Genetic Disorders ◽

Genetic Diseases ◽

Diagnostic Testing ◽

Positive Family History ◽

Clinical Diagnostics ◽

Whole Genome ◽

Exon Duplication ◽

The Impact

Several patients with rare genetic disorders remain undiagnosed following comprehensive diagnostic testing using whole-exome sequencing (WES). In these patients, pathogenic genetic variants may reside in intronic or regulatory regions or they may emerge through mutational mechanisms not detected by WES. For this reason, we implemented whole-genome sequencing (WGS) in routine clinical diagnostics of patients with undiagnosed genetic disorders and report on the outcome in 30 patients. Criteria for consideration included (1) negative WES, (2) a high likelihood of a genetic cause for the disorders, (3) positive family history, (4) detection of large blocks of homozygosity or (5) detection of a single pathogenic variant in a gene associated with recessive conditions. We successfully discovered a causative genetic variant in 6 cases, a retrotranspositional event in the APC gene, non-coding variants in the intronic region of the OTC gene and the promotor region of the UFM1 gene, repeat expansion in the RFC1 gene and a single exon duplication in the CNGB3 gene. We also discovered one coding variant, an indel, which was missed by variant caller during WES data analysis. Our study demonstrates the impact of WGS in the group of patients with undiagnosed genetic diseases after WES in the clinical setting and the diversity of mutational mechanisms discovered, which would remain undetected using other methods.

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