scholarly journals Linked-Read sequencing resolves complex structural variants

2017 ◽  
Author(s):  
Sarah Garcia ◽  
Stephen Williams ◽  
Andrew Wei Xu ◽  
Jill Herschleb ◽  
Patrick Marks ◽  
...  
Keyword(s):  
Chromosomal Rearrangements ◽  
Copy Number Variants ◽  
Cost Effective ◽  
Sequencing Depth ◽  
Structural Variants ◽  
Single Nucleotide Variants ◽  
Candidate Sequence ◽  
Full Spectrum ◽  
Short Reads ◽  
Complex Structural

SummaryLarge genomic structural variants (>50bp) are important contributors to disease, yet they remain one of the most difficult types of variation to accurately ascertain, in part because they tend to cluster in duplicated and repetitive regions, but also because the various signals for these events can be challenging to detect with short reads. Clinically, aCGH and karyotype remain the most commonly used assays for genome-wide structural variant (SV) detection, though there is clear potential benefit to an NGS-based assay that accurately detects both SVs and single nucleotide variants. Linked-Read sequencing is a relatively simple, fast, and cost-effective method that is applicable to both genome and targeted assays. Linked-Reads are generated by performing haplotype-level dilution of long input DNA molecules into >1 million barcoded partitions, generating barcoded short reads within those partitions, and then performing short read sequencing in bulk. We performed 30x Linked-Read genome sequencing on a set of 23 samples with known balanced or unbalanced SVs. Twenty-seven of the 29 known events were detected and another event was called as a candidate. Sequence downsampling was performed on a subset to determine the lowest sequence depth required to detect variations. Copy-number variants can be called with as little as 1-2x sequencing depth (5-10Gb) while balanced events require on the order of 10x coverage for variant calls to be made, although specific signal is clearly present at 1-2x sequencing depth. In addition to detecting a full spectrum of variant types with a single test, Linked-Read sequencing provides base-level resolution of breakpoints, enabling complete resolution of even the most complex chromosomal rearrangements.

scholarly journals Targeted long-read sequencing resolves complex structural variants and identifies missing disease-causing variants

2020 ◽  
Author(s):  
Danny E. Miller ◽  
Arvis Sulovari ◽  
Tianyun Wang ◽  
Hailey Loucks ◽  
Kendra Hoekzema ◽  
...  
Keyword(s):  
Copy Number ◽  
Genetic Diagnosis ◽  
Clinical Testing ◽  
Structural Variants ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Pathogenic Variants ◽  
Long Read ◽  
Repeat Expansions ◽  
Complex Structural

ABSTRACTBACKGROUNDDespite widespread availability of clinical genetic testing, many individuals with suspected genetic conditions do not have a precise diagnosis. This limits their opportunity to take advantage of state-of-the-art treatments. In such instances, testing sometimes reveals difficult-to-evaluate complex structural differences, candidate variants that do not fully explain the phenotype, single pathogenic variants in recessive disorders, or no variants in specific genes of interest. Thus, there is a need for better tools to identify a precise genetic diagnosis in individuals when conventional testing approaches have been exhausted.METHODSTargeted long-read sequencing (T-LRS) was performed on 33 individuals using Read Until on the Oxford Nanopore platform. This method allowed us to computationally target up to 100 Mbp of sequence per experiment, resulting in an average of 20x coverage of target regions, a 500% increase over background. We analyzed patient DNA for pathogenic substitutions, structural variants, and methylation differences using a single data source.RESULTSThe effectiveness of T-LRS was validated by detecting all genomic aberrations, including single-nucleotide variants, copy number changes, repeat expansions, and methylation differences, previously identified by prior clinical testing. In 6/7 individuals who had complex structural rearrangements, T-LRS enabled more precise resolution of the mutation, which led, in one case, to a change in clinical management. In nine individuals with suspected Mendelian conditions who lacked a precise genetic diagnosis, T-LRS identified pathogenic or likely pathogenic variants in five and variants of uncertain significance in two others.CONCLUSIONST-LRS can accurately predict pathogenic copy number variants and triplet repeat expansions, resolve complex rearrangements, and identify single-nucleotide variants not detected by other technologies, including short-read sequencing. T-LRS represents an efficient and cost-effective strategy to evaluate high-priority candidate genes and regions or to further evaluate complex clinical testing results. The application of T-LRS will likely increase the diagnostic rate of rare disorders.

scholarly journals Genomic and transcriptomic somatic alterations of hepatocellular carcinoma in non-cirrhotic livers

2021 ◽  
Author(s):  
Zachary L Skidmore ◽  
Jason Kunisaki ◽  
Yiing Lin ◽  
Kelsy C Cotto ◽  
Erica K Barnell ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Copy Number Variants ◽  
Genomic Analysis ◽  
Structural Variants ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Tert Promoter Mutations ◽  
Somatic Alterations ◽  
Differential Gene ◽  
Promoter Mutations

Background: Liver cancer is the second leading cause of cancer-related deaths worldwide. Hepatocellular carcinoma (HCC) risk factors include chronic hepatitis, cirrhosis, and alcohol abuse, whereby tumorigenesis is induced through inflammation and subsequent fibrotic response. However, a subset of HCC arises in non-cirrhotic livers. We characterized the genomic and transcriptomic landscape of non-cirrhotic HCC to identify features underlying the disease's development and progression. Methods: Whole genome and transcriptome sequencing was performed on 30 surgically resectable tumors comprised of primarily of non-cirrhotic HCC and adjacent normal tissue. Using somatic variants, capture reagents were created and employed on an additional 87 cases of mixed cirrhotic/non-cirrhotic HCC. Cases were analyzed to identify viral integrations, single nucleotide variants (SNVs), insertions and deletions (INDELS), copy number variants, loss of heterozygosity, gene fusions, structural variants, and differential gene expression. Results: We detected 3,750 SNVs/INDELS and extensive CNVs and expression changes. Recurrent TERT promoter mutations occurred in >52% of non-cirrhotic discovery samples. Frequently mutated genes included TP53, CTNNB1, and APOB. Cytochrome P450 mediated metabolism was significantly downregulated. Structural variants were observed at MACROD2, WDPCP, and NCKAP5 in >20% of samples. Furthermore, NR1H4 fusions involving gene partners EWSR1, GNPTAB, and FNIP1 were detected and validated in 2 non-cirrhotic samples. Conclusion: Genomic analysis can elucidate mechanisms that may contribute to non-cirrhotic HCC tumorigenesis. The comparable mutational landscape between cirrhotic and non-cirrhotic HCC supports previous work suggesting a convergence at the genomic level during disease progression. It is therefore possible genomic-based treatments can be applied to both HCC subtypes with progressed disease.

scholarly journals Childhood intellectual disability and parents' mental health: integrating social, psychological and genetic influences

2020 ◽  
pp. 1-8 ◽  
Author(s):  
Kate Baker ◽  
Rory T. Devine ◽  
Elise Ng-Cordell ◽  
F. Lucy Raymond ◽  
Claire Hughes ◽  
...  
Keyword(s):  
Mental Health ◽  
Intellectual Disability ◽  
Chromosomal Rearrangements ◽  
Copy Number Variants ◽  
Genetic Diagnosis ◽  
Well Being ◽  
Parental Distress ◽  
Single Nucleotide Variants ◽  
Behavioural Difficulties ◽  
The Uk

Background Intellectual disability has a complex effect on the well-being of affected individuals and their families. Previous research has identified multiple risk and protective factors for parental mental health, including socioeconomic circumstances and child behaviour. Aims This study explored whether genetic cause of childhood intellectual disability contributes to parental well-being. Method Children from across the UK with intellectual disability due to diverse genetic causes were recruited to the IMAGINE-ID study. Primary carers completed the Development and Well-being Assessment, including a measure of parental distress (Everyday Feeling Questionnaire). Genetic diagnoses were broadly categorised into aneuploidy, chromosomal rearrangements, copy number variants (CNVs) and single nucleotide variants. Results Compared with the UK general population, IMAGINE-ID parents (n = 888) reported significantly elevated emotional distress (Cohen's d = 0.546). Within-sample variation was related to recent life events and the perceived impact of children's difficulties. Impact was predicted by child age, physical disability, autistic characteristics and other behavioural difficulties. Genetic diagnosis also predicted impact, indirectly influencing parental well-being. Specifically, CNVs were associated with higher impact, not explained by CNV inheritance, neighbourhood deprivation or family structure. Conclusions The mental health of parents caring for a child with intellectual disability is influenced by child and family factors, converging on parental appraisal of impact. We found that genetic aetiologies, broadly categorised, also influence impact and thereby family risks. Recognition of these risk factors could improve access to support for parents, reduce their long-term mental health needs and improve well-being of individuals with intellectual disability.

scholarly journals Rapid and highly-specific generation of targeted DNA sequencing libraries enabled by linking capture probes with universal primers

2018 ◽  
Author(s):  
Joel Pel ◽  
Amy Leung ◽  
Wendy W. Y. Choi ◽  
Milenko Despotovic ◽  
W. Lloyd Ung ◽  
...  
Keyword(s):  
High Performance ◽  
Cost Effective ◽  
Repeated Sequence ◽  
Pcr Primers ◽  
Sequencing Depth ◽  
Targeted Sequencing ◽  
Universal Primers ◽  
Single Nucleotide Variants ◽  
Target Capture ◽  
Sequencing Cost

AbstractTargeted Next Generation Sequencing (NGS) is being adopted increasingly broadly in many research, commercial and clinical settings. Currently used target capture methods, however, typically require complex and lengthy (sometimes multi-day) workflows that complicates their use in certain applications. In addition, small panels for high sequencing depth applications such as liquid biopsy typically have low on-target rates, resulting in unnecessarily high sequencing cost.We have developed a novel targeted sequencing library preparation method, named Linked Target Capture (LTC), which replaces typical multi-day target capture workflows with a single-day, combined ‘target-capture-PCR’ workflow. This approach uses physically linked capture probes and PCR primers and is expected to work with panel sizes from 100 bp to >10 Mbp. It reduces the time and complexity of the capture workflow, eliminates long hybridization and wash steps and enables rapid library construction and target capture. High on-target read fractions are achievable due to repeated sequence selection in the target-capture-PCR step, thus lowering sequencing cost.We have demonstrated this technology on sample types including cell-free DNA (cfDNA) and formalin-fixed, paraffin-embedded (FFPE) derived DNA, capturing a 35-gene pan-cancer panel, and therein detecting single nucleotide variants, copy number variants, insertions, deletions and gene fusions. With the integration of unique molecular identifiers (UMIs), variants as low as 0.25% abundance were detected, limited by input mass and sequencing depth. Additionally, sequencing libraries were prepared in less than eight hours from extracted DNA to loaded sequencer, demonstrating that LTC holds promise as a broadly applicable tool for rapid, cost-effective and high performance targeted sequencing.

scholarly journals Molecular Diagnosis of Muscular Dystrophy Patients in Western Indian Population: A Comprehensive Mutation Analysis Using Amplicon Sequencing

2021 ◽  
Vol 12 ◽  
Author(s):  
Komal M. Patel ◽  
Arpan D. Bhatt ◽  
Krati Shah ◽  
Bhargav N. Waghela ◽  
Ramesh J. Pandit ◽  
...  
Keyword(s):  
Copy Number Variants ◽  
Cost Effective ◽  
Amplicon Sequencing ◽  
Large Deletion ◽  
Diagnostic Strategy ◽  
Single Nucleotide Variants ◽  
Time Saving ◽  
Large Deletions ◽  
Diagnosis Rate ◽  
Next Generation Sequencing Ngs

Muscular Dystrophies (MDs) are a group of inherited diseases and heterogeneous in nature. To date, 40 different genes have been reported for the occurrence and/or progression of MDs. This study was conducted to demonstrate the application of next-generation sequencing (NGS) in developing a time-saving and cost-effective diagnostic method to detect single nucleotide variants (SNVs) and copy number variants (CNVs) in a single test. A total of 123 cases clinically suspected of MD were enrolled in this study. Amplicon panel-based diagnosis was carried out for 102 (DMD/BMD) cases and the results were further screened using multiplex ligation-dependent probe amplification (MLPA). Whilst in the case of LGMD (N = 19) and UMD (N = 2), only NGS panel-based analysis was carried out. We identified the large deletions in 74.50% (76/102) of the cases screened with query DMD or BMD. Further, the large deletion in CAPN3 gene (N = 3) and known SNV mutations (N = 4) were identified in LGMD patients. Together, the total diagnosis rate for this amplicon panel was 70.73% (87/123) which demonstrated the utility of panel-based diagnosis for high throughput, affordable, and time-saving diagnostic strategy. Collectively, present study demonstrates that the panel based NGS sequencing could be superior over to MLPA.

scholarly journals Molecular Inversion Probe-Based Sequencing of USH2A Exons and Splice Sites as a Cost-Effective Screening Tool in USH2 and arRP Cases

2021 ◽  
Vol 22 (12) ◽  
pp. 6419
Author(s):  
Janine Reurink ◽  
Adrian Dockery ◽  
Dominika Oziębło ◽  
G. Jane Farrar ◽  
Monika Ołdak ◽  
...  
Keyword(s):  
Screening Tool ◽  
Usher Syndrome ◽  
Copy Number Variants ◽  
Genetic Diagnosis ◽  
Cost Effective ◽  
Single Nucleotide Variants ◽  
Effective Screening ◽  
Pathogenic Variants ◽  
Future Therapy ◽  
Molecular Inversion Probe

A substantial proportion of subjects with autosomal recessive retinitis pigmentosa (arRP) or Usher syndrome type II (USH2) lacks a genetic diagnosis due to incomplete USH2A screening in the early days of genetic testing. These cases lack eligibility for optimal genetic counseling and future therapy. USH2A defects are the most frequent cause of USH2 and are also causative in individuals with arRP. Therefore, USH2A is an important target for genetic screening. The aim of this study was to assess unscreened or incompletely screened and unexplained USH2 and arRP cases for (likely) pathogenic USH2A variants. Molecular inversion probe (MIP)-based sequencing was performed for the USH2A exons and their flanking regions, as well as published deep-intronic variants. This was done to identify single nucleotide variants (SNVs) and copy number variants (CNVs) in 29 unscreened or partially pre-screened USH2 and 11 partially pre-screened arRP subjects. In 29 out of these 40 cases, two (likely) pathogenic variants were successfully identified. Four of the identified SNVs and one CNV were novel. One previously identified synonymous variant was demonstrated to affect pre-mRNA splicing. In conclusion, genetic diagnoses were obtained for a majority of cases, which confirms that MIP-based sequencing is an effective screening tool for USH2A. Seven unexplained cases were selected for future analysis with whole genome sequencing.

scholarly journals Next-Generation Molecular Investigations in Lysosomal Diseases: Clinical Integration of a Comprehensive Targeted Panel

Diagnostics ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 294
Author(s):  
Bénédicte Sudrié-Arnaud ◽  
Sarah Snanoudj ◽  
Ivana Dabaj ◽  
Hélène Dranguet ◽  
Lenaig Abily-Donval ◽  
...  
Keyword(s):  
Performance Metrics ◽  
Age At Onset ◽  
Copy Number Variants ◽  
Cost Effective ◽  
Diagnostic Procedures ◽  
Next Generation ◽  
Single Nucleotide Variants ◽  
Bioinformatics Analyses ◽  
Clinical Complications ◽  
Lysosomal Disorders

Diagnosis of lysosomal disorders (LDs) may be hampered by their clinical heterogeneity, phenotypic overlap, and variable age at onset. Conventional biological diagnostic procedures are based on a series of sequential investigations and require multiple sampling. Early diagnosis may allow for timely treatment and prevent clinical complications. In order to improve LDs diagnosis, we developed a capture-based next generation sequencing (NGS) panel allowing the detection of single nucleotide variants (SNVs), small insertions and deletions, and copy number variants (CNVs) in 51 genes related to LDs. The design of the LD panel covered at least coding regions, promoter region, and flanking intronic sequences for 51 genes. The validation of this panel consisted in testing 21 well-characterized samples and evaluating analytical and diagnostic performance metrics. Bioinformatics pipelines have been validated for SNVs, indels and CNVs. The clinical output of this panel was tested in five novel cases. This capture-based NGS panel provides an average coverage depth of 474× which allows the detection of SNVs and CNVs in one comprehensive assay. All the targeted regions were covered above the minimum required depth of 30×. To illustrate the clinical utility, five novel cases have been sequenced using this panel and the identified variants have been confirmed using Sanger sequencing or quantitative multiplex PCR of short fluorescent fragments (QMPSF). The application of NGS as first-line approach to analyze suspected LD cases may speed up the identification of alterations in LD-associated genes. NGS approaches combined with bioinformatics analyses, are a useful and cost-effective tool for identifying the causative variations in LDs.

scholarly journals Rare Germline Variants in Chordoma-Related Genes and Chordoma Susceptibility

Cancers ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 2704
Author(s):  
Sally Yepes ◽  
Nirav N. Shah ◽  
Jiwei Bai ◽  
Hela Koka ◽  
Chuzhong Li ◽  
...  
Keyword(s):  
Internal Control ◽  
Susceptibility Gene ◽  
Copy Number Variants ◽  
Bone Cancer ◽  
Chinese Patients ◽  
European Ancestry ◽  
Unknown Etiology ◽  
Loss Of Function ◽  
Single Nucleotide Variants ◽  
Pathogenic Variants

Background: Chordoma is a rare bone cancer with an unknown etiology. TBXT is the only chordoma susceptibility gene identified to date; germline single nucleotide variants and copy number variants in TBXT have been associated with chordoma susceptibility in familial and sporadic chordoma. However, the genetic susceptibility of chordoma remains largely unknown. In this study, we investigated rare germline genetic variants in genes involved in TBXT/chordoma-related signaling pathways and other biological processes in chordoma patients from North America and China. Methods: We identified variants that were very rare in general population and internal control datasets and showed evidence for pathogenicity in 265 genes in a whole exome sequencing (WES) dataset of 138 chordoma patients of European ancestry and in a whole genome sequencing (WGS) dataset of 80 Chinese patients with skull base chordoma. Results: Rare and likely pathogenic variants were identified in 32 of 138 European ancestry patients (23%), including genes that are part of notochord development, PI3K/AKT/mTOR, Sonic Hedgehog, SWI/SNF complex and mesoderm development pathways. Rare pathogenic variants in COL2A1, EXT1, PDK1, LRP2, TBXT and TSC2, among others, were also observed in Chinese patients. Conclusion: We identified several rare loss-of-function and predicted deleterious missense variants in germline DNA from patients with chordoma, which may influence chordoma predisposition and reflect a complex susceptibility, warranting further investigation in large studies.

scholarly journals Structural variant detection in cancer genomes: computational challenges and perspectives for precision oncology

2021 ◽  
Vol 5 (1) ◽  
Author(s):  
Ianthe A. E. M. van Belzen ◽  
Alexander Schönhuth ◽  
Patrick Kemmeren ◽  
Jayne Y. Hehir-Kwa
Keyword(s):  
Intratumor Heterogeneity ◽  
Precision Oncology ◽  
Single Nucleotide Variants ◽  
Full Spectrum ◽  
Single Nucleotide ◽  
Sequencing Technologies ◽  
Cancer Genomes ◽  
Genomic Aberrations

AbstractCancer is generally characterized by acquired genomic aberrations in a broad spectrum of types and sizes, ranging from single nucleotide variants to structural variants (SVs). At least 30% of cancers have a known pathogenic SV used in diagnosis or treatment stratification. However, research into the role of SVs in cancer has been limited due to difficulties in detection. Biological and computational challenges confound SV detection in cancer samples, including intratumor heterogeneity, polyploidy, and distinguishing tumor-specific SVs from germline and somatic variants present in healthy cells. Classification of tumor-specific SVs is challenging due to inconsistencies in detected breakpoints, derived variant types and biological complexity of some rearrangements. Full-spectrum SV detection with high recall and precision requires integration of multiple algorithms and sequencing technologies to rescue variants that are difficult to resolve through individual methods. Here, we explore current strategies for integrating SV callsets and to enable the use of tumor-specific SVs in precision oncology.

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