Complex structural variants in Mendelian disorders: identification and breakpoint resolution using short- and long-read genome sequencing

Sanchis‐Juan, Alba; Stephens, Jonathan; French, Courtney; Gleadall, Nicholas; Mégy, Karyn; Penkett, Christopher J.; Shamardina, Olga; Stirrups, Kathleen; Delon, Isabelle; Dewhurst, Eleanor; Dolling, Helen; Erwood, Marie; Grozeva, Detelina; Stefanucci, Luca; Arno, Gavin; Webster, Andrew R.; Cole, Trevor; Austin, Topun; Branco, Ricardo Garcia; Ouwehand, Willem H.; Raymond, F. Lucy; Carss, Keren

doi:10.1186/s13073-018-0606-6

Cited by 125 publications

(114 citation statements)

References 51 publications

(78 reference statements)

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“…Firstly, long reads improve our ability to resolve complex structural variants, as shown with the chromosome 16 example (Figure 4). Complex SVs are common in cancer and resolving them can be critical for interpreting pathogenicity 18,29 . Secondly, we also used long read information to phase a somatic mutation (p.Arg249Met in TP53) and large deletion encompassing the same gene, confirming that they were in trans.…”

Section: Discussionmentioning

confidence: 99%

“…Thorough comparative studies have shown that long reads reduce the number of `dark' or `camouflaged' regions of the genome 26 and improve the sensitivity of structural variant (SV) detection 27 . Of course, both technologies have their pros and cons, but with fast turn-around times and lower start-up costs, and despite higher error rates of >10%, ONT WGS has already been used to resolve SVs in clinical cases 28,29 . Within cancer research specifically, low coverage ONT (Nanopore) WGS has been used for same-day diagnosis of brain tumours 30 , while targeted approaches SHORT VS LONG-READ GENOME SEQUENCING FOR SOMATIC VARIANT DETECTION 4 have been developed for detecting BCR-ABL1 fusion transcripts 31 , analysing prognostically relevant genes in chronic lymphocytic leukaemia 32,33 , and sequencing the entirety of BRCA1 34 .…”

Section: Introductionmentioning

confidence: 99%

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Short and long-read genome sequencing methodologies for somatic variant detection; genomic analysis of a patient with diffuse large B-cell lymphoma

Roberts

Lopopolo

Pagnamenta

et al. 2020

Preprint

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Recent advances in throughput and accuracy mean that the Oxford Nanopore Technologies (ONT) PromethION platform is a now a viable solution for genome sequencing. Much of the validation of bioinformatic tools for this long-read data has focussed on calling germline variants (including structural variants). Somatic variants are outnumbered many-fold by germline variants and their detection is further complicated by the effects of tumour purity/subclonality. Here, we evaluate the extent to which Nanopore sequencing enables genome-wide detection and analysis of somatic variation. We do this through sequencing tumour and germline genomes for a patient with diffuse B-cell lymphoma and comparing results with 150bp short-read sequencing of the same samples. Calling germline single nucleotide variants (SNVs) from the long-read data achieved good specificity and sensitivity.However, results of somatic SNV calling highlight the need for the development of specialized joint calling algorithms. We find the comparative performance of different tools varies significantly between structural variant types, and suggest long reads are especially advantageous for calling large somatic deletions and duplications. Finally, we highlight the utility of long reads for phasing clinically relevant variants, confirming that a somatic 1.6Mb deletion and a p.(Arg249Met) mutation involving TP53 are oriented in trans.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Short and long-read genome sequencing methodologies for somatic variant detection; genomic analysis of a patient with diffuse large B-cell lymphoma

Roberts

Lopopolo

Pagnamenta

et al. 2020

Preprint

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“…Variant annotation was performed with Variant Effect Predictor (58), which included allelic population frequencies from gnomAD (22) and deleteriousness scores from CADD (59), Sift (60) and Polyphen-2 (61). Structural variants were also identified by Manta (62) and Canvas (63) algorithms, as described previously (64,65). Trio analysis focused on de novo and rare biallelic variant discovery unrestricted by a gene list.…”

Section: Genomic Analysismentioning

confidence: 99%

Structural analysis of pathogenic missense mutations in GABRA2 and identification of a novel de novo variant in the desensitization gate

Sanchis‐Juan

Hasenahuer

Baker

et al. 2019

Preprint

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AbstractCys-loop receptors are vital for controlling neuronal excitability in the brain and their dysfunction results in numerous neurological disorders. Recently, six de novo missense variants in GABRA2 gene, a member of this family, have been associated with early infantile epileptic encephalopathy (EIEE) and intellectual disability with seizures. Here, using whole-genome sequencing we identified a de novo missense variant in GABRA2 gene in a patient with EIEE and developmental delay. We perform protein structural analysis of the seven variants and show that all the mutations are in the transmembrane domain, either close to the desensitization gate, the activation gate or in inter-subunit interfaces. Further investigations demonstrated that the majority of pathogenic variants reported are at equivalent positions in other Cys-loop receptors, emphasizing the importance of these residues for the adequate function of the receptor. Also, a comparison of the distribution of the mutations in all the Cys-loop receptors showed that pathogenic variants are more common in the transmembrane helices, more specifically in the M2 helix, highlighting the importance of this segment. Our study expands the clinical spectrum of individuals with pathogenic missense mutations in GABRA2, defines the regions where pathogenic mutations are in the protein structure, and highlights the value of considering sequence, evolutionary, and structural information from other Cys-loop receptors as a strategy for variant interpretation of novel missense mutations in GABRA2.

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“…Structural variations are implicated in the development of many human diseases 1,2 and account for most of the genetic variation in the human population 3,4 . Structural variants (SVs), defined as genomic alterations greater than 50 bp 5 , can functionally affect cellular physiology by forming genetic lesions which may lead to gene dysregulation or novel gene-fusions, driving the development of diseases such as cancer 6,7 , Mendelian disorders 8,9 , and complex diseases 10 . SVs can exist as different classes including deletion, duplication, insertion, inversion and translocation.…”

Section: Introductionmentioning

confidence: 99%

NanoVar: Accurate Characterization of Patients’ Genomic Structural Variants Using Low-Depth Nanopore Sequencing

Tham

Tirado-Magallanes

Goh

et al. 2019

Preprint

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Despite the increasing relevance of structural variants (SV) in the development of many human diseases, progress in novel pathological SV discovery remains impeded, partly due to the challenges of accurate and routine SV characterization in patients. The recent advent of third-generation sequencing (3GS) technologies brings promise for better characterization of genomic aberrations by virtue of having longer reads. However, the applications of 3GS are restricted by their high sequencing error rates and low sequencing throughput. To overcome these limitations, we present NanoVar, an accurate, rapid and low-depth (4X) 3GS SV caller utilizing long-reads generated by Oxford Nanopore Technologies. NanoVar employs split-reads and hard-clipped reads for SV detection and utilizes a neural network classifier for true SV enrichment. In simulated data, NanoVar demonstrated the highest SV detection accuracy (F1 score = 0.91) amongst other long-read SV callers using 12 gigabases (4X) of sequencing data. In patient samples, besides the detection of genomic aberrations, NanoVar also uncovered many normal alternative sequences or alleles which were present in healthy individuals. The low sequencing depth requirements of NanoVar enable the use of Nanopore sequencing for accurate SV characterization at a lower sequencing cost, an approach compatible with clinical studies and largescale SV-association research. ________________________________________________________________________________________

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Complex structural variants in Mendelian disorders: identification and breakpoint resolution using short- and long-read genome sequencing

Cited by 125 publications

References 51 publications

Short and long-read genome sequencing methodologies for somatic variant detection; genomic analysis of a patient with diffuse large B-cell lymphoma

Short and long-read genome sequencing methodologies for somatic variant detection; genomic analysis of a patient with diffuse large B-cell lymphoma

Structural analysis of pathogenic missense mutations in GABRA2 and identification of a novel de novo variant in the desensitization gate

NanoVar: Accurate Characterization of Patients’ Genomic Structural Variants Using Low-Depth Nanopore Sequencing

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