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

My Linh Thibodeau, Kieran O'Neill, Katherine Dixon, Caralyn Reisle, Karen L. Mungall, Martin Krzywinski, Yaoqing Shen, Howard J. Lim, Dean Cheng, Kane Tse, Tina Wong, Eric Chuah (+18 others)
2020 Genetics in Medicine  
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. 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
more » ... tome sequencing. Candidate SVs were subsequently assessed by Oxford Nanopore long-read sequencing. 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. 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.
doi:10.1038/s41436-020-0880-8 pmid:32624572 pmcid:PMC7605438 fatcat:tasunsvz2jgvtdez5bsnntc24y

Co-occurrence of anaerobic bacteria in colorectal carcinomas

René L Warren, Douglas J Freeman, Stephen Pleasance, Peter Watson, Richard A Moore, Kyla Cochrane, Emma Allen-Vercoe, Robert A Holt
2013 Microbiome  
Numerous cancers have been linked to microorganisms. Given that colorectal cancer is a leading cause of cancer deaths and the colon is continuously exposed to a high diversity of microbes, the relationship between gut mucosal microbiome and colorectal cancer needs to be explored. Metagenomic studies have shown an association between Fusobacterium species and colorectal carcinoma. Here, we have extended these studies with deeper sequencing of a much larger number (n = 130) of colorectal
more » ... and matched normal control tissues. We analyzed these data using co-occurrence networks in order to identify microbe-microbe and host-microbe associations specific to tumors. Results: We confirmed tumor over-representation of Fusobacterium species and observed significant co-occurrence within individual tumors of Fusobacterium, Leptotrichia and Campylobacter species. This polymicrobial signature was associated with over-expression of numerous host genes, including the gene encoding the pro-inflammatory chemokine Interleukin-8. The tumor-associated bacteria we have identified are all Gram-negative anaerobes, recognized previously as constituents of the oral microbiome, which are capable of causing infection. We isolated a novel strain of Campylobacter showae from a colorectal tumor specimen. This strain is substantially diverged from a previously sequenced oral Campylobacter showae isolate, carries potential virulence genes, and aggregates with a previously isolated tumor strain of Fusobacterium nucleatum. Conclusions: A polymicrobial signature of Gram-negative anaerobic bacteria is associated with colorectal carcinoma tissue.
doi:10.1186/2049-2618-1-16 pmid:24450771 pmcid:PMC3971631 fatcat:3otqseyofrctdkaz4gqyymqj2i

Loss of CIC promotes mitotic dysregulation and chromosome segregation defects [article]

Suganthi Chittaranjan, Jungeun Song, Susanna Y Chan, Stephen Dongsoo Lee, Shiekh Tanveer Ahmad, William Brothers, Richard D Corbett, Alessia Gagliardi, Amy Lum, Annie Moradian, Stephen Pleasance, Robin Coope (+9 others)
2019 bioRxiv   pre-print
CIC is a transcriptional repressor inactivated by loss-of-function mutations in several cancer types, including gliomas, lung cancers, and gastric adenocarcinomas. CIC alterations and/or loss of CIC activity have been associated with poorer outcomes and more aggressive phenotypes across cancer types, which is consistent with the notion that CIC functions as a tumour suppressor across a wide range of contexts. Results: Using mammalian cells lacking functional CIC, we found that CIC deficiency
more » ... associated with chromosome segregation (CS) defects, resulting in chromosomal instability and aneuploidy. These CS defects were associated with transcriptional dysregulation of spindle assembly checkpoint and cell cycle regulators. We also identified novel CIC interacting proteins, including core members of the SWI/SNF complex, and showed that they cooperatively regulated the expression of genes involved in cell cycle regulation. Finally, we showed that loss of CIC and ARID1A cooperatively increased CS defects and reduced cell viability. Conclusions: Our study ascribes a novel role to CIC as an important regulator of the cell cycle and demonstrates that loss of CIC can lead to chromosomal instability and aneuploidy in human and murine cells through defects in CS, providing insight into the underlying mechanisms of CIC's increasingly apparent role as a "pan-cancer" tumour suppressor.
doi:10.1101/533323 fatcat:3llhwcsvsbgstkvpf2vipwfx3u

Largest Complete Mitochondrial Genome of a Gymnosperm, Sitka Spruce (Picea sitchensis), Indicates Complex Physical Structure [article]

Shaun D Jackman, Lauren Coombe, Rene L Warren, Heather Kirk, Eva Trinh, Tina McLeod, Stephen Pleasance, Pawan Pandoh, Yongjun Zhao, Robin J Coope, Jean Bousquet, Joerg Bohlmann (+2 others)
2019 bioRxiv   pre-print
Plant mitochondrial genomes vary widely in size. Although many plant mitochondrial genomes have been sequenced and assembled, the vast majority are of angiosperms, and few are of gymnosperms. Most plant mitochondrial genomes are smaller than a megabase, with a few notable exceptions. We have sequenced and assembled the 5.5 Mbp mitochondrial genome of Sitka spruce (Picea sitchensis), the largest complete mitochondrial genome of a gymnosperm. We sequenced the whole genome using Oxford Nanopore
more » ... ION, and then identified contigs of mitochondrial origin assembled from these long reads. The assembly graph shows a multipartite genome structure, composed of one smaller 168 kbp circular segment of DNA, and a larger 5.4 Mbp component with a branching structure. The assembly graph gives insight into a putative complex physical genome structure, and its branching points may represent active sites of recombination.
doi:10.1101/601104 fatcat:idboteyojnftfglt4zhmw6na7m

Removing orbital debris with lasers

Claude R. Phipps, Kevin L. Baker, Stephen B. Libby, Duane A. Liedahl, Scot S. Olivier, Lyn D. Pleasance, Alexander Rubenchik, James E. Trebes, E. Victor George, Bogdan Marcovici, James P. Reilly, Michael T. Valley
2012 Advances in Space Research  
Orbital debris in low Earth orbit (LEO) are now sufficiently dense that the use of LEO space is threatened by runaway collisional cascading. A problem predicted more than thirty years ago, the threat from debris larger than about 1 cm demands serious attention. A promising proposed solution uses a high power pulsed laser system on the Earth to make plasma jets on the objects, slowing them slightly, and causing them to re-enter and burn up in the atmosphere. In this paper, we reassess this
more » ... ch in light of recent advances in low-cost, light-weight modular design for large mirrors, calculations of laser-induced orbit changes and in design of repetitive, multi-kilojoule lasers, that build on inertial fusion research. These advances now suggest that laser orbital debris removal (LODR) is the most cost-effective way to mitigate the debris problem. No other solutions have been proposed that address the whole problem of large and small debris. A LODR system will have multiple uses beyond debris removal. International cooperation will be essential for building and operating such a system.
doi:10.1016/j.asr.2012.02.003 fatcat:f6vxh5vww5hqthtu5ojr5qdn4q

Assembly of the Complete Sitka Spruce Chloroplast Genome Using 10X Genomics' GemCode Sequencing Data

Lauren Coombe, René L. Warren, Shaun D. Jackman, Chen Yang, Benjamin P. Vandervalk, Richard A. Moore, Stephen Pleasance, Robin J. Coope, Joerg Bohlmann, Robert A. Holt, Steven J. M. Jones, Inanc Birol (+1 others)
2016 PLoS ONE  
The linked read sequencing library preparation platform by 10X Genomics produces barcoded sequencing libraries, which are subsequently sequenced using the Illumina short read sequencing technology. In this new approach, long fragments of DNA are partitioned into separate micro-reactions, where the same index sequence is incorporated into each of the sequencing fragment inserts derived from a given long fragment. In this study, we exploited this property by using reads from index sequences
more » ... ated with a large number of reads, to assemble the chloroplast genome of the Sitka spruce tree (Picea sitchensis). Here we report on the first Sitka spruce chloroplast genome assembled exclusively from P. sitchensis genomic libraries prepared using the 10X Genomics protocol. We show that the resulting 124,049 base pair long genome shares high sequence similarity with the related white spruce and Norway spruce chloroplast genomes, but diverges substantially from a previously published P. sitchensis-P. thunbergii chimeric genome. The use of reads from high-frequency indices enabled separation of the nuclear genome reads from that of the chloroplast, which resulted in the simplification of the de Bruijn graphs used at the various stages of assembly.
doi:10.1371/journal.pone.0163059 pmid:27632164 pmcid:PMC5025161 fatcat:b2icsnujm5fmze2pjjwrcgnuwi

Continuous flow fast atom bombardment with packed microcolumns: A comparison of precolumn versus coaxial matrix delivery

Stephen Pleasance, Pierre Thibault, M. Arthur Moseley, Leesa J. Deterding, Kenneth B. Tomer, J. W. Jorgenson
1990 Journal of the American Society for Mass Spectrometry  
The effect of adding glycerol to the mobile phase on the chromatographic separation of peptides has been investigated using a continuous flow fast atom bombardment (CFFAB) interface coupled with commercial packed microcolumns (25 cm x 320 pm id.). In a comparative study using a UV detector, it was found that chromatographic peak broadening progressively increased with increasing percentage of glycerol in the mobile phase. In the liquid chromatographic FAB mass spectrometric analysis, this
more » ... is compounded by the dynamic mixing of the column effluent on the probe. Improvements of 25-155% in the overall separation efficiencies were obtained by introducing the matrix independently to the probe tip via a coaxial arrangement. Application of this coaxial CFFAB is demonstrated by the analysis of peptide mixtures and tryptic digests.
doi:10.1016/1044-0305(90)85007-9 pmid:24248825 fatcat:zhde7stp3rajfeawrovxsdrjfm

Organellar Genomes of White Spruce (Picea glauca): Assembly and Annotation

Shaun D. Jackman, René L. Warren, Ewan A. Gibb, Benjamin P. Vandervalk, Hamid Mohamadi, Justin Chu, Anthony Raymond, Stephen Pleasance, Robin Coope, Mark R. Wildung, Carol E. Ritland, Jean Bousquet (+3 others)
2015 Genome Biology and Evolution  
The genome sequences of the plastid and mitochondrion of white spruce (Picea glauca) were assembled from whole-genome shotgun sequencing data using ABySS. The sequencing data contained reads from both the nuclear and organellar genomes, and reads of the organellar genomes were abundant in the data as each cell harbors hundreds of mitochondria and plastids. Hence, assembly of the 123-kb plastid and 5.9-Mb mitochondrial genomes were accomplished by analyzing data sets primarily representing low
more » ... verage of the nuclear genome. The assembled organellar genomes were annotated for their coding genes, ribosomal RNA, and transfer RNA. Transcript abundances of the mitochondrial genes were quantified in three developmental tissues and five mature tissues using data from RNA-seq experiments. C-to-U RNA editing was observed in the majority of mitochondrial genes, and in four genes, editing events were noted to modify ACG codons to create cryptic AUG start codons. The informatics methodology presented in this study should prove useful to assemble organellar genomes of other plant species using whole-genome shotgun sequencing data.
doi:10.1093/gbe/evv244 pmid:26645680 pmcid:PMC4758241 fatcat:73qfjmhc35amlc64ozqj4nqjci

Comprehensive identification of essential Staphylococcus aureus genes using Transposon-Mediated Differential Hybridisation (TMDH)

Roy R Chaudhuri, Andrew G Allen, Paul J Owen, Gil Shalom, Karl Stone, Marcus Harrison, Timothy A Burgis, Michael Lockyer, Jorge Garcia-Lara, Simon J Foster, Stephen J Pleasance, Sarah E Peters (+2 others)
2009 BMC Genomics  
In recent years there has been an increasing problem with Staphylococcus aureus strains that are resistant to treatment with existing antibiotics. An important starting point for the development of new antimicrobial drugs is the identification of "essential" genes that are important for bacterial survival and growth. We have developed a robust microarray and PCR-based method, Transposon-Mediated Differential Hybridisation (TMDH), that uses novel bioinformatics to identify transposon inserts in
more » ... enome-wide libraries. Following a microarray-based screen, genes lacking transposon inserts are re-tested using a PCR and sequencing-based approach. We carried out a TMDH analysis of the S. aureus genome using a large random mariner transposon library of around a million mutants, and identified a total of 351 S. aureus genes important for survival and growth in culture. A comparison with the essential gene list experimentally derived for Bacillus subtilis highlighted interesting differences in both pathways and individual genes. We have determined the first comprehensive list of S. aureus essential genes. This should act as a useful starting point for the identification of potential targets for novel antimicrobial compounds. The TMDH methodology we have developed is generic and could be applied to identify essential genes in other bacterial pathogens.
doi:10.1186/1471-2164-10-291 pmid:19570206 pmcid:PMC2721850 fatcat:dwwm2kivh5fdpfln352jjdh5ii

An evaluation of atmospheric pressure ionization techniques for the analysis of N-methyl carbamate pesticides by liquid chromatography mass spectrometry

Stephen Pleasance, Joseph F. Anacleto, M.Ruth Bailey, David H. North
1992 Journal of the American Society for Mass Spectrometry  
In each case, after removal of the solvent, the residue was taken to PLEASANCE IZ AL. ).  ... 
doi:10.1016/1044-0305(92)87066-8 pmid:24243049 fatcat:ppuv7uuyd5bzdema2dws6bp5yu

Comprehensive Identification of Salmonella enterica Serovar Typhimurium Genes Required for Infection of BALB/c Mice

Roy R. Chaudhuri, Sarah E. Peters, Stephen J. Pleasance, Helen Northen, Chrissie Willers, Gavin K. Paterson, Danielle B. Cone, Andrew G. Allen, Paul J. Owen, Gil Shalom, Dov J. Stekel, Ian G. Charles (+2 others)
2009 PLoS Pathogens  
Genes required for infection of mice by Salmonella Typhimurium can be identified by the interrogation of random transposon mutant libraries for mutants that cannot survive in vivo. Inactivation of such genes produces attenuated S. Typhimurium strains that have potential for use as live attenuated vaccines. A quantitative screen, Transposon Mediated Differential Hybridisation (TMDH), has been developed that identifies those members of a large library of transposon mutants that are attenuated.
more » ... H employs custom transposons with outward-facing T7 and SP6 promoters. Fluorescently-labelled transcripts from the promoters are hybridised to whole-genome tiling microarrays, to allow the position of the transposon insertions to be determined. Comparison of microarray data from the mutant library grown in vitro (input) with equivalent data produced after passage of the library through mice (output) enables an attenuation score to be determined for each transposon mutant. These scores are significantly correlated with bacterial counts obtained during infection of mice using mutants with individual defined deletions of the same genes. Defined deletion mutants of several novel targets identified in the TMDH screen are effective live vaccines.
doi:10.1371/journal.ppat.1000529 pmid:19649318 pmcid:PMC2712085 fatcat:velm7zsggrffbncq3zhdogd6p4

Fluorouracil sensitivity in a head and neck squamous cell carcinoma with a somatic DPYD structural variant

Elisa Majounie, Kathleen Wee, Laura M. Williamson, Martin R. Jones, Erin Pleasance, Howard J. Lim, Cheryl Ho, Daniel J. Renouf, Stephen Yip, Steven J.M. Jones, Marco A. Marra, Janessa Laskin
2019 Molecular Case Studies  
Head and neck squamous cell carcinoma (HNSCC) is one of the most common cancers worldwide and represents a heterogeneous group of tumours, the majority of which are treated with a combination of surgery, radiation and chemotherapy. Fluoropyrimidine (5-FU) and its oral pro-drug, capecitabine, are commonly prescribed treatments for several solid tumour types including HNSCC. 5-FU-associated toxicity is observed in approximately 30% of treated patients and is largely caused by germline
more » ... s in DPYD which encodes dihydropyrimidine dehydrogenase (DPD), a key enzyme of 5-FU catabolism and deactivation. Although the association of germline DPYD alterations with toxicity is well-described, the potential contribution of somatic DPYD alterations to 5-FU sensitivity has not been explored. In a patient with metastatic HNSCC, in-depth genomic and transcriptomic integrative analysis on a biopsy from a metastatic neck lesion revealed alterations in genes that are associated with 5-FU uptake and metabolism. These included a novel somatic structural variant resulting in a partial deletion affecting DPYD, a variant of unknown significance affecting SLC29A1 and homozygous deletion of MTAP. There was no evidence of deleterious germline polymorphisms that have been associated with 5-FU toxicity, indicating a potential vulnerability of the tumour to 5-FU therapy. The discovery of the novel DPYD variant led to the initiation of 5-FU treatment that resulted in a rapid response lasting 17 weeks, with subsequent relapse due to unknown resistance mechanisms. This suggests that somatic alterations present in this tumour may serve as markers for tumour sensitivity to 5-FU, aiding in selection of personalized treatment strategies.
doi:10.1101/mcs.a004713 pmid:31871216 pmcid:PMC6996515 fatcat:kkpg2h3xbvactgi7aeagxku7by

The pivotal role of sampling recurrent tumors in the precision care of patients with tumors of the central nervous system

Derek Wong, Yaoqing Shen, Adrian B. Levine, Erin Pleasance, Martin Jones, Karen Mungall, Brian Thiessen, Brian Toyota, Janessa Laskin, Steven J.M. Jones, Marco A. Marra, Stephen Yip
2019 Molecular Case Studies  
Effective management of brain and spine tumors relies on a multidisciplinary approach encompassing surgery, radiation, and systemic therapy. In the era of personalized oncology, the latter is complemented by various molecularly targeting agents. Precise identification of cellular targets for these drugs requires comprehensive profiling of the cancer genome coupled with an efficient analytic pipeline, leading to an informed decision on drug selection, prognosis, and confirmation of the original
more » ... athological diagnosis. Acquisition of optimal tumor tissue for such analysis is paramount and often presents logistical challenges in neurosurgery. Here, we describe the experience and results of the Personalized OncoGenomics (POG) program with a focus on tumors of the central nervous system (CNS). Patients with recurrent CNS tumors were consented and enrolled into the POG program prior to accrual of tumor and matched blood followed by whole-genome and transcriptome sequencing and processing through the POG bioinformatic pipeline. Sixteen patients were enrolled into POG. In each case, POG analyses identified genomic drivers including novel oncogenic fusions, aberrant pathways, and putative therapeutic targets. POG has highlighted that personalized oncology is truly a multidisciplinary field, one in which neurosurgeons must play a vital role if these programs are to succeed and benefit our patients.
doi:10.1101/mcs.a004143 pmid:31371350 pmcid:PMC6672021 fatcat:fxi235uddjfhvpgnqfhvhs2efm

Subclonal phylogenetic structures in cancer revealed by ultra-deep sequencing

P. J. Campbell, E. D. Pleasance, P. J. Stephens, E. Dicks, R. Rance, I. Goodhead, G. A. Follows, A. R. Green, P. A. Futreal, M. R. Stratton
2008 Proceedings of the National Academy of Sciences of the United States of America  
During the clonal expansion of cancer from an ancestral cell with an initiating oncogenic mutation to symptomatic neoplasm, the occurrence of somatic mutations (both driver and passenger) can be used to track the on-going evolution of the neoplasm. All subclones within a cancer are phylogenetically related, with the prevalence of each subclone determined by its evolutionary fitness and the timing of its origin relative to other subclones. Recently developed massively parallel sequencing
more » ... s promise the ability to detect rare subclones of genetic variants without a priori knowledge of the mutations involved. We used ultra-deep pyrosequencing to investigate intraclonal diversification at the Ig heavy chain locus in 22 patients with B-cell chronic lymphocytic leukemia. Analysis of a non-polymorphic control locus revealed artifactual insertions and deletions resulting from sequencing errors and base substitutions caused by polymerase misincorporation during PCR amplification. We developed an algorithm to differentiate genuine haplotypes of somatic hypermutations from such artifacts. This proved capable of detecting multiple rare subclones with frequencies as low as 1 in 5000 copies and allowed the characterization of phylogenetic interrelationships among subclones within each patient. This study demonstrates the potential for ultra-deep resequencing to recapitulate the dynamics of clonal evolution in cancer cell populations. Results Deep Resequencing of the IGH Rearrangement in CLL. We developed a nested PCR approach to amplify the clone-specific IGH VDJ
doi:10.1073/pnas.0801523105 pmid:18723673 pmcid:PMC2529122 fatcat:j6prbw5lpnh7hagumzlrunhbsm

Clinical and cost outcomes following genomics‐informed treatment for advanced cancers

Deirdre Weymann, Samantha Pollard, Brandon Chan, Emma Titmuss, Alexandra Bohm, Janessa Laskin, Steven J. M. Jones, Erin Pleasance, Jessica Nelson, Alexandra Fok, Howard Lim, Aly Karsan (+7 others)
2021 Cancer Medicine  
Stephen Yip is an advisory board member for and has received travel allowance from Amgen, AstraZeneca, Bayer, Norvatis, and Roche. Dean A. Regier has received speaking honoraria from Roche Canada.  ... 
doi:10.1002/cam4.4076 pmid:34152087 pmcid:PMC8335838 fatcat:ewwukajwsjhgfoz5bzibjs6yuu
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