Holt lab

The Holt Lab uses cutting edge tools and methodologies to investigate the biology of cancer from several different angles. Focusing on the immune system, the group has used deep sequencing to survey T cell repertoire diversity at the resolution of individual clonotypes and are now using these methodologies to explore the role of T cells in cancer. They are also working to develop cancer immunotherapies using engineered T cells to selectively deliver cytotoxic payloads to bolster the anti-cancer immune response and to enhance tumour cell killing. The group employs their expertise in DNA sequencing and computational analyses to investigate the role of infectious agents in cancer development and were the first to demonstrate a strong link between the pathogen Fusobacterium nucleatum and colorectal cancer. Finally, they apply deep sequencing technologies to identify the spectrum of mutations in various cancer types, with a particular focus on tumour evolution and the identification of antigens for cancer vaccines.

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Location

CRC

We are located at Canada's Michael Smith Genome Sciences Centre, part of the British Columbia Cancer Research Institute.

Address: 
675 West 10th Avenue 
Vancouver, British Columbia 
V5Z 1L3 

Projects

Immunogenetics

the Holt Lab is using deep sequencing methods to explore the role of T cells in cancer, and how to enhance the anti-cancer immune response. They are particularly focused on developing new sequence-based approaches to T cell antigen discovery and characterization.

Cancer Genomes

Dr. Holt’s lab is using deep sequencing and novel computational methods to identify the spectrum of somatic mutations in various cancers, with a particular focus on tumour evolution and the identification of antigens for cancer vaccines.

Synthetic immunology

It has been recognized for nearly three decades that patients with tumours that are strongly infiltrated by T cells, in particular cytotoxic T cells, have better outcomes. We use computational approaches and targeted immunoassays in the lab to gain insights into the nature of the anti-cancer T cell response, and to determine how and why it varies among healthy individuals and among cancer patients. These studies inform our programs for pre-clinical and clinical development of genetically engineered T cell therapies, including chimeric antigen receptor (CAR-T) and recombinant T cell receptor (rTCR) therapies for cancer.

Selected Publications

Modulation of the Host Cell Transcriptome and Epigenome by Fusobacterium nucleatum

mbio
Cody A Despins, Scott D Brown, Avery V Robinson, Andrew J Mungall, Emma Allen-Vercoe, Robert A Holt

Fusobacterium nucleatum is a ubiquitous opportunistic pathogen with an emerging role as an oncomicrobe in colorectal cancer and other cancer settings. F. nucleatum can adhere to and invade host cells in a manner that varies across F. nucleatum strains and host cell phenotypes. Here, we performed pairwise cocultures between three F. nucleatum strains and two immortalized primary host cell types (human colonic epithelial [HCE] cells and human carotid artery endothelial [HCAE] cells) followed by transcriptome sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) to investigate transcriptional and epigenetic host cell responses. We observed that F. nucleatum-induced host cell transcriptional modulation involves strong upregulation of genes related to immune migration and inflammatory processes, such as TNFCXCL8CXCL1, and CCL20. Furthermore, we identified genes strongly upregulated in a cell line-specific manner. In HCE cells, overexpressed genes included UBD and DUOX2/DUOXA2, associated with p53 degradation-mediated proliferation and intestinal reactive oxygen species (ROS) production, respectively. In HCAE cells, overexpressed genes included EFNA1 and LIF, two genes commonly upregulated in colorectal cancer and associated with poor patient outcomes, and PTGS2 (COX2), a gene associated with the protective effect of aspirin in the colorectal cancer setting. Interestingly, we also observed downregulation of numerous histone modification genes upon F. nucleatum exposure. We used the ChIP-seq data to annotate chromatin states genome wide and found significant chromatin remodeling following F. nucleatum exposure in HCAE cells, with increased frequencies of active enhancer and low-signal/quiescent states. Thus, our results highlight increased inflammation and chemokine gene expression as conserved host cell responses to F. nucleatum exposure and extensive host cell epigenomic changes specific to host cell type. IMPORTANCE Fusobacterium nucleatum is a bacterium normally found in the healthy oral cavity but also has an emerging role in colorectal cancer and other cancer settings. The host-microbe interactions of F. nucleatum and its involvement in tumor initiation, progression, and treatment resistance are not fully understood. We explored host cell changes that occur in response to F. nucleatum. We identified key genes differentially expressed in response to various conditions of F. nucleatum exposure and determined that the conserved host cell response to F. nucleatum was dominated by increased inflammation and chemokine gene expression. Additionally, we found extensive host cell epigenomic changes as a novel aspect of host modulation associated with F. nucleatum exposure. These results extend our understanding of F. nucleatum as an emerging pathogen and highlight the importance of considering strain heterogeneity and host cell phenotypic variation when exploring pathogenic mechanisms of F. nucleatum.

Hematologists’ barriers and enablers to screening and recruiting patients to a chimeric antigen receptor (CAR) T cell therapy trial: a theory-informed interview study

Trials
Gisell Castillo, Manoj Lalu, Sarah Asad, Madison Foster, Natasha Kekre, Dean Fergusson, Terry Hawrysh, Harold Atkins, Kednapa Thavorn, Joshua Montroy, Stuart Schwartz, Robert Holt, Raewyn Broady, Justin Presseau, GO CART team

Background: Novel therapies often fail to reach the bedside due to low trial recruitment rates. Prior to conducting one of the first chimeric antigen receptor (CAR) T cell therapy trials in Canada, we used the Theoretical Domains Framework, a novel tool for identifying barriers and enablers to behavior change, to identify physician-related barriers and enablers to screening and recruiting patients for an early phase immunotherapy trial.

Methods: We conducted interviews with hematologists across Canada and used a directed content analysis to identify relevant domains reflecting the key factors that may affect screening and recruitment.

Results: In total, we interviewed 15 hematologists. Physicians expressed "cautious hope"; while expressing safety, feasibility, and screening criteria concerns, 14 out of 15 hematologists intended to screen for the trial (domains: knowledge, goals, beliefs about consequences, intentions). Physicians underscored the "challenging contexts," identifying resources, workload, forgetting, and patient wait times to receive CAR T cells as key practical barriers to screening (domains: environmental context and resources, memory, attention and decision-making, behavioral regulation). They also highlighted "variability in roles and procedures" that may lead to missed trial candidates (domain: social and professional role). Left unaddressed, these barriers may undermine trial recruitment.

Conclusions: This study is among the first to use the Theoretical Domains Framework from the physician perspective to identify recruitment challenges to early phase trials and demonstrates the value of this approach for identifying barriers to screening and recruitment that may not otherwise have been elicited. This approach can optimize trial procedures and may serve to inform future promising early phase cancer therapy trials.

Trial registration: ClinicalTrials.gov Identifier: NCT03765177 . Registered on December 5, 2018.

A survey of Fusobacterium nucleatum genes modulated by host cell infection

Microbial Genomics
Kyla Cochrane, Avery V Robinson, Robert A Holt, Emma Allen-Vercoe

Here, we report comprehensive transcriptomic profiles from Fusobacterium nucleatum under conditions that mimic the first stages of bacterial infection in a highly differentiated adenocarcinoma epithelial cell line. Our transcriptomic in vitro adenocarcinoma approach allows us to measure the expression dynamics and regulation of bacterial virulence and response factors in real time, and is a novel strategy for clarifying the role of F. nucleatum infection in colorectal cancer (CRC) progression. Our data show that: (i) infection alters metabolic and functional pathways in F. nucleatum, allowing the bacterium to adapt to the host-imposed milieu; (ii) infection also stimulates the expression of genes required to help induce and promote a hypoxic and inflammatory microenvironment in the host; and (iii) F. nucleatum invasion occurs by a haematogenous route of infection. Our study identifies novel gene targets from F. nucleatum that are activated during invasion and which may aid in determining how this species invades and promotes disease within the human gastrointestinal tract. These invasion-specific genes may be useful as biomarkers for CRC progression in a host and could also assist in the development of new diagnostic tools and treatments (such as vaccines or small molecule drug targets), which will be able to combat infection and inflammation in the host while circumventing the potential problem of F. nucleatum tolerization.

Characteristics of TCR Repertoire Associated With Successful Immune Checkpoint Therapy Responses

Frontiers In Immunology
Joel Kidman, Nicola Principe, Mark Watson, Timo Lassmann, Robert A Holt, Anna K Nowak, Willem Joost Lesterhuis, Richard A Lake, Jonathan Chee

Immunotherapies have revolutionized cancer treatment. In particular, immune checkpoint therapy (ICT) leads to durable responses in some patients with some cancers. However, the majority of treated patients do not respond. Understanding immune mechanisms that underlie responsiveness to ICT will help identify predictive biomarkers of response and develop treatments to convert non-responding patients to responding ones. ICT primarily acts at the level of adaptive immunity. The specificity of adaptive immune cells, such as T and B cells, is determined by antigen-specific receptors. T cell repertoires can be comprehensively profiled by high-throughput sequencing at the bulk and single-cell level. T cell receptor (TCR) sequencing allows for sensitive tracking of dynamic changes in antigen-specific T cells at the clonal level, giving unprecedented insight into the mechanisms by which ICT alters T cell responses. Here, we review how the repertoire influences response to ICT and conversely how ICT affects repertoire diversity. We will also explore how changes to the repertoire in different anatomical locations can better correlate and perhaps predict treatment outcome. We discuss the advantages and limitations of current metrics used to characterize and represent TCR repertoire diversity. Discovery of predictive biomarkers could lie in novel analysis approaches, such as network analysis of amino acids similarities between TCR sequences. Single-cell sequencing is a breakthrough technology that can link phenotype with specificity, identifying T cell clones that are crucial for successful ICT. The field of immuno-sequencing is rapidly developing and cross-disciplinary efforts are required to maximize the analysis, application, and validation of sequencing data. Unravelling the dynamic behavior of the TCR repertoire during ICT will be highly valuable for tracking and understanding anti-tumor immunity, biomarker discovery, and ultimately for the development of novel strategies to improve patient outcomes.

A survey of genes modulated by host cell infection.

Microbial genomics, 2020
Cochrane, Kyla, Robinson, Avery V, Holt, Robert A, Allen-Vercoe, Emma
Here, we report comprehensive transcriptomic profiles from under conditions that mimic the first stages of bacterial infection in a highly differentiated adenocarcinoma epithelial cell line. Our transcriptomic adenocarcinoma approach allows us to measure the expression dynamics and regulation of bacterial virulence and response factors in real time, and is a novel strategy for clarifying the role of infection in colorectal cancer (CRC) progression. Our data show that: (i) infection alters metabolic and functional pathways in , allowing the bacterium to adapt to the host-imposed milieu; (ii) infection also stimulates the expression of genes required to help induce and promote a hypoxic and inflammatory microenvironment in the host; and (iii) invasion occurs by a haematogenous route of infection. Our study identifies novel gene targets from that are activated during invasion and which may aid in determining how this species invades and promotes disease within the human gastrointestinal tract. These invasion-specific genes may be useful as biomarkers for CRC progression in a host and could also assist in the development of new diagnostic tools and treatments (such as vaccines or small molecule drug targets), which will be able to combat infection and inflammation in the host while circumventing the potential problem of tolerization.

Rapid selection and identification of functional CD8+ T cell epitopes from large peptide-coding libraries.

Nature communications, 2019
Sharma, Govinda, Rive, Craig M, Holt, Robert A
Cytotoxic CD8{{sup}}+{{/sup}} T cells recognize and eliminate infected or malignant cells that present peptide epitopes derived from intracellularly processed antigens on their surface. However, comprehensive profiling of specific major histocompatibility complex (MHC)-bound peptide epitopes that are naturally processed and capable of eliciting a functional T cell response has been challenging. Here, we report a method for deep and unbiased T cell epitope profiling, using in vitro co-culture of CD8{{sup}}+{{/sup}} T cells together with target cells transduced with high-complexity, epitope-encoding minigene libraries. Target cells that are subject to cytotoxic attack from T cells in co-culture are isolated prior to apoptosis by fluorescence-activated cell sorting, and characterized by sequencing the encoded minigenes. We then validate this highly parallelized method using known murine T cell receptor/peptide-MHC pairs and diverse minigene-encoded epitope libraries. Our data thus suggest that this epitope profiling method allows unambiguous and sensitive identification of naturally processed and MHC-presented peptide epitopes.

Risks and Benefits of Chimeric Antigen Receptor T-Cell (CAR-T) Therapy in Cancer: A Systematic Review and Meta-Analysis.

Transfusion medicine reviews, 2019
Grigor, Emma J M, Fergusson, Dean, Kekre, Natasha, Montroy, Joshua, Atkins, Harold, Seftel, Matthew D, Daugaard, Mads, Presseau, Justin, Thavorn, Kednapa, Hutton, Brian, Holt, Robert A, Lalu, Manoj M
Promising efficacy results of chimeric antigen receptor (CAR) T-cell therapy have been tempered by safety considerations. Our objective was to comprehensively summarize the efficacy and safety of CAR-T cell therapy in patients with relapsed or refractory hematologic or solid malignancies. MEDLINE, Embase, and the Cochrane Register of Controlled Trials (inception - November 21, 2017). Interventional studies investigating CAR-T cell therapy in patients with malignancies were included. Our primary outcome of interest was complete response (defined as the absence of detectable cancer). Two independent reviewers extracted relevant data, assessed risk of bias, and graded the quality of evidence using established methods. A total of 42 hematological malignancy studies and 18 solid tumor studies met were included (913 participants). Of 486 evaluable hematologic patients, 54.4% [95% CI, 42.5%-65.9%] experienced complete response in 27 CD19 CAR-T cell therapy studies. Of 65 evaluable hematologic patients, 24.4% [95% CI, 9.4%-50.3%] experienced complete response in seven non-CD19 CAR-T cell therapy studies. Cytokine release syndrome was experienced by 55.3% [95% CI, 40.3%-69.4%] of patients and neurotoxicity 37.2% [95% CI, 28.6%-46.8%] of patients with hematologic malignancies. Of 86 evaluable solid tumor patients, 4.1% [95% CI, 1.6%-10.6%] experienced complete response in eight CAR-T cell therapy studies. Limitations include heterogeneity of study populations, as well as high risk of bias of included studies. There was a strong signal for efficacy of CAR-T cell therapy in patients with CD19+ hematologic malignancies and no overall signal in solid tumor trials published to date. These results will help inform patients, physicians, and other stakeholders of the benefits and risks associated with CAR-T cell therapy.

Twenty-Seven Tamoxifen-Inducible iCre-Driver Mouse Strains for Eye and Brain, Including Seventeen Carrying a New Inducible-First Constitutive-Ready Allele.

Genetics, 2019
Korecki, Andrea J, Hickmott, Jack W, Lam, Siu Ling, Dreolini, Lisa, Mathelier, Anthony, Baker, Oliver, Kuehne, Claudia, Bonaguro, Russell J, Smith, Jillian, Tan, Chin-Vern, Zhou, Michelle, Goldowitz, Daniel, Deussing, Jan M, Stewart, A Francis, Wasserman, Wyeth W, Holt, Robert A, Simpson, Elizabeth M
To understand gene function, the cre/loxP conditional system is the most powerful available for temporal and spatial control of expression in mouse. However, the research community requires more cre recombinase expressing transgenic mouse strains (cre-drivers) that restrict expression to specific cell types. To address these problems, a high-throughput method for large-scale production that produces high-quality results is necessary. Further, endogenous promoters need to be chosen that drive cell type specific expression, or we need to further focus the expression by manipulating the promoter. Here we test the suitability of using knock-ins at the docking site 5' of for rapid development of numerous cre-driver strains focused on expression in adulthood, using an improved cre tamoxifen inducible allele (icre/ERT2), and testing a novel inducible-first, constitutive-ready allele (icre/f3/ERT2/f3). In addition, we test two types of promoters either to capture an endogenous expression pattern (MaxiPromoters), or to restrict expression further using minimal promoter element(s) designed for expression in restricted cell types (MiniPromoters). We provide new cre-driver mouse strains with applicability for brain and eye research. In addition, we demonstrate the feasibility and applicability of using the locus 5' of for the rapid generation of substantial numbers of cre-driver strains. We also provide a new inducible-first constitutive-ready allele to further speed cre-driver generation. Finally, all these strains are available to the research community through The Jackson Laboratory.

Neoantigen characteristics in the context of the complete predicted MHC class I self-immunopeptidome.

Oncoimmunology
Brown, Scott D, Holt, Robert A
The self-immunopeptidome is the repertoire of all self-peptides that can be presented by the combination of MHC variants carried by an individual, defined by their HLA genotype. Each MHC variant presents a distinct set of self-peptides, and the number of peptides in a set is variable. Subjects carrying MHC variants that present fewer self-peptides should also present fewer mutated peptides, resulting in decreased immune pressure on tumor cells. To explore this, we predicted peptide-MHC binding values using all unique 8-11mer human peptides in the human proteome and all available HLA class I allelic variants, for a total of 134 billion unique peptide--MHC binding predictions. From these predictions, we observe that most peptides are able to be presented by relatively few (< 250) MHC, while some can be presented by upwards of 1,500 different MHC. There is substantial overlap among the repertoires of peptides presented by different MHC and no relationship between the number of peptides presented and HLA population frequency. Nearly 30% of self-peptides are presentable by at least one MHC, leaving 70% of the human peptidome unsurveyed by T cells. We observed similar distributions of predicted self-immunopeptidome sizes in cancer subjects compared to controls, and within the pan-cancer population, predicted self-immunopeptidome size combined with mutational load to predict survival. Self-immunopeptidome analysis revealed evidence for tumor immunoediting and identified specific peptide positions that most influence immunogenicity. Because self-immunopeptidome size is defined by HLA genotypes and approximates neoantigen load, HLA genotyping could offer a rapid predictive biomarker for response to immunotherapy.

A library-based screening method identifies neoantigen-reactive T cells in peripheral blood prior to relapse of ovarian cancer.

Oncoimmunology
Martin, Spencer D, Wick, Darin A, Nielsen, Julie S, Little, Nicole, Holt, Robert A, Nelson, Brad H
Mutated cancer antigens, or neoantigens, represent compelling immunological targets and appear to underlie the success of several forms of immunotherapy. While there are anecdotal reports of neoantigen-specific T cells being present in the peripheral blood and/or tumors of cancer patients, effective adoptive cell therapy (ACT) against neoantigens will require reliable methods to isolate and expand rare, neoantigen-specific T cells from clinically available biospecimens, ideally prior to clinical relapse. Here, we addressed this need using "mini-lines", large libraries of parallel T cell cultures, each originating from only 2,000 T cells. Using small quantities of peripheral blood from multiple time points in an ovarian cancer patient, we screened over 3.3 × 10{{sup}}6{{/sup}} CD8{{sup}}+{{/sup}} T cells by ELISPOT for recognition of peptides corresponding to the full complement of somatic mutations (n = 37) from the patient's tumor. We identified ten T cell lines which collectively recognized peptides encoding five distinct mutations. Six of the ten T cell lines recognized a previously described neoantigen from this patient (HSDL1{{sup}}L25V{{/sup}}), whereas the remaining four lines recognized peptides corresponding to four other mutations. Only the HSDL1{{sup}}L25V{{/sup}}-specific T cell lines recognized autologous tumor. HSDL1{{sup}}L25V{{/sup}}-specific T cells comprised at least three distinct clonotypes and could be identified and expanded from peripheral blood 3-9 months prior to the first tumor recurrence. These T cells became undetectable at later time points, underscoring the dynamic nature of the response. Thus, neoantigen-specific T cells can be expanded from small volumes of blood during tumor remission, making pre-emptive ACT a plausible clinical strategy.

Fusobacterium nucleatum infection is prevalent in human colorectal carcinoma.

Genome research, 2012
Castellarin, Mauro, Warren, René L, Freeman, J Douglas, Dreolini, Lisa, Krzywinski, Martin, Strauss, Jaclyn, Barnes, Rebecca, Watson, Peter, Allen-Vercoe, Emma, Moore, Richard A, Holt, Robert A
An estimated 15% or more of the cancer burden worldwide is attributable to known infectious agents. We screened colorectal carcinoma and matched normal tissue specimens using RNA-seq followed by host sequence subtraction and found marked over-representation of Fusobacterium nucleatum sequences in tumors relative to control specimens. F. nucleatum is an invasive anaerobe that has been linked previously to periodontitis and appendicitis, but not to cancer. Fusobacteria are rare constituents of the fecal microbiota, but have been cultured previously from biopsies of inflamed gut mucosa. We obtained a Fusobacterium isolate from a frozen tumor specimen; this showed highest sequence similarity to a known gut mucosa isolate and was confirmed to be invasive. We verified overabundance of Fusobacterium sequences in tumor versus matched normal control tissue by quantitative PCR analysis from a total of 99 subjects (p = 2.5 × 10(-6)), and we observed a positive association with lymph node metastasis.

Profiling the T-cell receptor beta-chain repertoire by massively parallel sequencing.

Genome research, 2009
Freeman, J Douglas, Warren, René L, Webb, John R, Nelson, Brad H, Holt, Robert A
T-cell receptor (TCR) genomic loci undergo somatic V(D)J recombination, plus the addition/subtraction of nontemplated bases at recombination junctions, in order to generate the repertoire of structurally diverse T cells necessary for antigen recognition. TCR beta subunits can be unambiguously identified by their hypervariable CDR3 (Complement Determining Region 3) sequence. This is the site of V(D)J recombination encoding the principal site of antigen contact. The complexity and dynamics of the T-cell repertoire remain unknown because the potential repertoire size has made conventional sequence analysis intractable. Here, we use 5'-RACE, Illumina sequencing, and a novel short read assembly strategy to sample CDR3(beta) diversity in human T lymphocytes from peripheral blood. Assembly of 40.5 million short reads identified 33,664 distinct TCR(beta) clonotypes and provides precise measurements of CDR3(beta) length diversity, usage of nontemplated bases, sequence convergence, and preferences for TRBV (T-cell receptor beta variable gene) and TRBJ (T-cell receptor beta joining gene) gene usage and pairing. CDR3 length between conserved residues of TRBV and TRBJ ranged from 21 to 81 nucleotides (nt). TRBV gene usage ranged from 0.01% for TRBV17 to 24.6% for TRBV20-1. TRBJ gene usage ranged from 1.6% for TRBJ2-6 to 17.2% for TRBJ2-1. We identified 1573 examples of convergence where the same amino acid translation was specified by distinct CDR3(beta) nucleotide sequences. Direct sequence-based immunoprofiling will likely prove to be a useful tool for understanding repertoire dynamics in response to immune challenge, without a priori knowledge of antigen.

The genome sequence of the malaria mosquito Anopheles gambiae.

Science (New York, N.Y.), 2002
Holt, Robert A, Subramanian, G Mani, Halpern, Aaron, Sutton, Granger G, Charlab, Rosane, Nusskern, Deborah R, Wincker, Patrick, Clark, Andrew G, Ribeiro, José M C, Wides, Ron, Salzberg, Steven L, Loftus, Brendan, Yandell, Mark, Majoros, William H, Rusch, Douglas B, Lai, Zhongwu, Kraft, Cheryl L, Abril, Josep F, Anthouard, Veronique, Arensburger, Peter, Atkinson, Peter W, Baden, Holly, de Berardinis, Veronique, Baldwin, Danita, Benes, Vladimir, Biedler, Jim, Blass, Claudia, Bolanos, Randall, Boscus, Didier, Barnstead, Mary, Cai, Shuang, Center, Angela, Chaturverdi, Kabir, Christophides, George K, Chrystal, Mathew A, Clamp, Michele, Cravchik, Anibal, Curwen, Val, Dana, Ali, Delcher, Art, Dew, Ian, Evans, Cheryl A, Flanigan, Michael, Grundschober-Freimoser, Anne, Friedli, Lisa, Gu, Zhiping, Guan, Ping, Guigo, Roderic, Hillenmeyer, Maureen E, Hladun, Susanne L, Hogan, James R, Hong, Young S, Hoover, Jeffrey, Jaillon, Olivier, Ke, Zhaoxi, Kodira, Chinnappa, Kokoza, Elena, Koutsos, Anastasios, Letunic, Ivica, Levitsky, Alex, Liang, Yong, Lin, Jhy-Jhu, Lobo, Neil F, Lopez, John R, Malek, Joel A, McIntosh, Tina C, Meister, Stephan, Miller, Jason, Mobarry, Clark, Mongin, Emmanuel, Murphy, Sean D, O'Brochta, David A, Pfannkoch, Cynthia, Qi, Rong, Regier, Megan A, Remington, Karin, Shao, Hongguang, Sharakhova, Maria V, Sitter, Cynthia D, Shetty, Jyoti, Smith, Thomas J, Strong, Renee, Sun, Jingtao, Thomasova, Dana, Ton, Lucas Q, Topalis, Pantelis, Tu, Zhijian, Unger, Maria F, Walenz, Brian, Wang, Aihui, Wang, Jian, Wang, Mei, Wang, Xuelan, Woodford, Kerry J, Wortman, Jennifer R, Wu, Martin, Yao, Alison, Zdobnov, Evgeny M, Zhang, Hongyu, Zhao, Qi, Zhao, Shaying, Zhu, Shiaoping C, Zhimulev, Igor, Coluzzi, Mario, della Torre, Alessandra, Roth, Charles W, Louis, Christos, Kalush, Francis, Mural, Richard J, Myers, Eugene W, Adams, Mark D, Smith, Hamilton O, Broder, Samuel, Gardner, Malcolm J, Fraser, Claire M, Birney, Ewan, Bork, Peer, Brey, Paul T, Venter, J Craig, Weissenbach, Jean, Kafatos, Fotis C, Collins, Frank H, Hoffman, Stephen L
Anopheles gambiae is the principal vector of malaria, a disease that afflicts more than 500 million people and causes more than 1 million deaths each year. Tenfold shotgun sequence coverage was obtained from the PEST strain of A. gambiae and assembled into scaffolds that span 278 million base pairs. A total of 91% of the genome was organized in 303 scaffolds; the largest scaffold was 23.1 million base pairs. There was substantial genetic variation within this strain, and the apparent existence of two haplotypes of approximately equal frequency ("dual haplotypes") in a substantial fraction of the genome likely reflects the outbred nature of the PEST strain. The sequence produced a conservative inference of more than 400,000 single-nucleotide polymorphisms that showed a markedly bimodal density distribution. Analysis of the genome sequence revealed strong evidence for about 14,000 protein-encoding transcripts. Prominent expansions in specific families of proteins likely involved in cell adhesion and immunity were noted. An expressed sequence tag analysis of genes regulated by blood feeding provided insights into the physiological adaptations of a hematophagous insect.

The sequence of the human genome.

Science (New York, N.Y.), 2001
Venter, J C, Adams, M D, Myers, E W, Li, P W, Mural, R J, Sutton, G G, Smith, H O, Yandell, M, Evans, C A, Holt, R A, Gocayne, J D, Amanatides, P, Ballew, R M, Huson, D H, Wortman, J R, Zhang, Q, Kodira, C D, Zheng, X H, Chen, L, Skupski, M, Subramanian, G, Thomas, P D, Zhang, J, Gabor Miklos, G L, Nelson, C, Broder, S, Clark, A G, Nadeau, J, McKusick, V A, Zinder, N, Levine, A J, Roberts, R J, Simon, M, Slayman, C, Hunkapiller, M, Bolanos, R, Delcher, A, Dew, I, Fasulo, D, Flanigan, M, Florea, L, Halpern, A, Hannenhalli, S, Kravitz, S, Levy, S, Mobarry, C, Reinert, K, Remington, K, Abu-Threideh, J, Beasley, E, Biddick, K, Bonazzi, V, Brandon, R, Cargill, M, Chandramouliswaran, I, Charlab, R, Chaturvedi, K, Deng, Z, Di Francesco, V, Dunn, P, Eilbeck, K, Evangelista, C, Gabrielian, A E, Gan, W, Ge, W, Gong, F, Gu, Z, Guan, P, Heiman, T J, Higgins, M E, Ji, R R, Ke, Z, Ketchum, K A, Lai, Z, Lei, Y, Li, Z, Li, J, Liang, Y, Lin, X, Lu, F, Merkulov, G V, Milshina, N, Moore, H M, Naik, A K, Narayan, V A, Neelam, B, Nusskern, D, Rusch, D B, Salzberg, S, Shao, W, Shue, B, Sun, J, Wang, Z, Wang, A, Wang, X, Wang, J, Wei, M, Wides, R, Xiao, C, Yan, C, Yao, A, Ye, J, Zhan, M, Zhang, W, Zhang, H, Zhao, Q, Zheng, L, Zhong, F, Zhong, W, Zhu, S, Zhao, S, Gilbert, D, Baumhueter, S, Spier, G, Carter, C, Cravchik, A, Woodage, T, Ali, F, An, H, Awe, A, Baldwin, D, Baden, H, Barnstead, M, Barrow, I, Beeson, K, Busam, D, Carver, A, Center, A, Cheng, M L, Curry, L, Danaher, S, Davenport, L, Desilets, R, Dietz, S, Dodson, K, Doup, L, Ferriera, S, Garg, N, Gluecksmann, A, Hart, B, Haynes, J, Haynes, C, Heiner, C, Hladun, S, Hostin, D, Houck, J, Howland, T, Ibegwam, C, Johnson, J, Kalush, F, Kline, L, Koduru, S, Love, A, Mann, F, May, D, McCawley, S, McIntosh, T, McMullen, I, Moy, M, Moy, L, Murphy, B, Nelson, K, Pfannkoch, C, Pratts, E, Puri, V, Qureshi, H, Reardon, M, Rodriguez, R, Rogers, Y H, Romblad, D, Ruhfel, B, Scott, R, Sitter, C, Smallwood, M, Stewart, E, Strong, R, Suh, E, Thomas, R, Tint, N N, Tse, S, Vech, C, Wang, G, Wetter, J, Williams, S, Williams, M, Windsor, S, Winn-Deen, E, Wolfe, K, Zaveri, J, Zaveri, K, Abril, J F, Guigó, R, Campbell, M J, Sjolander, K V, Karlak, B, Kejariwal, A, Mi, H, Lazareva, B, Hatton, T, Narechania, A, Diemer, K, Muruganujan, A, Guo, N, Sato, S, Bafna, V, Istrail, S, Lippert, R, Schwartz, R, Walenz, B, Yooseph, S, Allen, D, Basu, A, Baxendale, J, Blick, L, Caminha, M, Carnes-Stine, J, Caulk, P, Chiang, Y H, Coyne, M, Dahlke, C, Mays, A, Dombroski, M, Donnelly, M, Ely, D, Esparham, S, Fosler, C, Gire, H, Glanowski, S, Glasser, K, Glodek, A, Gorokhov, M, Graham, K, Gropman, B, Harris, M, Heil, J, Henderson, S, Hoover, J, Jennings, D, Jordan, C, Jordan, J, Kasha, J, Kagan, L, Kraft, C, Levitsky, A, Lewis, M, Liu, X, Lopez, J, Ma, D, Majoros, W, McDaniel, J, Murphy, S, Newman, M, Nguyen, T, Nguyen, N, Nodell, M, Pan, S, Peck, J, Peterson, M, Rowe, W, Sanders, R, Scott, J, Simpson, M, Smith, T, Sprague, A, Stockwell, T, Turner, R, Venter, E, Wang, M, Wen, M, Wu, D, Wu, M, Xia, A, Zandieh, A, Zhu, X
A 2.91-billion base pair (bp) consensus sequence of the euchromatic portion of the human genome was generated by the whole-genome shotgun sequencing method. The 14.8-billion bp DNA sequence was generated over 9 months from 27,271,853 high-quality sequence reads (5.11-fold coverage of the genome) from both ends of plasmid clones made from the DNA of five individuals. Two assembly strategies-a whole-genome assembly and a regional chromosome assembly-were used, each combining sequence data from Celera and the publicly funded genome effort. The public data were shredded into 550-bp segments to create a 2.9-fold coverage of those genome regions that had been sequenced, without including biases inherent in the cloning and assembly procedure used by the publicly funded group. This brought the effective coverage in the assemblies to eightfold, reducing the number and size of gaps in the final assembly over what would be obtained with 5.11-fold coverage. The two assembly strategies yielded very similar results that largely agree with independent mapping data. The assemblies effectively cover the euchromatic regions of the human chromosomes. More than 90% of the genome is in scaffold assemblies of 100,000 bp or more, and 25% of the genome is in scaffolds of 10 million bp or larger. Analysis of the genome sequence revealed 26,588 protein-encoding transcripts for which there was strong corroborating evidence and an additional approximately 12,000 computationally derived genes with mouse matches or other weak supporting evidence. Although gene-dense clusters are obvious, almost half the genes are dispersed in low G+C sequence separated by large tracts of apparently noncoding sequence. Only 1.1% of the genome is spanned by exons, whereas 24% is in introns, with 75% of the genome being intergenic DNA. Duplications of segmental blocks, ranging in size up to chromosomal lengths, are abundant throughout the genome and reveal a complex evolutionary history. Comparative genomic analysis indicates vertebrate expansions of genes associated with neuronal function, with tissue-specific developmental regulation, and with the hemostasis and immune systems. DNA sequence comparisons between the consensus sequence and publicly funded genome data provided locations of 2.1 million single-nucleotide polymorphisms (SNPs). A random pair of human haploid genomes differed at a rate of 1 bp per 1250 on average, but there was marked heterogeneity in the level of polymorphism across the genome. Less than 1% of all SNPs resulted in variation in proteins, but the task of determining which SNPs have functional consequences remains an open challenge.

Staff

Dr. Joan Shellard

Staff Scientist

Dr. Eric Yung

Staff Scientist
Lab Manager

Dr. Scott Brown

Research Associate
Clinical Informatics Analyst

Dr. Craig Rive

Research Associate

Lisa Dreolini

Research Assistant

Nasrin Mawji

Genome Sciences Technologist

Mhairi Sigrist, PhD, PMP

Projects Team Leader

Postdoctoral Fellows

Dr. James Round

Postdoctoral Fellow

Dr. Govinda Sharma

Post-Doctoral Fellow

Dr. Sophie Sneddon

Post-Doctoral Fellow

Trainees

Cody Despins

Graduate Student

Nicole Knoetze

Graduate Student

Christopher May

Graduate Student

Michelle Sag

Student Researcher

Open Positions

Postdoctoral Fellow

Canada’s Michael Smith Genome Sciences Centre (GSC)

Today’s Research. Tomorrow’s Medicine.

The GSC is a department of the BC Cancer Research Institute and a high-throughput genome sequencing facility. We are leaders in genomics, proteomics and bioinformatics in pursuit of novel treatment strategies for cancers and other diseases.

Among the world’s first genome centres to be established within a cancer clinic, for more than two decades our scientists and innovators have been designing and deploying cutting-edge technologies to benefit health and advance clinical research.

Among the GSC’s most significant accomplishments are the first publication to demonstrate the use of whole-genome sequencing to inform cancer treatment planning, the first published sequence of the SARS coronavirus genome and major contributions to the first physical map of the human genome as part of the Human Genome Project.

By joining the GSC you will become part of an exceptional and diverse team of scientists, clinicians, experts and professionals operating at the leading edge of clinical research. We look for people who share our core values—science, timeliness, respect—to join us on our mission to use genome science for the betterment of health and society.

Summary

The Holt Laboratory at BC Cancer is looking for a qualified candidate to aid in the preclinical and clinical development of novel cancer vaccines and cellular immunotherapies. The candidate will work with local, national and international partners, collaborators and service providers in academia, industry, government and not-for-profit organizations.

Qualifications

  • A recent PhD, and competency with standard methods and approaches in microbiology and immunology, or a closely related field.
  • Familiarity with cancer immunology and immunotherapy 
  • Bioinformatics skills and the ability to analyse large genomics and  proteomics data sets
  • Familiarity with regulatory aspects of preclinical and clinical research methodology
  • Excellent verbal and written communication skills
  • Ability to work in a team environment. 

This position will begin ASAP.  This is a one-year term appointment subject to annual renewal.  Salary will be commensurate with qualifications and experience.

Application

Please submit a detailed cover letter and resume to bcgscjobs@bcgsc.ca, using PDF_01553_2021_10_04_Holt Lab in the subject line of your email.

This posting will remain online until filled.

Due to current COVID-19 restrictions, the position would require working remotely within British Columbia on a temporary basis with an ability to come to the normal workplace within reasonable notice. This restriction would be re-evaluated after finalization of reopening plans.

As per the current Public Health Order, full vaccination against COVID-19 is a condition of employment with PHSA as of October 26, 2021.

We believe that equity, diversity and inclusivity are essential for the advancement of human knowledge and science.

We welcome all applicants and provide all employees with equal opportunity for advancement, regardless of race, colour, ancestry, place of origin, political belief, religion, marital status, family status, physical or mental disability, sex, sexual orientation, gender identity or expression, age, conviction of a criminal or summary conviction offence unrelated to their employment.

All qualified candidates are encouraged to apply; however, Canadian citizens and permanent residents will be given priority.

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