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• Ultra-long sequencing for contiguous haplotype resolution of the human immunoglobulin heavy chain locus

  Genetic diversity within the human immunoglobulin heavy chain (IGH) locus influences the expressed antibody repertoire and susceptibility to infectious and autoimmune diseases. However, repetitive sequences and complex structural variation pose significant challenges for large-scale characterization. Here, we introduce a method that combines Oxford Nanopore ultra-long sequencing and adaptive sampling with a bioinformatic pipeline to produce haplotype-resolved, annotated IGH assemblies. Notably, our strategy overcomes prior limitations in phasing resolution, enabling single-contig haplotype assemblies that span the entire IGH locus. We applied this method to four individuals and validated the accuracy of the IGH assemblies using Pacific Biosciences HiFi reads, demonstrating near-complete sequence congruence, detecting only indels with a high degree of confidence. Moreover, when applied to the reference material HG002, our pipeline revealed no base differences and a limited number of indels compared with the Telomere-to-Telomere genome benchmark across the IGH region. Importantly, in the four individuals, our approach uncovered 30 novel alleles and previously uncharacterized large structural variants, including a 120 kb duplication spanning IGHE to IGHA1 and an expanded seven-copy IGHV3-23 gene haplotype. These findings underscore the power of our method to resolve the full complexity of the IGH locus and uncover previously unrecognized variants that may affect immune function and disease susceptibility. Thus, our method provides a strong basis for future immunological research and translational applications.

  Study EGAS50000001042

• Delineating pediatric brain tumor progression using single-nuclei sequencing

  Pediatric brain cancers are the most common solid tumors in children with varying survival rates depending on molecular features of cancer cells and associated microenvironment. Several studies have used single-cell RNA sequencing to define transcriptional programs active in cancer cells and highlighted supportive roles of non-cancerous cells embedded in these tumors. Although application of single-cell RNA sequencing technology has expanded our understanding of intratumoral heterogeneity and underlying transcriptional mechanisms in brain tumors at diagnosis, mechanisms of disease remains elusive. Therefore, the purpose of this proposal is to characterize cellular and transcriptional states of malignant and non-malignant compartments at primary diagnosis and at relapse. In this study, we performed single-nuclei sequencing of 42 patient samples taken at various disease stages (initial diagnosis, progressive disease, and relapsed disease) from 20 patients with low-grade glioma or Atypical Teratoid Rhabdoid Tumor (ATRT). Bulk whole genome and RNA-seq data for these samples are available from the Children’s Brain Tumor Network and the Gabriella Miller Kids First Data Resource Center. Findings from our study will help define the mechanistic shifts of tumor and non-cancerous cells over course of disease progression. Collectively with the single-cell Pediatric Cancer Atlas funded, this project lays the foundation of an important data set focused on lethal relapsed cancers that can continue to be expanded to identify cancer-intrinsic and extrinsic microenvironmental factors supporting recurrence of pediatric brain tumors.

  Study EGAS50000001288

• UK10K_RARE_HYPERCHOL

  In the UK10K project we propose a series of complementary genetic approaches to find new low frequency/rare variants contributing to disease phenotypes. These will be based on obtaining the genome wide sequence of 4000 samples from the TwinsUK and ALSPAC cohorts (at 6x sequence coverage), and the exome sequence (protein coding regions and related conserved sequence) of 6000 samples selected for extreme phenotypes. Our studies will focus primarily on cardiovascular-related quantitative traits, obesity and related metabolic traits, neurodevelopmental disorders and a limited number of extreme clinical phenotypes that will provide proof-of-concept for future familial trait sequencing. We will analyse directly quantitative traits in the cohorts and the selected traits in the extreme samples, and also use imputation down to 0.1% allele frequency to extend the analyses to further sample sets with genome wide genotype data. In each case we will investigate indels and larger structural variants as well as SNPs, and use statistical methods that combine rare variants in a locus or pathway as well as single-variant approaches.Familial Hypercholesterolemia is a condition where the affected person has a consistently high level of LDL which can lead to early clogging of the coronary arteries. All patients selected for this study will have been found not to carry the common APOB and PCSK9 mutations, and to have no detectable LDLR mutations by testing for 18 common mutations. For further information with regard to this cohort please contact Steve Humphries(steve.humphries@ucl.ac.uk).

  Study EGAS00001000129

• Identification_of_synthetic_lethal_genes_by_CRISPR_Cas9_library

  Sequencing is performed on PCR products from the amplification of gRNA segments introduced into cell lines (some are commercial, some are derived by collaborators, as described in HMDMC 13/076). Cellular DNA or RNA are not sequenced. This data is defined as "Other Biological/Biochemical Assay Data" under the WTSI datasharing guidelines and consequently will be released at the time of publication and/or through faculty websites. SEQUENCING DETAILS: in October 2016 3 DNA samples will be submitted for PCR amplification and purification through CGP pipeline using Thompson's custom primer set and PCR settings and sequencing with Hiseq using a custom protocol (with 3 forward reads to read sgRNA1, sgRNA2 and the index, optimized with Illumina Bespoke Sanger team); read length 50bp. The following set of samples will be submitted in later 2016 and ealry 2017 as cell pellet to be processed through CGP Cas9 pipeline (Thompson's oligos and protocols) and sequences on Hiseq (same custom sequencing protocol described above) We are utilizing a custom CRISPR/Cas9 library to identify synthetic lethal gene pairs. Cells will be transduced with LV expressing Cas9, then infected with LV expressign the library of 42'000 sgRNA pairs. Cell pellet will be collected from the cell population at different timepoints. The samples will be processed by CGP pipeline customized for the specific protocol and then sequenced to identify the 19bp of the sgRNA and quantify their representation.

  Study EGAS00001002117

• Initial whole genome sequencing of plasma cell neoplasms in First Responders exposed to the World Trade Center attack of September 11, 2001

  The World Trade Center (WTC) attack of September 11, 2001 created an unprecedented environmental exposure to known and suspected carcinogens. High incidence of multiple myeloma (MM) and precursor conditions has been reported among first responders to the WTC disaster. To expand on our prior screening studies, and to characterize the genomic impact of the exposure to known and potential carcinogens in the WTC debris, we were motivated to perform whole genome sequencing (WGS) of WTC first responders and recovery workers who developed a plasma cell disorder after the attack. PATIENTS AND METHODS: We performed WGS of 9 CD138-positive bone marrow mononuclear samples from patients who were diagnosed with plasma cell disorders after the WTC disaster. RESULTS: No significant differences were observed in comparing the post-WTC driver and mutational signatures landscape with 110 previously published WGS from 56 patients with MM and the CoMMpass WGS cohort (n=752). Leveraging constant activity of the single base substitution mutational signatures 1 and 5 over time, we estimated that tumor-initiating chromosomal gains were windowed to both pre- and post-WTC exposure. CONCLUSIONS: Although limitations in sample size preclude any definitive conclusions, our findings suggest that the observed increased incidence of plasma cell neoplasms in this population is due to complex and heterogeneous effects of the WTC exposure that may have initiated or contributed to progression of malignancy.

  Study EGAS00001004467

• Oxidative phosphorylation is a key ontogenetic feature of monocyte immunometabolism promoting myeloid differentiation after birth

  Neonates primarily rely on innate immunity, yet their inflammatory responses to microbes, particularly those of monocytes, are usually restricted compared to adults. This is controversially interpreted as immaturity increasing the risk of sepsis or essential programming allowing immune adaptation after birth. Which changes the cellular immunometabolism undergoes after birth and its role in these concepts are poorly defined. Here, we applied transcriptomic, metabolic, and immunological approaches and found that monocytes exhibit an inverse ontogenetic balance of immunometabolism. Our data show that glycolysis in monocytes increases within the first year of life and fuels their inflammatory responsiveness. In contrast, oxidative phosphorylation (OXPHOS) is high in neonatal monocytes supporting myeloid differentiation but declines only gradually during the first five years of life. Treatment of neonatal monocytes with lipopolysaccharide induces an adult-like immunometabolic phenotype. Ketogenic diet restricts glycolysis in adult monocytes, but cannot reactivate OXPHOS and revive a neonatal-like immunometabolic phenotype, suggesting that neonatal metabolism is hardwired and cannot be restored by simple dietary changes. Transcriptional network and population-wide human variation analyses identified E2F1, MYB, STAT1 and FLI1 as important regulators of age-dependent cell functions and related energy demands in human monocytes. Collectively, our findings show that restricted glycolysis and increased OXPHOS are a physiological programming in neonatal monocytes. Premature switching to an adult-like metabolism could untimely enhance inflammatory responses and disrupt important myeloid differentiation, whereas microbial challenges accumulating during childhood seem to induce an essential gradual metabolic switch.

  Study EGAS00001007555

• Spatio-temporal Profiling of a Rhabdoid Tumor Case Study

  Background Immune checkpoint inhibition (ICI) has revolutionized oncology, offering extended survival and long-term remission in previously incurable cancers. While highly effective in tumors with high mutational burden, lowly mutated cancers, including pediatric malignancies, present low response rate and limited predictive biomarkers. Patients and methods We present a framework for the identification and validation of tumor-reactive T cells as a biomarker to quantify ICI efficacy and as candidates for a personalized TCR-T cell therapy. Therefore, we profiled a pediatric malignant rhabdoid tumor patient with complete remission after ICI therapy using deep single-cell T cell receptor (TCR) repertoire sequencing of the tumor microenvironment (TME) and the peripheral blood. Results Tracking T cell dynamics longitudinally from the tumor to cells in circulation over a time course of 12 months revealed a systemic response and durable clonal expansion of tumor-resident and ICI-induced TCR clonotypes. We functionally validated tumor reactivity of TCRs identified from the TME and the blood by co-culturing patient-derived tumor cells with TCR-engineered autologous T cells. Here, we observed unexpectedly high frequencies of tumor-reactive TCR clonotypes in the TME and confirmed T cell dynamics in the blood post-ICI to predict tumor-reactivity. Conclusion These findings strongly support spatio-temporal tracking of T cell activity in response to ICI to inform therapy efficacy and to serve as a source of tumor-reactive TCRs for personalized TCR-T designs.

  Study EGAS00001008174

• Identification of Somatic Changes in Tumors from Fanconi Anemia Patients

  This study focused on the identification of genomic signatures present in tumors from Fanconi anemia patients. Fanconi anemia is a rare hereditary disease caused by defects in DNA interstrand crosslink repair. The primary patient phenotypes are bone marrow failure and cancer predisposition. We concentrated on squamous cell carcinomas and used whole genome Illumina sequencing (22 tumor samples and 12 normal samples) , whole exome sequencing (37 tumor samples and 33 normal samples), PacBio long-read whole-genome (nine tumor and four normal samples),10X linked-read whole-genome sequencing, 10x single-cell/nuclei 3’ RNA sequencing (three tumor samples), 10x Visium spatial transcriptomics (one tumor sample), EPIC 850K methylation array (six samples), and bulk RNAseq (six tumor samples) to identify somatic changes present in tumors. The study aimed to identify changes that drive tumorigenesis in Fanconi anemia, give insight into the early development and aggressive nature of these tumors, and suggest therapeutic avenues for these patients. Another goal was to compare the genomic changes identified in tumors from Fanconi anemia patients with sporadic head and neck cancers. This study demonstrated that the tumors from Fanconi anemia individuals are generally not driven by HPV. Instead, they acquire mutations in the p53 tumor suppressor. The main characteristic of the tumors is the presence of a high number of structural variants in the form of small deletions, unbalanced translocations, and fold-back inversions, which often form complex rearrangements. These rearrangements result in amplifications and deletions of a multitude of oncogenes and tumor suppressors, respectively. The study also shows that lack of the Fanconi anemia DNA repair pathway leads to an epithelial-to-mesenchymal transition in a mouse model and in human samples that may influence the behavior of tumors in Fanconi anemia. Finally, comparing genomic changes in Fanconi anemia and sporadic tumors of the head and neck, we propose that the structural variants present in sporadic cancer result in a functional deficiency of the Fanconi anemia DNA repair pathway.

  Study phs002652

• UK10K NEURO IOP COLLIER

  In the UK10K project we propose a series of complementary genetic approaches to find new low frequency/rare variants contributing to disease phenotypes. These will be based on obtaining the genome wide sequence of 4000 samples from the TwinsUK and ALSPAC cohorts (at 6x sequence coverage), and the exome sequence (protein coding regions and related conserved sequence) of 6000 samples selected for extreme phenotypes. Our studies will focus primarily on cardiovascular-related quantitative traits, obesity and related metabolic traits, neurodevelopmental disorders and a limited number of extreme clinical phenotypes that will provide proof-of-concept for future familial trait sequencing. We will analyse directly quantitative traits in the cohorts and the selected traits in the extreme samples, and also use imputation down to 0.1% allele frequency to extend the analyses to further sample sets with genome wide genotype data. In each case we will investigate indels and larger structural variants as well as SNPs, and use statistical methods that combine rare variants in a locus or pathway as well as single-variant approaches. The Genetics and Psychosis (GAP) set consists of samples from subjects with schizophrenia, ascertained as a new-onset sample. This set is of UK origin, with data on cognition, brain imaging and other endophenotypes. The Maudsley twin series consists of probands ascertained from the Maudsley Twin Register, defined as patients of multiple birth who had suffered psychotic symptoms. This set is of UK origin, with data on cognition, brain imaging and other endophenotypes, with DNA available from an MZ or DZ affected or unaffected co-twin. The Maudsley family study (MFS) consists of over 250 families who have a history of schizophrenia or bipolar disorder Within the Maudsley Family Study, biological markers of psychosis include neuropsychological tests, Evoked Response Potentials Tests (ERPs), MRI scans, dermatoglyphics and eye tracking. Early risk factors for psychosis and clinical symptoms are also investigated. This set is of UK origin, with DNA available from both affected and unaffected relatives in many of the probands. For further information on this study please contact David Collier (david.collier@kcl.ac.uk).

  Study EGAS00001000121

• Neoantigen-Targeted CD8+ T-Cell Responses 1 With PD-1 Blockade Therapy

  Background: The primary target of T-cell responses induced against cancer cells are peptides derived from non-synonymous mutations presented by HLA (neoantigens). However, the large diversity of HLA alleles and restricted availability of clinical samples have limited the study of the specificity and the evolution over time of the neoantigen-specific T cell responses induced after treatment with immune checkpoint blockade (ICB) immunotherapy. Methods: We applied a newly developed neoepitope-specific T cell isolation technology to perform a longitudinal landscape analysis of the neoepitope-specific T cells in peripheral blood and tumor from 11 patients with metastatic melanoma; 7 with response and 4 with no response to ICB. Briefly, first using computational prediction, the putative neoantigens in each patient were ranked based on affinity for the patient's HLA and expression levels. Then hundreds of barcoded capture reagents consisting of the patient HLA class I subtypes loaded with the corresponding predicted neoantigen were made and used to isolate neoepitope-specific T cells. Once isolated, the TCRs and the barcodes were sequenced. Finally, the tumor reactivity of the isolated neoepitope-specific TCRs (neoTCRs) was assessed upon co-culture of autologous melanoma cell lines from each patient with primary human T cells expressing the neoTCRs generated using a CRISPR-based non-viral gene editing to replace the endogenous TCRs. Results: The tumor mutation burden ranged between 3507 and 177 for patients with response to therapy and 977 to 31 for patients without response to therapy. We screened a median of 172 predicted neoantigen-HLA per patient across their 6 HLA molecules and isolated neoTCRs in all 11 patients. The number of mutations targeted ranged between 13 and 1. We assessed the neoTCR-tumor reactivity in samples from 3 patients with response and 3 patients without response. 39 of the 62 neoTCRs demonstrated specific recognition and cytotoxicity to patient-matched melanoma cell lines. Multiple T cell clonotypes recognized a limited number of mutations in 7 patients with response (median of 41 different neoTCR clonotypes per patient), and the T cell specificities were recurrently detected in the blood and the tumor over time. Samples from 4 patients without response to therapy also demonstrated neoepitope-specific T cell responses in blood and tumor to a similarly restricted number of mutations, but lacked TCR polyclonality (median 6.5 neoTCR clonotypes per patient) and were not recurrently detected in sequential samples. Conclusion: Effective ICB therapy is associated with polyclonal neoepitope-specific T-cell responses in the tumor and blood that recognize a limited number of immunodominant mutations and are recurrently detected over time.

  Study phs003153