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• Whole exome sequencing of a representative cohort of AML

  The Genomic DNA Clean & Concentrator kit (ZYMO Research) was used to remove EDTA from the DNA samples. Sample libraries were prepared using 100 ng of input according to the KAPA HyperPlus Kit (Roche) using Unique Dual Index adapters (Integrated DNA Technologies, Inc.). Exomes were captured using the SeqCap EZ MedExome (Roche Nimblegen) according to SeqCap EZ HyperCap Library v1.0 Guide (Roche) with the xGen Universal blockers – TS Mix (Integrated DNA Technologies, Inc.). The amplified captured sample libraries were paired-end sequenced (2x100 bp) on the Novaseq 6000 platform (Illumina) and aligned to the hg19 reference genome using the Burrows-Wheeler Aligner (BWA)3, v0.7.15-r1140.

  Dataset EGAD00001007580

• RNA-seq study of human long-term and short-term hematopoietic stem cells from umblical cord blood with lentiviral overexpression of S1PR3

  To investigate the molecular and biological pathways altered by S1PR3OE in human hematopoietic stem cells (HSC), we performed RNA-sequencing (RNA-seq) of LT- and ST-HSC 3 days after transduction with control or S1PR3 overexpression (OE) lentiviral vectors. LT-HSC and ST-HSC from 3 pool of CB lin- were FACS-purified, cells were prestimulated for 4 hours and transduced with lentiviral vectors. At day 3, 2000-5300 BFP+ cells were FACS-purified for RNA isolation with a PicoPure kit. We were able to isolate only 1600-1800 BFP+ cells from LT-HSC control samples as opposed to 4000-5400 BFP+ cells from S1PR3OE samples. Thus, we pooled all control BFP+ LT-HSC cells into one sample for RNA-seq analysis. BFP- LT-HSC from control vector transduction were purified from CB1 as an additional LT-HSC control. Nextera libraries generated from 10 ng RNA from 5 LT-HSC samples (2 controls, 3 S1PR3OE) and 6 ST-HSC samples (3 controls, 3 S1PR3OE) were subjected to 125 bp, paired-end RNA-sequencing on the Illumina HiSeq 2500 with an average of 50 million reads/sample at the Center for Applied Genomics, Sick Kids Hospital.

  Dataset EGAD00001006582

• 59 CLPD-NK cases WGS & WTS data

  For the cohort of 59 samples, we performed TruSeq DNA PCR-Free whole-genome sequencing library preparation according to manufacturer’s instructions (llumina, ILMN, San Diego, CA) on the automated NGS Star liquid handling platform (Hamilton, Bonaduz, Switzerland) followed by 2x150 bp paired-end sequencing on the HiSeqX or NovaSeq6000 (ILMN). An average coverage of >100x was achieved. For whole transcriptome analysis, the TruSeq Total Stranded RNA kit was used, starting with 250 ng of total RNA, to generate RNA libraries following the manufacturer’s recommendations (ILMN). 2x100bp paired-end reads were sequenced on the NovaSeq 6000 with a median of 50 mio. reads per sample (ILMN).

  Dataset EGAD00001008558

• Common early-life exposure of the colorectal epithelium to the microbiome-derived mutagen colibactin

  Colibactin is a mutagen produced by bacterial strains in the colorectal microbiomes of some healthy people. It generates somatic mutations characterised by the mutational signatures SBS88 and ID18 in normal human colorectal epithelial cells in vivo. Increasing prevalence of colibactin mutagenesis during the 20th century is hypothesised to have caused recent international increases in early-onset colorectal cancer incidence. Here, we establish the early-life prevalence, timing, mutation burdens and distribution through the colon of colibactin mutagenesis by sequencing normal colorectal epithelial cell genomes to detect SBS88 and ID18 in children and adolescents from the UK and Denmark biopsied between 2016 and 2024. Colibactin mutagenesis was found in ~40% individuals. It starts soon after birth and often ceases by age five years, although some children show longer exposures. Mutation burdens are higher in the left than right colon and show substantial variation between exposed individuals, in some causing >100-fold increases in total mutation loads compared to unexposed children of the same age. Therefore, colorectal mutagenesis by colibactin is currently common in some populations, occurs during early childhood and can produce markedly elevated somatic mutation burdens. The future public health implications of this widespread mutagen exposure will depend on clarification of its role in colorectal cancer causation.

  Dataset EGAD00001015830

• scRNA-seq of HSPC treated with gemcitabine and carboplatin

  4 samples of 2000000 HSPCs in 2 ml media each were then prepared and treated for 24 hours: carboplatin-high: 150 µg/ml carboplatin carboplatin-low: 18.75 µg/ml carboplatin gemcitabine: 25 ng/ml gemcitabine control: no drug Single-cells were subsequently extracted using the ddSEQ™ Single-Cell Isolator (Bio-Rad) and later sequenced using the SureCell™ Whole Transcriptome Analysis 3' Library Prep Kit (Illumina) on the NextSeq 500 System (Illumina) using the NextSeq 500/550 High Output Kit v2.5 150 Cycles (Illumina) all following the manufacturer's instructions. The raw FASTQ-files are available as read 1 and read 2 files from 4 lanes for each sample giving a total of 16 FASTQ-files. The FASTQ-files were also processed following the ddSeeker (Romagnoli, D., et al., BMC Genomics 2018, doi:10.1186/s12864-018-5249-x) and Drop-seq (Macosko, E.Z., et al., Cell 2015, doi:10.1016/j.cell.2015.05.002) protocols for processing scRNA-seq data to yield the final digital gene expression (dge) for each cell of each sample. This has resulted in one dge text file and one dge summary text file per sample. These 8 text-files with dge data are found in the zip-compressed analysis data-file.

  Dataset EGAD00001006080

• A comprehensive proteogenomic pipeline for neoantigen discovery to advance personalized cancer immunotherapy

  The accurate identification and prioritization of antigenic peptides is crucial for the development of personalized cancer immunotherapies. Publicly available pipelines to predict clinical neoantigens do not allow direct integration of mass spectrometry immunopeptidomics data, which can uncover antigenic peptides derived from various canonical and non-canonical sources. To address this, we present an end-to-end clinical proteogenomic pipeline, called NeoDisc, that combines state-of-the-art publicly available and in-house software for immunopeptidomics, genomics and transcriptomics with in-silico tools for the identification, prediction, and prioritization of tumor-specific and immunogenic antigens from multiple sources, including neoantigens, viral antigens and high-confidence tumor-specific antigens.

  Study EGAS50000000228

• Whole exome sequencing of tumors from the Precision Medicine Program in Pediatric and Adolescent Patients with Recurrent Malignancies

  Sequencing libraries were constructed according to standard procedures from 600 ng of tumor and paired constitutional DNA. WES was captured using Agilent SureSelect V5 (50 Mb), Clinical Research Exome (54 Mb) kit, SureSelect XT human All exon CRE version 1 or 2, or Twist Human Core Exome Enrichment System. Sequencing of subsequent libraries was performed using Illumina sequencers (Next-Seq 500 or Hiseq 2000/2500/4000) in 75 bp paired-end mode, aiming for a mean depth of coverage of 100x.

  Dataset EGAD00001010928

• Genomic Origins and Admixture in Latinos (GOAL)

  The GOAL Study was designed to comprehensively evaluate the genomic architecture of diverse Hispanic/Latino individuals with origins from the Caribbean, Central and South America, and to understand the impact of this diversity on genetic disease studies. Population structure and admixture are key confounders in genome-wide association and medical resequencing studies. In particular, accounting for difference in ancestry among cases and controls, both in terms of genomic and geographic location, is critical for proper analysis and interpretation of studies with multi- and trans-ethnic samples. Genomic studies of Hispanics/Latinos, the largest and fastest growing minority group in the US, reveal that they are a highly genetically heterogeneous admixed group with immense variation among individuals and populations in the proportions of African, European, and Native American ancestry. Knowledge of the underlying complex genetic structure of US Hispanic/Latino and Caribbean populations is, therefore, essential to ensuring robustness of genotype-phenotype associations and understanding the medical relevance of associated variants across diverse populations in the US and throughout the Americas. Furthermore, since much is known about the African and European migrations into the Americas over the past 500 years, population genetic studies of Hispanics/Latinos serve as an excellent model for developing novel algorithms and approaches for characterizing fine-scale genetic structure of admixed populations, in general. This project extends current studies of population genetic structure in US Hispanics/Latinos by densely genotyping parent-offspring triads from individuals (sampled in the US) of Caribbean-descent from six Latin American countries: Puerto Rico, Cuba, Dominican Republic, Haiti, Honduras and Colombia. This study will provide immediate insights and new statistical methods to improve study design and genetic analysis for medical genomic studies in Hispanics/Latinos, other complex admixed groups, and multi- and trans-ethnic studies.

  Study phs000750

• NHLBI TOPMed: GOLDN Epigenetic Determinants of Lipid Response to Dietary Fat and Fenofibrate

  The GOLDN study was initiated to assess how genetic factors interact with environmental (diet and drug) interventions to influence blood levels of triglycerides and other atherogenic lipid species and inflammation markers (registered at clinicaltrials.gov, number NCT00083369). The study recruited Caucasian participants primarily from three-generational pedigrees from two NHLBI Family Heart Study (FHS) field centers (Minneapolis, MN and Salt Lake City, UT). Only families with at least two siblings were recruited and only participants who did not take lipid-lowering agents (pharmaceuticals or nutraceuticals) for at least 4 weeks prior to the initial visit were included. The diet intervention followed the protocol of Patsch et al. (1992). The whipping cream (83% fat) meal had 700 Calories/m2 body surface area (2.93 mJ/m2 body surface area): 3% of calories were derived from protein (instant nonfat dry milk) and 14% from carbohydrate (sugar). The ratio of polyunsaturated to saturated fat was 0.06 and the cholesterol content of the average meal was 240 mg. The mixture was blended with ice and flavorings. Blood samples were drawn immediately before (fasting) and at 3.5 and 6 hours after consuming the high-fat meal. The diet intervention was administered at baseline as well as after a 3-week treatment with 160 mg micronized fenofibrate. Participants were given the option to complete one or both (diet and drug) interventions. Of all participants, 1079 had phenotypic data and provided appropriate consent, and underwent whole genome sequencing through the Trans-Omics for Precision Medicine (TOPMed) program. Comprehensive phenotypic and pedigree data for GOLDN study participants are available through dbGaP phs000741.

  Study phs001359

• GEnetics of Nephropathy - an International Effort (GENIE) GWAS of Diabetic Nephropathy in the UK GoKinD and All-Ireland Cohorts

  Diabetic kidney disease, or diabetic nephropathy (DN), is one of the leading causes of end-stage renal disease in the United States and worldwide. DN is a common complication of long-standing type 1 and type 2 diabetes. The clinical course is characterized by development of proteinuria and gradual loss of kidney function. Although existing treatments that decrease proteinuria have been shown to moderately abate progression of diabetic kidney disease, many affected patients, who do not die from cardiovascular disease, go on to develop terminal renal failure, necessitating costly renal replacement therapies, such as dialysis and renal transplantation. Type 1 diabetes (T1D) can have its onset in childhood and affected individuals often develop end-stage renal disease in early adulthood, leading to further loss of quality of life. The genetic basis of the disease is not well understood. The GENIE (GEnetics of Nephropathy an International Effort) consortium was initiated to perform the most comprehensive and well powered DN susceptibility genome wide association study (GWAS) analysis to date, using the largest collection of type 1 diabetics with and without kidney disease across four study cohorts. The UK-ROI samples were genotyped as part of this project. UK-ROI Sample Description The UK-ROI collection consists of samples derived from the Republic of Ireland (Dr. Catherine Godson, PI, at University College, Dublin, Ireland) and the United Kingdom (Warren 3 and Genetics of Kidneys in Diabetes UK, UK GoKinD, Dr. Alexander P. Maxwell, PI, at Queen's University of Belfast, UK). All study subjects met the inclusion criteria: white individuals with T1D, diagnosed before 31 years of age, whose parents and grandparents were born in the British Isles.

  Study phs000389

• Characterization of Human Transcriptome by Computational and HTS Approaches

  We have deeply sequenced total RNA and whole exome of six different post-mortem human snap-frozen tissues (brain, liver, lung, striated muscle, kidney, heart) from three unrelated healthy Caucasian individuals (males, aged 47-54) with the aim to characterize post-transcriptional events due to alternative splicing and RNA editing. In addition, to facilitate the detection of RNA editing sites we have also resequenced the entire genome of each individual. Data are also used to investigate tissue-specific abundance of mitochondrial DNA from exomes and its correlation with mitochondrial transcription, mass and respiratory activity. Tissues were obtained from Cureline (South San Francisco, CA, USA). DNA and RNA were purified using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) and the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany), respectively. Exome capture was performed using the TruSeq Exome Enrichment Kit (Illumina, San Diego, CA) and the sequencing was carried out at IGA Technology Services in Udine (Italy) on Illumina HiSeq 2000 (generating for each tissue approximately 40 millions of 100bp paired-end reads). Strand-oriented RNA reads (2x100bp) were generated using the TruSeq Stranded Total RNA Sample Prep Kit (Illumina, San Diego, CA, USA) and sequenced at IGA Technology Services in Udine (Italy) on Illumina HiSeq 2000 platform. Whole genome resequencing was performed at Personal Genomics in Italy on an Illumina HiSeq 2000 platform (2x100bp) with a mean coverage of 30X. MiRNA-Seq was instead performed at IBBE Institute (Italy) using the TruSeq Small RNA Sample Prep Kit on Illumina MiSeq platform. Preliminary bioinformatics analyses have been performed to check quality using FASTQC software and inconsistent read regions have been trimmed using trim_galore. All reads were mapped onto the human reference genome (version hg19) using GSNAP.

  Study phs000870

• National Cancer Institute (NCI) Non-Hodgkin Lymphoma Genome-wide Association Study (GWAS)

  Within the framework of the International Lymphoma Epidemiology Consortium (InterLymph) Consortium and NCI-sponsored Cohort Consortium, investigators from 22 studies came together to conduct a genome-wide association study (GWAS) of non-Hodgkin lymphoma (NHL). The goal of the study was to identify common genetic variants associated with the risk of NHL. As described previously (Berndt SI et al., Nature Genetics, 2013, PMID: 23770605 ), cases and controls from 22 studies, including nine prospective cohort studies, eight population-based case-control studies, and five clinic or hospital-based case-control studies were included in this effort. Cases of NHL were ascertained from cancer registries, clinics or hospitals, or through self-report verified by medical and pathology reports. The phenotype information for all NHL cases was reviewed centrally at the InterLymph Data Coordinating Center and harmonized according to the hierarchical classification proposed by the InterLymph Pathology Working Group based on the World Health Organization (WHO) classification (2008). Controls were frequency matched to cases on age and gender by study, and the study was limited to subjects of European ancestry. Participants were genotyped using the Illumina Omni Express. The NCI Non-Hodgkin Lymphoma GWAS is utilized in the following dbGaP sub-studies. To view genotypes and derived variables collected in these sub-studies, please click on the following sub-studies below or in the "Sub-studies" box located on the right hand side of this top-level study page phs000801 NCI Non-Hodgkin Lymphoma GWAS. phs000802 NCI Non-Hodgkin Lymphoma GWAS: CLL phs000818 NCI Non-Hodgkin Lymphoma GWAS: Controls phs000889 NCI Non-Hodgkin Lymphoma GWAS: DLBCL phs000890 NCI Non-Hodgkin Lymphoma GWAS: FL phs000891 NCI Non-Hodgkin Lymphoma GWAS: MZL

  Study phs000801

• Non-coding RNAs Activated by the Wnt/Beta-catenin Signaling Pathway in Hepatoblastoma

  Hepatoblastoma (HB) is the most common pediatric liver tumor, affecting mostly children under 3 years of age. This rare tumor represents 1% of all pediatric cancers. Genetic studies have shown that HB is characterized by high frequency mutations of the CTNNB1 gene encoding beta-catenin (around 75%) and relative genomic stability. Here we have analyzed the transcriptional profile of 21 HBs compared to matched non-tumor livers by Cap Analysis of Gene Expression (CAGE), which provides accurate and quantitative profiling of all transcripts. CAGE analysis revealed strong upregulation of known Wnt target coding genes in most tumors analyzed, consistent with previous transcriptomic studies. To better define the Wnt-dependent transcriptional landscape of HB, we integrated CAGE data with TCF4 ChIP-seq data from a CTNNB1-mutated cancer cell line and with the FANTOM5 genomic coordinates of TCF/LEF binding motifs. Both TCF/LEF binding motifs and ChIP-seq peaks were strongly enriched in the immediate upstream region, not only for protein-coding genes, but also for non-coding transcripts. Among the selected 112 top Wnt target genes at FDR<1.0E-6 and fold change>8, we found clear over-representation (66%) of distant transcription start sites (TSSs) representing lncRNAs and enhancer RNAs, which raises the question of their role in HB pathogenesis. Analysis of the 112 promoters using CAGEd-oPOSSUM confirmed the predominant involvement of Tcf/Lef transcription factors, together with HNF4G, GATA2, Sox3 and Ets-related genes. Finally, the 112 Wnt target signature defined 3 tumor subclasses, T1, T2 and T3, characterized by progressive alteration of the non-coding part of the transcriptome and significant differences in clinical behavior.

  Study phs001433

• Intraductal transplantation models of human pancreatic ductal adenocarcinoma reveal progressive transition of molecular subtypes

  Pancreatic ductal adenocarcinoma (PDAC) is the most lethal common malignancy, with little improvement in patient outcomes over the past few decades. Recently, subtypes of pancreatic cancer with different prognoses have been elaborated. However, the inability to model these subtypes has precluded mechanistic investigation of their origins. Here, we present an orthotopic xenotransplantation model of human patient-derived PDAC organoids in which organoids are grafted via direct injection into the pancreatic duct of immunocompromised (NSG) mice. Tumors that arise from these intraductally grafted organoids (IGOs) enable the distinction of the two main PDAC subtypes: intraepithelial neoplasms from this model progress in an indolent or invasive manner representing the classical or basal-like subtypes of PDAC, respectively, depending on which organoid they originate from. RNA-sequencing was used to characterize the tumors that resulted from IGO xenografts, and to compare the fast- and slow-progressing IGO tumors. Tumors generated using a traditional orthotopic xenograft model, in which organoids are injected into the parenchyma of the pancreas (i.e. orthotopically grafted organoids, or OGOs), were also sequenced for comparison with the IGO tumors. To further characterize the model, laser-capture microdissection followed by RNA-sequencing was used to compare intraductal to invasive lesions resulting from IGOs. To better understand the connection between KRAS signaling and the fast-progressing phenotype, RNA-sequencing was used to compare cultured organoids in which oncogenic KRAS (GeneID: 3845) was hyperactivated to control organoids, and to compare laser-capture microdissected lesions derived from IGOs of KRAS-hyperactivated organoids to lesions derived from control organoids. To ascertain copy number, low-coverage, whole genome DNA-sequencing was performed on organoids cultured in vitro, and on laser-capture microdissected lesions derived from IGO tumors.

  Study phs002045

• Resistance to Checkpoint Blockade Therapy Through Inactivation of Antigen Presentation

  Although immune checkpoint blockade (CPB) leads to prolonged responses in 15-40% of patients with metastatic melanoma, treatment refractory disease and progression after initial response remain major causes of mortality. While predictors of response have been reported, the common mechanisms of both primary and acquired resistance are poorly understood. To identify mechanisms of resistance and examine the evolving landscape in response to CPB, we performed whole exome sequencing (WES), immunohistochemistry (IHC), and RNA-sequencing (RNAseq) of longitudinal tumor biopsies from 17 metastatic melanoma patients treated with various CPB therapies. We found no significant changes in both mutational and neoantigen loads over time between responders and nonresponders. However, we identified abnormalities in one gene, beta-2-microglobulin (B2M), an essential component of MHC Class I antigen presentation, that were present in samples during disease progression but not regression. In total, we identified B2M aberrations in 29.4% of patients, including multiple early frameshift mutations, loss of heterozygosity (LOH) overlapping B2M, and absence of tumor-specific B2M protein expression. Additional defects in the antigen presentation and IFNγ pathways were identified but were not restricted to progressing lesions in our cohort. In two independent cohorts of 105 and 38 melanoma patients treated with ipilimumab (anti-CTLA4) and pembrolizumab (anti-PD1) respectively, we found that B2M LOH was enriched 3-fold in nonresponders (~30%) vs. responders (~10%) and associated with poorer overall survival (log-rank p=0.01, p=0.006). Loss of both copies of B2M was found only in nonresponders. We also found evidence for association of LOH overlapping IFNGR1 with poorer overall survival exclusively in the anti-PD1 cohort. Thus, B2M loss is likely a common mechanism of primary and acquired resistance to therapies targeting CTLA4 or PD-1.

  Study phs001427

• The Role of Germline Mutation and Parental Age in Autism Spectrum Disorders

  <p>We will apply whole genome sequencing of trio families to determine how patterns of germline mutation throughout the genome determine risk for Autism Spectrum Disorder (ASD). We will investigate the nature intrinsic hypermutability and the extrinsic forces, such as paternal age, that influence rates of germline mutation. We will accomplish these goals through the following specific aims: Specific Aim 1 will characterize germline de novo mutations (DNMs) by whole genome sequencing in families. These studies will identify and validate ~8,000 de novo point mutations and structural variants in trios and controls to determine the parent of origin of DNMs. Specific Aim 2 will identify hot spots for germline mutation based on the regional density of DNMs in the genome, and determine the effects of DNA sequence features on rates of mutation. We will determine the association of mutation hotspots with ASD in the discovery sample and in genomic datasets from an independent sample of 2700 cases and 2700 controls. Specific Aim 3 will characterize the effects of extrinsic factors, including parental age and environment, on genome-wide rates of mutation. We will quantify the effect of paternal age on pathogenic and neutral alleles in sperm and investigate whether some DNMs confer a germline selective advantage. The findings of this study will provide fundamental insights into the genetic basis of autism risk and the genetic mechanism of the observed parental age effects in ASD. We will identify genes that confer significant risk for autism, and we will determine how intrinsic properties of the genome interact with extrinsic forces to determining risk for disease in offspring.

  Study phs001164

• The Human Pancreas Analysis Program (HPAP)

  The past decade has seen a dramatic improvement in our ability to phenotype and molecularly profile human tissues with unprecedented resolution at the genomic, epigenomic, protein, and functional levels. The Human Pancreas Analysis Program (HPAP), part of the Human Islet Research Network and supported by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) through multiple NIH grants, is performing deep phenotyping of the human endocrine pancreas to better understand the cellular and molecular events that precede and lead to beta-cell loss and/or dysfunction in type 1 diabetes (T1D) and type 2 diabetes (T2D) as well as to accumulate, analyze, and distribute high-value data sets to the diabetes research community through the HPAP PANC-DB database (and additional details provided at that site). To this end, HPAP employs state-of-the-art technologies to perform comprehensive analyses of pancreas biology as it pertains to organ donors with T1D, autoantibody-positive donors without diabetes, donors with T2D, and control donors. Pancreas procurement and analyses take advantage of the expertise and extensive network of Organ Procurement Organizations (OPOs) and autoantibody screening centers established through the JDRF/The Lenona M. and Harry B. Helmsley Charitable Trust (HCT) – funded nPOD program. In contrast to nPOD, the major product provided by HPAP is not archived biomaterial subject to broad distribution, but rather, the delivery of extensive and high-quality molecular data sets to the diabetes research community in order to facilitate further discovery. Together, nPOD and HPAP are complementary programs that assist the diabetes community and afford the maximal opportunity for advancing knowledge about the pathogenesis of T1D and T2D.

  Study phs002465

• Chromatin Landscape of BET Inhibitor-Treated CD8+ T-cells from Chronic Lymphocytic Leukemia Patients

  The pan-BET (bromodomain and extra-terminal) inhibitor, OPN-51107 (OPN5) was found to alleviate features of T-cell exhaustion in models of chronic lymphocytic leukemia (CLL). To evaluate if OPN5 treatment could relieve fixed chromatin states associated with terminal T-cell dysfunction, ATAC-sequencing was performed on CLL patient T-cells. Peripheral blood mononuclear cells (PBMCs) from 4 treatment-naïve CLL patients were treated ex vivo with 0.5 μM OPN5 or equivalent vehicle (DMSO) in the presence of T-cell activating stimuli (10 μg/mL anti-CD3 and 5 μg/mL anti-CD28) for 16 hours. Live CD8+ T-cells were then isolated via fluorescence-activated cell sorting, and approximately 100,000 viable CD8+ T-cells were subjected to ATAC-sequencing. Libraries were prepared using the Active Motif ATAC-seq kit and underwent paired-end sequencing (2 x 50 bp, 100 cycles) on an Illumina NovaSeq 6000 SP sequencer. Analysis of this data revealed that OPN5 treatment induces a unique chromatin landscape in CLL patient T-cells that is less like that of terminally exhausted T-cells and more like that of effector and memory T-cells. In particular, OPN5 treatment reduced chromatin accessibility at promoter regions of terminal exhaustion-associated genes (e.g., CD101, CCR5, LAG3, BATF3) and increased chromatin accessibility at promoter regions of genes associated with progenitor T-cell function (e.g., TCF7, IL2, CXCR5, IFNG). Individual raw sequencing (fastq) files and processed bigwig files (aligned to the hg38 human reference genome), as well as bigwig files for grouped OPN5- and vehicle-treated samples (aggregate of all 4 patient samples per treatment group) are available via authorized access request through dbGaP.

  Study phs003613

• Genomic Profiling of Pediatric B-cell Acute Lymphoblastic Leukemia

  Pediatric leukemia is the most common type of childhood cancer and, despite tremendous progress in treatment, remains the second leading cause of cancer deaths in children. B-cell acute lymphoblastic leukemia (B-ALL) is the most common type of pediatric leukemia. Recent studies using transcriptional profiling of large cohorts of pediatric B-ALL have shown that patients can be grouped into transcriptional subtypes that are often defined by specific driver mutations. Interestingly, many of these mutational subtypes are defined by gene fusions or rearrangements that drive altered expression of transcription factors (ETV6-RUNX1, PAX5, DUX4, TCF3-PBX1, ZNF384) or in chromatin regulatory machinery (KMT2A), suggesting that transcriptional dysregulation is a major driver of B-ALL. While the transcriptional distinctions are clear, the relationship between patients in terms of their chromatin regulatory landscape remains unclear. To address this, we profiled gene expression, open chromatin, and 3D genome folding in patient derived B-ALL samples. We identified that at the level of 3D genome organization, many chromatin interactions and looping events are shared across mutational subtypes, suggesting that shared transcriptional programs, in addition to group specific driver mutations, possibly regulate the chromatin landscape in B-ALL. Using RNA-seq and ATAC-seq data, we were able to identify "latent" signatures of expression related to non-mutant transcription factors (TFs) that also contribute to expression heterogeneity across TF subtypes. These are related to patient specific patterns of 3D genome folding, and in some cases, are predictive of disease outcomes. This study helps shed light on the chromatin landscape in B-ALL and furthers our understanding of the interactions between diverse mutant TFs that drive B-ALL and non-mutant TFs that help shape the B-ALL chromatin landscape.

  Study phs003226

• Impact of Genetic Variation on Response to GO Therapy in COG-AML Trials AAML03P1 and AAML0531

  The 340 de novo Acute Myeloid Leukemia (AML) patients (ages 1 month to 21 years) were enrolled in COG-AAML03P1 (NCT00070174). Everyone received standard chemotherapy regimen of ara-C, daunorubicin and etoposide (ADE) with addition of one 3 mg/m2 dose of Gemtuzumab Ozogamicin (GO) in induction 1 as well as in intensification II phase. The 1022 de novo AML patients (ages 0–30 years) were enrolled in COG-AAML0531 (NCT01407757). They were randomly assigned to receive either the standard ADE regimen (ADE arm, n = 511) or with the addition of one 3 mg/m2 dose of GO, during induction I as well as intensification II phase (ADE+GO arm, n = 511). Detailed study design, treatment regimen, and clinical outcomes of these two trials have been previously published (Cooper TM, Franklin J, Gerbing RB, et al., PMID:21766293 and Gamis AS, Alonzo TA, Meshinchi S, et al., PMID:25092781). The current study used genomic DNA from 1,225 pediatric patients treated in these two trials with 470 patients treated with standard chemotherapy in COG-AAML0531 (ADE arm) and 755 patients treated with addition of GO to standard therapy in COG-AAML03P1 and COG-AAML0531 trials (ADE+GO arm). The 132 SNPs in 42 genes within DNA-damage repair (DDR) pathways or genes implicated in mediating calicheamicin were selected for genotyping. These SNPs were genotyped using the Sequenom platform at the Biomedical Genomics Center, University of Minnesota. All SNPs had a call rate of more than 0.98 and were in accordance with the Hardy–Weinberg equilibrium. These genotypes were used to test for association with clinical endpoints as defined in the COG-AAML03P1 and COG-AAML0531 trials.

  Study phs003490

• CCR8-targeted specific depletion of clonally expanded Treg cells in tumor tissues evokes potent tumor immunity with long-lasting memory

  Foxp3-expressing CD25+CD4+ regulatory T cells (Tregs) are abundantly infiltrating into tumor tissues, hindering effective anti-tumor immunity. Here, assuming that such tumor Tregs were clonally expanding by recognizing tumor-associated antigens, we attempted to characterize them by their gene expression profiling and T-cell receptor (TCR) clonotyping at the single-cell level. We found that they predominantly expressed the chemokine receptor CCR8: ~40% of tumor Tregs, less than 10% of Tregs in other tissues, and few tumor-infiltrating conventional T cells expressed CCR8 in mice and humans. CCR8+ tumor Tregs were phenotypically highly differentiated and functionally stable. One injection of anti-CCR8 mAb with high antibody-dependent cellular cytotoxic activity was indeed able to specifically deplete clonally expanded tumor Tregs and thereby eradicate established tumors in mice. The depletion predominantly expanded tumor-infiltrating CD8+ effector T cells, which were different in activated or exhausted patterns from those seen after immune checkpoint blockade by anti-PD-1 mAb. The treatment also evoked potent secondary immune responses against the same tumor cell line inoculated several months after tumor eradication, indicating that the anti-CCR8-induced memory-type tumor-reactive effector T cells were, once induced, resistant to recovered Treg suppression. In contrast to such evocation of potent tumor immunity, anti-CCR8 treatment scarcely elicited discernable autoimmunity in mice, contrasting with a similar treatment for systemic whole Treg depletion, which induced histologically and serologically evident autoimmunity. Thus, temporary and specific depletion of clonally expanded Tregs in tumor tissues by cell-depleting anti-CCR8 mAb is sufficient to evoke potent tumor immunity with long-lasting memory and without serious autoimmunity.

  Study JGAS000312

• WGS of cell-free DNA derived from plasma of patients with pediatric sarcoma and healthy controls, and lcWGS/RRBS of matched tumor tissue

  This dataset contains: i) 241 deep (median 12x) whole-genome sequencing profiles of 95 patients with Ewing sarcoma, 31 patients with other pediatric sarcomas, and 22 additional profiles from healthy controls. Sequencing was performed on a NovaSeq 6000 instrument using the NovaSeq S4 2x100 bp configuration. In addition, pilot experiments for 18 cfDNA samples were performed using Illumina HiSeq 2000/2500 machines (2x75 bp configuration). Data is provided as .fastq.gz files (2 files, .R1 and .R2, per sample). i) Low coverage whole-genome-sequencing on 43 tumor biopsy samples from patients with Ewing sarcoma with matched cfDNA samples. The samples were sequenced using a NovaSeq 6000 instrument with the NovaSeq S4 1x100 bp configuration. Data are provided as unmapped (raw) .bam files. iii) Reduced-representation bisulfite sequencing data for 38 tumor biopsy samples from patients with Ewing sarcoma with matched cfDNA samples, and 2 control samples. RRBS libraries were sequenced on Illumina HiSeq 2000/2500 machines. Data are provided as unmapped (raw) .bam files.

  Dataset EGAD00001007080

• An integrative model of pathway convergence in genetically heterogeneous blast crisis chronic myeloid leukemia (CML)

  Targeted therapies against the BCR-ABL1 kinase have revolutionized treatment of chronic phase (CP) chronic myeloid leukemia (CML). In contrast, management of blast crisis (BC) CML remains challenging because BC cells acquire complex molecular alterations that confer stemness features to progenitor populations and resistance to BCR-ABL1 tyrosine kinase inhibitors. Comprehensive models of BC transformation have proved elusive because of the rarity and genetic heterogeneity of BC, but are important for developing biomarkers predicting BC progression and effective therapies. To better understand BC, we performed an integrated multiomics analysis of 74 CP and BC samples using whole-genome and exome sequencing, transcriptome and methylome profiling, and chromatin immunoprecipitation followed by high-throughput sequencing. Employing pathway-based analysis, we found the BC genome was significantly enriched for mutations affecting components of the polycomb repressive complex (PRC) pathway. While transcriptomically, BC progenitors were enriched and depleted for PRC1- and PRC2-related gene sets respectively. By integrating our data sets, we determined that BC progenitors undergo PRC-driven epigenetic reprogramming toward a convergent transcriptomic state. Specifically, PRC2 directs BC DNA hypermethylation, which in turn silences key genes involved in myeloid differentiation and tumor suppressor function via so-called epigenetic switching, whereas PRC1 represses an overlapping and distinct set of genes, including novel BC tumor suppressors. On the basis of these observations, we developed an integrated model of BC that facilitated the identification of combinatorial therapies capable of reversing BC reprogramming (decitabine+PRC1 inhibitors), novel PRC-silenced tumor suppressor genes (NR4A2), and gene expression signatures predictive of disease progression and drug resistance in CP.

  Study EGAS00001001751

• Comparison of capture-based method for transcriptome profiling of formalin-fixed paraffin embedded tumor samples

  Background: The need for fresh frozen (FF) tissue limits implementing RNA sequencing (RNA-seq) in the clinic. The majority of clinical samples are stored as formalin-fixed, paraffin-embedded (FFPE) tissues. Exome capture platforms have been developed for RNA-seq from FFPE samples. However, these methods have not been systematically compared. Methods: Transcriptomic analysis of 32 FFPE tumor samples from 11 patients was performed using three exome capture-based methods: Agilent SureSelect V6, TWIST NGS Exome, and IDT XGen Exome Research Panel. We compared these methods to TruSeq RNA-seq of fresh frozen (FF-TruSeq) tumor samples from the same patients. We assessed the recovery of clinically relevant biological features. Results: The Spearman's correlation coefficients between the global expression profiles of the three capture-based methods from FFPE and matched FF-TruSeq were high (rho = 0.72-0.9, p < 0.05). A significant correlation between the expression of key immune genes between individual capture-based methods and FF-TruSeq (rho = 0.76-0.88, p < 0.05) was observed. All exome capture-based methods reliably detected outlier expression of actionable genes, including ERBB2, MET, NTRK1, and PPARG. In urothelial cancer samples, the Agilent assay was associated with the highest molecular subtype concordance with FF-TruSeq (Cohen's k = 0.7, p < 0.01). Both Agilent and IDT detected all the clinically relevant fusions that were initially identified in FF-TruSeq. Conclusion: All exome capture-based methods had comparable performance and concordance with FF-TruSeq. By enabling the interrogation of FFPE tumor samples, our findings will enable the implementation of RNA-seq in the clinic to guide precision oncology approaches.

  Study EGAS00001005255

• Single-cell decoding of drug induced transcriptomic reprogramming in triple negative breast cancers

  Background The encoding of cell intrinsic drug resistance states in breast cancer reflects the contributions of genomic and non-genomic variations and requires accurate estimation of clonal fitness from co-measurement of transcriptomic and genomic data. Somatic copy number (CN) variation is the dominant mutational mechanism leading to transcriptional variation and notably contributes to platinum chemotherapy resistance cell states. Here, we deploy time series measurements of triple negative breast cancer (TNBC) single-cell transcriptomes, along with co-measured single-cell CN fitness, identifying genomic and transcriptomic mechanisms in drug associated transcriptional cell states. Results We present scRNA-seq data (53,641 filtered cells) from serial passaging TNBC patient-derived xenograft (PDX) experiments spanning 2.5 years, matched with genomic single-cell CN data from the same samples. Our findings reveal distinct clonal responses within TNBC tumors exposed to platinum. Clones with high drug fitness undergo clonal sweeps and show subtle transcriptional reversion, while those with weak fitness exhibit dynamic transcription upon drug withdrawal. Pathway analysis highlights convergence on epithelial-mesenchymal transition and cytokine signaling, associated with resistance. Furthermore, pseudotime analysis demonstrates hysteresis in transcriptional reversion, indicating generation of new intermediate transcriptional states upon platinum exposure. Conclusions Within a polyclonal tumor, clones with strong genotype-associated fitness under platinum remained fixed, minimizing transcriptional reversion upon drug withdrawal. Conversely, clones with weaker fitness display non-genomic transcriptional plasticity. This suggests CN-associated and CN-independent transcriptional states could both contribute to platinum resistance. The dominance of genomic or non-genomic mechanisms within polyclonal tumors has implications for drug sensitivity, restoration and re-treatment strategies.

  Study EGAS00001007242