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• Multi-modal spatial characterization of tumor-immune microenvironments in diffuse large B-cell lymphoma

  We have leveraged spatial transcriptomics together with proteomic and genomic analysis to investigate the spatial immunology and pathobiology of DLBCL. We define distinct cellular niches that differ in frequency across tumors, related to both Epstein-Barr virus (EBV) infection status and the anatomical site of the tumor, and show that residence within different cellular niches is associated with divergent functional states of T-cells and tumor B-cells due to different patterns of cell-cell communication. This provides a framework for understanding stereotyped patterns of spatial organization in DLBCL and immunological interactions associated with heterogeneity in immune cell infiltration and function across patients.

  Study EGAS50000001146

• Insertion Site Analysis of T cells in a CD19-CAR Study on the T-SCM effect in B-cell malignancies

  Integration site analysis (ISA) was performed using a previously described non-restrictive vector-genome Junction amplification method combined with high-throughput sequencing59. Briefly, amplified gDNA was fragmented by sonication to generate ~1000 bp fragments and ligated to a linker cassette (LC) with a known sequence. Two rounds of exponential PCRs (35 cycles each) were conducted using primers specific to the vector long terminal repeats (LTRs) and the LCs. A final PCR step introduced Illumina adapter sequences and a unique index for each sample, enabling multiplexing for next-generation sequencing on the Illumina NextSeq 2000. A set of 24 fusion PCR primers was designed to bind the vector LTR, and a set of 24 fusion PCR primers designed to bind the LC (all primers were purchased from Integrated DNA Technology). Fusion PCR products were purified, quantified, and pooled in an equimolar fashion. Prepared pooled libraries were quantified and loaded onto the Illumina NextSeq 2000. Sequencing reads were demultiplexed based on the sample-specific indices and analysis of sequencing reads including mapping and annotation to the human genome (hg18 version) was performed using the IS-Seq bioinformatic pipeline.

  Dataset EGAD00001015540

• Microbiome

  16S-sequencing: All stool samples were processed according to the standards of the Flemish Gut Flora Project (Falony et al., 2016). All samples were processed together and sequenced in as a single batch (dual index run DI23R22) which included positive controls (PC, identical stool sample), negative controls (PCR-mix) and ‘blanks’ or negative controls of the extraction (no sample). In addition, a Runella strain (RS) was included to check for potential cross-contamination, as this strain is not present in human samples. The V4 region of the 16S rRNA gene was amplified with the primer pair 515F and 806R (GTGYCAGCMGCCGCGGTAA and GGACTACNVGGGTWTCTAAT, resp.), modified to contain a barcode sequence between each primer and the Illumina adaptor sequences to produce dual- barcoded libraries (Tito et al., 2019). Sequencing was performed on the Illumina MiSeq. Sequences were processed using the LotuS (Hildebrand et al., 2014) and DADA2 pipeline (Callahan et al., 2016) using RDP 16 as the reference taxonomy.

  Dataset EGAD50000002027

• Emory University African American Vaginal, Oral, and Gut Microbiome in Pregnancy Cohort Study

  The Emory University African American Microbiome in Pregnancy Cohort Study prospectively enrolls African American women between 18-40 years of age without chronic medical conditions into a longitudinal follow-up study that involves completing microbiome sampling and data collection at two time points in pregnancy (8-14 weeks gestation and 24-30 weeks gestation). The study is investigating the role of the oral, vaginal, and gut microbiome on preterm birth and other pregnancy outcomes as well as biobehavioral factors that shape the microbiome among this socio-demographically diverse group of women. Microbiome sampling involves the oral, vaginal, and gut sites. Biobehavioral factors that are being explored for their association with microbiome composition at the three body sites include measures of sociodemographic status as well as diet and nutrition, infectious and stress exposures.

  Study phs001865

• Longitudinal Study of Vaginal Flora

  This prospective observational study aims to advance our knowledge of the natural history and abnormal alteration in vaginal microbial flora, and to identify risk factors that are associated with these changes. A total of 5500 healthy, non-pregnant women of reproductive age will be enrolled and followed up in Birmingham, Alabama, for one year (baseline visit plus once every three months afterwards, totaling 5 visits). Detailed information on sociodemographic status, medical history, hygiene practice, sexual behavior and psychosocial stress will be collected through in-person interviews. Routine gynecological and dental examinations will be carried out during the study visits. Samples of vaginal secretion and blood will be collected. Laboratory tests to quantify vaginal microbial flora and to identify infection of bacterial vaginosis as well as common sexually transmitted diseases will be conducted. Future submissions may contain metadata or molecular data from additional subjects.

  Study phs002367

• BLUEPRINT Hematopoietic Stem/Progenitor Cell Methylomes

  An optimized whole genome bisulfite sequencing protocol (µWGBS, Farlik et al. 2015 Cell Reports) was used to establish DNA methylation profiles of FACS-purified stem and progenitor cells types of the human blood lineage. Most cell types were sorted from the peripheral blood of three donors with eight pools of ten cells, two pools of 50 cells, and one pool of 1000 cells. HSCs and MPPs were also sorted from fetal liver (HSCs only), cord blood, and bone marrow. Single-cell methylomes using the scWGBS protocol (Farlik et al. 2015 Cell Reports) were established for six cell types (HSC, MPP, CMP, GMP, CLP, MLP0). Gene expression profiling using the Smart-seq2 protocol (Picelli et al. 2013 Nature Methods) was done for nine stem/progenitor cell types (HSC, MPP, CMP, GMP, CLP, MLP0, MLP1, MLP2, MLP3).

  Study EGAS00001002070

• RNA-seq sequence data from DNMT3A microcephalic dwarfism patients.

  RNA-seq data generated from cells from control individuals and individuals with de novo DNMT3A variants causing microcephalic dwarfism.

  Dataset EGAD00001004471

• DNMT3A MOPD patient ChIP-seq data

  ChIP-seq data from controls and patients with de novo DNMT3A mutations resulting in microcephalic primordial dwarfism.

  Dataset EGAD00001004473

• Human Pilocytic Astrocytoma Single Cell RNA Sequencing

  Pilocytic astrocytoma (PA), the most common childhood brain tumor, is a low-grade glioma with a single driver BRAF rearrangement. Here, we perform scRNAseq in six PAs using methods that enabled detection of the rearrangement. When compared to higher-grade gliomas, a strikingly higher proportion of the PA cancer cells exhibit a differentiated, astrocyte-like phenotype. A smaller proportion of cells exhibit a progenitor-like phenotype with evidence of proliferation. These express a mitogen-activated protein kinase (MAPK) program that was absent from higher-grade gliomas. Immune cells, especially microglia, comprise 40% of all cells in the PAs and account for differences in bulk expression profiles between tumor locations and subtypes. These data indicate that MAPK signaling is restricted to relatively undifferentiated cancer cells in PA, with implications for investigational therapies directed at this pathway. Note that 931 out of 1234 samples in the Sequence Read Archive (SRA) passed QC and have transcript count data available from the Broad Institute Single Cell Portal. The transcript count data can be found at https://singlecell.broadinstitute.org/single_cell/study/SCP271/pilocytic-astrocytoma-single-cell-rna-seq#study-download

  Study phs001854

• KRT17High/CXCL8+ Tumor Cells Display Both Classical and Basal Features and Regulate Myeloid Infiltration in the Pancreatic Cancer Microenvironment

  Recent studies have uncovered the co-existence of basal and classical subtypes within pancreatic ductal adenocarcinoma (PDAC). We sought to characterize these intermediary cells using single-cell RNA sequencing of PDAC. We performed subtyping on 18 single-cell RNA sequencing human PDAC samples to identify multiple intermediary subtypes. We compared single-cell profiling of patient-matched blood and tumor samples to measure changes in the local and systemic immune microenvironments in CXCL8-high tumors vs. CXCL8-low tumors. Finally, we leveraged longitudinally patient-matched blood over the course of chemotherapy. In our studies we identified a cluster of KRT17high intermediary cancer cells that uniquely express high levels of CXCL8 and other cytokines which in turn influence the myeloid compartment both locally and systemically. We utilized sequencing data from dbGaP study accession phs002070. In addition to that, we included sequencing from new specimens which are deposited to this submission, consisting of one additional tumor specimen and 5 PBMC samples, sequenced after chemotherapy (longitudinally matched to PBMC samples in phs002071.v1.p1). FASTQ files of these sequencing runs are available through this dbGaP submission.

  Study phs003436