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• Development of Computational Approaches for Cell Hashing in scRNA-Seq

  As a test of cell overloading after cell hashing, a sample of peripheral blood mononuclear cells (PBMC) from a single subject was separated into naive T cells, memory T cells, and non-T cell PBMC populations using fluorescence activated cell sorting (FACS). Each population was separated into two technical replicates, then stained with BioLegend TotalSeq-A antibodies. All 6 samples were pooled, then loaded into 6 wells of a 10X Genomics Chromium 3' RNA-seq v3 chip at increasing cell counts: 16,000 (k), 24k, 32k, 48k, 64, and 80k cells per well. We also included a single sample that was not hashed or pooled, as well as a single sample that was stained with hashing antibodies, but not pooled in two separate wells.

  Study phs002695

• Determining off-target effects of splice-switching antisense oligonucleotides using short read RNAseq in neuronally differentiated human induced pluripotent stem cells

  Splice-switching antisense oligonucleotides (AONs) are designed to modulate pre-mRNA splicing, thereby for instance restoring protein expression or modifying the eventual protein to restore its function or reduce its toxicity. To determine off-target effects, RNA from neuronally differentiated human induced pluripotent stem cells transfected with splice-switching AONs was sequenced.

  Study EGAS50000001222

• Single cell multi-omics analysis of chromothriptic medulloblastoma highlights genomic and transcriptomic consequences of genome instability

  Chromothripsis is a form of genome instability, whereby a presumably single catastrophic event generates extensive genomic rearrangements of one or few chromosome(s). However, little is known about the heterogeneity of chromothripsis across different clones from the same tumor, as well as changes in response to treatment. We analyzed single-cell genomic and transcriptomic alterations linked with chromothripsis in human p53-deficient medulloblastoma (n=7). We reconstructed the order of somatic events, identified early alterations likely linked to chromothripsis and depicted the contribution of chromothripsis to malignancy. We characterized subclonal variation of chromothripsis and its effects on double-minute chromosomes, cancer drivers and putatively druggable targets. Furthermore, we highlighted the causative role and the fitness consequences of specific rearrangements in neural progenitors.

  Study EGAS00001005410

• scRNA-seq and scTCR-seq from 7 MF patients

  Dataset containing scRNA and scTCR sequencing of 7 patients with cutaneous T cell lymphoma. Sequencing was performed on Illumina NextSeq 550, HiSeq 4000 and NovaSeq 6000. The sequencing was always paired.

  Dataset EGAD50000000332

• Successful immune checkpoint blockade in a patient with advanced stage microsatellite unstable biliary tract cancer (H021)

  Cancers acquire multiple somatic mutations that can lead to the generation of immunogenic mutation-induced neoantigens. These neoantigens can be recognized by the host’s immune system. However, continuous stimulation of immune cells against tumor antigens can lead to immune cell exhaustion, which allows uncontrolled outgrowth of tumor cells. Recently, immune checkpoint inhibitors have emerged as a novel approach to overcome immune cell exhaustion and re-activate anti-tumor immune responses. In particular, antibodies blocking the exhaustion-mediating programmed death receptor (PD-1)/PD-L1 pathway have shown clinical efficacy. The effects were particularly pronounced in tumors with DNA mismatch repair deficiency and a high mutational load, which typically occur in the colon and endometrium. Here, we report on a 24-year old woman diagnosed with extrahepatic cholangiocarcinoma who showed strong and durable response to the immune checkpoint inhibitor pembrolizumab, although treatment was initiated at an advanced stage of disease. The patient’s tumor displayed DNA mismatch repair deficiency and microsatellite instability (MSI), but lacked other features commonly discussed as predictors of response towards checkpoint blockade, such as PD-L1 expression or dense infiltration with cytotoxic T cells. Notably, high levels of HLA class I and II antigen expression were detected in the tumor, suggesting a potential causal relation between functionality of the tumor’s antigen presentation machinery and the success of immune checkpoint blockade. We suggest determining MSI status in combination with HLA class I and II antigen expression in tumors potentially eligible for immune checkpoint blockade even in the absence of conventional markers predictive for anti-PD-1/PD-L1 therapy and in entities not commonly linked to MSI phenotype. Further studies are required to determine the value of these markers for predicting the success of immune checkpoint blockade. (H021)

  Study EGAS00001002441

• Psoriasis PBMCs

  This DAC is for the purpose of controlling access to Psoriasis PBMC scRNA-seq data of the study Spermidine/spermine N1-acetyltransferase controls tissue-specific regulatory T cell function in chronic inflammation

  Dac EGAC50000000470

• COMET TCRseq raw data

  This is the raw TCRseq data for the manuscript T cell receptor repertoire sequencing reveals chemotherapy-driven clonal expansion in colorectal liver metastases.

  Dataset EGAD00001010269

• Hashed data from three immortalized fibroblastic reticular cells (iFRCs)

  Co-culture experiments of primary lymphoid and DLBCL-derived immortalized FRCs (iFRC) followed by scRNA-seq. iFRCs as well as lymphocytes from DLBCL samples (n=3) were harvested after 24h of incubation and different experimental conditions were multiplexed using in-house cell multiplexing oligonucleotides (CMOs; full list of barcodes provided in Table S9). Cells were incubated with CMOs at a final concentration of 1.8 μM for 20 minutes on ice, followed by four washes with PBS (centrifugation at 400 × g for 3 minutes at 4 °C). Single-cell RNA sequencing (scRNA-seq) and multiplexing libraries were prepared using the 10x Genomics Single Cell 3′ Gene Expression v4 assay, according to the manufacturer’s protocol. Sequencing was performed on an Illumina NovaSeq 6000 platform using paired-end 100 bp reads on an S4 flow cell.

  Dataset EGAD50000001779

• HV31 - Illumina PCR-free sequencing

  Illumina PCR-free sequencing data for individual HV31 generated using DNA from peripheral blood mononuclear cells, to a sequencing depth of ~44×. Sequencing was performed at the Wellcome Centre for Human Genetics on the Illumina Novaseq platform.

  Dataset EGAD00001007042

• Genetic landscape of pediatric Retinoblastoma

  Retinoblastoma is a pediatric cancer of the developing retina. All retinoblastomas are believed to initiate with biallelic inactivation of the RB1 gene. To identify subsequent genetic lesions in retinoblastoma, we performed whole genome sequencing of tumor and normal DNA of 4 children with retinoblastoma and one matched orthotopic xenograft. Both alleles of RB1 were inactivated in the tumor samples. 3 of the patients had sporadic retinoblastoma and one patient had inherited retinoblastoma. Overall, there were few single nucleotide changes in coding regions of the genome and some of the tumors had few chromosomal lesions. There were very few new genetic lesions in the xenograft compared to the primary tumor. These data suggest that the genome in retinoblastoma is more stable than previously believed and there are relatively few recurrent genetic lesions in known cancer pathways other than the RB1 pathway.

  Study EGAS00001000346