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• SCI-MAP: Single Cell Microgel embedded iPS-cells to map molecular variability of cell differentiation using a systems biology approach.

  In the SCI-MAP project we will map cell states encountered across a previously established 49 day in vitro chondrogenic differentiation protocol. We do this to optimise the differentiation process for therapeutic and in vitro modelling ends. We collect epigenetic open chromatin and phenotypic transcriptome data to determine cell states and so performed single nucleus sequencing in two modalities: RNA- and ATAC-sequencing. Data was collected in a time series experiment at 7 points: Days 0, 3, 6, 8, 14, 21, and 49, starting with the first pilot data from day 21 (D21).

  Study EGAS50000001041

• Glioblastoma stem cell lines RNA-seq (Guilhamon et al.)

  RNA was extracted from GSCs using the Qiagen RNeasy Plus kit. RNA sample quality was measured by Qubit (Life Technologies) for concentration and by Agilent Bioanalyzer for RNA integrity. All samples had RIN above 9. Libraries were prepared using the TruSeq Stranded mRNA kit (Illumina). Two hundred nanograms from each sample were purified for polyA tail containing mRNA molecules using poly-T oligo attached magnetic beads, then fragmented post-purification. The cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase and random primers. This is followed by second strand cDNA synthesis using RNase H and DNA Polymerase I. A single “A” base was added and adapter ligated followed by purification and enrichment with PCR to create cDNA libraries. Final cDNA libraries were verified by the Agilent Bioanalyzer for size and concentration quantified by qPCR. All libraries were pooled to a final concentration of 1.8nM, clustered and sequenced on the Illumina NextSeq500 as a pair-end 75 cycle sequencing run using v2 reagents to achieve a minimum of ~40 million reads per sample.

  Dataset EGAD00001006813

• Structural rearrangements generate cell-specific, gene-independent CRISPR-Cas9 loss of fitness effects.

  CRISPR-Cas9 genome editing is widely used to study gene function, from basic biology to biomedical research. Structural rearrangements are a ubiquitous feature of cancer cells and their impact on the functional consequences of CRISPR-Cas9 gene-editing has not yet been assessed. Utilizing CRISPR-Cas9 knockout screens for 250 cancer cell lines, we demonstrate that targeting structurally rearranged regions, in particular tandem or interspersed amplifications, is highly detrimental to cellular fitness in a gene independent manner. In contrast, amplifications caused by whole chromosomal duplications have little to no impact on fitness. This effect is cell line specific and dependent on the ploidy status. We devise a copy-number ratio metric that substantially improves the detection of gene-independent cell fitness effects in CRISPR-Cas9 screens. Furthermore, we develop a computational tool, called Crispy, to account for these effects on a single sample basis and provide corrected gene fitness effects. Our analysis demonstrates the importance of structural rearrangements in mediating the effect of CRISPR-Cas9-induced DNA damage, with implications for the use of CRISPR-Cas9 gene-editing in cancer cells.

  Dataset EGAD00001004124

• cfDNA analysis reveals relation of POLR1D amplification to bevacizumab resistance in colorectal cancer patients

  Bevacizumab is an approved anti-angiogenic drug for patients with metastasized colorectal cancer (mCRC) targeting VEGF. The survival benefit of anti-VEGF therapy in mCRC patients is limited to a few months and acquired resistance mechanisms are greatly unknown. Using plasma DNA, we studied the evolution of tumor genomes in a cohort of patients with mCRC (n=150) and observed a recurrent focal amplification (8.7% of cases) on chromosome 13q12.2. Analysis of TCGA data (n=619) suggested an association with later stages, which we confirmed by longitudinal plasma analyses. We defined the minimally amplified region and studied the mechanistic consequences of copy number gain of the involved genes. The amplification of one gene, POLR1D, impacted cell proliferation, resulting in upregulation of VEGFA, an important regulator of angiogenesis which has been implicated in the resistance to bevacizumab. In several patients, we observed the emergence of this 13q12.2 amplicon under bevacizumab treatment, which was invariably associated with evolution of therapy resistance. Hence, we describe a novel resistance mechanism against a widely applied treatment in mCRC patients which will impact clinical management .

  Dataset EGAD00001005761

• SINGLE CELL ANALYSIS OF IN VITRO ERYTHROPOIESIS

  We are developing a protocol to differentiate mouse and human induced pluripotent stem (IPS) and embryonic stem (ES) cells towards the haematopoietic pathway to generate erythrocytes in vitro. This system has many applications such as the study of the role of specific genes and human polymorphisms in infectious diseases such as malaria, as well as haematological diseases such as myelodysplastic syndrome. The nature of the in vitro differentiation process means that a heterogeneous population of cells is generated. In order to understand the types of cells produced with our protocol, we have performed a single cell analysis, which has the power to reveal the different populations of cells and their characteristics. For this, a cDNA library has been made that needs to be sequenced to obtain the gene expression profiles of the different cells. With this information we will be able to assess the quality of the differentiation protocol and improve it in order to produce better cells for the downstream applications.This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/

  Dataset EGAD00001000979

• MBD4 targeted sequencing

  We sequenced a gene of interest, MBD4, in both germline (n=1099) and tumor (n=192) sample from Uveal Melanoma patients.

  Study EGAS00001005012

• Tanzania dietary intervention study 2019-2020

  We performed a dietary intervention study comparing western diet, traditional diet and the supplementation of fermented banana beverage in a cohort of 66 individuals (22 in each study arm).

  Dataset EGAD50000000457

• Compound heterozygous variants in LAMC3 in association with posterior periventricular nodular heterotopia

  Periventricular nodular heterotopia is a malformation of cortical development characterized by nodules of abnormally migrated neurons.

  Study EGAS00001004793

• Discordant_Monozygotic_Twins_ALS (Epigenetics)

  We conducted a multi-omics study on a monozygotic twin pair discordant for amyotrophic lateral sclerosis. Data refers to 7 samples that are technical replicates from biological samples collected from the twins at two different time points (T1 and T2).

  Dataset EGAD00010002716

• WGS and RNA-Seq data from a GBM patient PT-DS9789

  WGS and RNA-Seq data from a GBM patient PT-DS9789

  Dataset EGAD00001004233