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• Scalable whole-genome single-cell library preparation without pre-amplification

  Single-cell genomics is critical for understanding cellular heterogeneity in cancer, but existing library preparation methods are expensive, require sample preamplification and introduce coverage bias. Here we describe direct library preparation (DLP), a robust, scalable, and high-fidelity method that uses nanoliter-volume transposition reactions for single-cell whole-genome library preparation without preamplification. We examined 782 cells from cell lines and triple-negative breast xenograft tumors. Low-depth sequencing, compared with existing methods, revealed greater coverage uniformity and more reliable detection of copy-number alterations. Using phylogenetic analysis, we found minor xenograft subpopulations that were undetectable by bulk sequencing, as well as dynamic clonal expansion and diversification between passages. Merging single-cell genomes in silico, we generated "bulk-equivalent" genomes with high depth and uniform coverage. Thus, low-depth sequencing of DLP libraries may provide an attractive replacement for conventional bulk sequencing methods, permitting analysis of copy number at the cell level and of other genomic variants at the population level.

  Study EGAS00001002170

• Cell-type eQTLs for single-cell Mendelian Randomisation

  The following study mapped eQTLs in the post-mortem human brain from snRNA-seq generated on 391 individuals. Following eQTL discovery and identification of state-specific eQTLs (dynamic associations interacting with disease), colocalisation and MR was performed integrating GWAS from 42 traits, for the identification of putatively causal genes with cell-type specific context. This study contains raw sequencing data and genotype for two datasets used in this work; - The MRC 60 dataset, which contains snRNA-seq generated on post-mortem brain from 60 control individuals (104 samples total, split across prefrontal cortex and hippocampus). Samples were pooled into 27 distinct pools, each of which was split across 4 lanes. Following sequencing and mapping using cellranger, demultiplexing was done using Demuxlet using the pool-specific genotype data (which contain sample IDs). - The Roche PD dataset, which is 10X multiome data generated on on 70 PD and 22 control individuals.

  Study EGAS50000000687

• Trans-ethnic genome-wide association study identifies 12 genetic loci influencing blood pressure and implicates a role for DNA methylation

  We carried out a trans-ethnic genome-wide association and replication study of blood pressure phenotypes amongst up to 320,251 individuals of East Asian, European and South Asian ancestry. We find genetic variants at 12 novel loci to be associated with blood pressure (P=3.9x10-11 to P=5.0x10-21). The sentinel blood pressure SNPs are enriched for association with DNA methylation at multiple nearby CpG sites, suggesting that at some of the loci identified DNA methylation may lie on the regulatory pathway linking sequence variation to blood pressure. The sentinel SNPs at the 12 novel loci point to genes involved in vascular smooth muscle (IGFBP3, KCNK3, PDE3A and PRDM6) and renal (ARHGAP24, OSR1, SLC22A7, TBX2) function. The novel and known genetic variants predict increased left ventricular mass, circulating levels of NT-proBNP, cardiovascular and all-cause mortality (P=0.04 to 8.6x10-6). Our results provide new evidence for the role of DNA methylation in blood pressure regulation.

  Study EGAS00001001427

• We describe a method to culture organoids from adult human kidney tissue and describe applications for the culture system.

  Adult Stem Cell (ASC)-derived organoids are 3D epithelial structures that recapitulate essential aspects of their organ of origin. We have developed conditions for the long-term growth of primary kidney epithelial organoids. Cultures can be established from mouse and human kidney tissue, as well as from urine and can be expanded for at least 20 passages (> 6 months). Chromosome numbers remain normal. Human organoids represent proximal as well as distal nephron segments, as evidenced by gene expression, immunofluorescence and tubular functional analyses. BK virus infection of organoids recapitulates in vivo phenomena. Organoids can be established from Wilms nephroblastoma. Kidney organoids from Cystic Fibrosis (CF) patient’s urine allowed ex vivo assessment of treatment efficacy. Finally, organoids cultured on microfluidic organ-on-a-chip plates adopt a tubular conformation and display active transport function. Adult kidney-derived organoids allow studies of hereditary, infectious and malignant kidney disease in a personalized fashion.

  Study EGAS00001002729

• RNA-seq profiling of NKX6.1 reporter iPSC lines for isolation and analysis of functionally relevant neuronal and pancreas populations

  We generated human induced pluripotent stem cell (iPSC) lines with a GFP reporter inserted in the endogenous NKX6.1 locus. Characterisation of the reporter lines demonstrated faithful GFP labelling of NKX6.1 expression during pancreas and motor neuron differentiation. We performed three independent in vitro differentiations towards the pancreatic endocrine lineage. We FACS-purified GFP positive and negative cells from stage 7 cultures, and generated Smart-Seq2 RNA-sequencing libraries for the pre-sorted cells, as well as the two GFP-sorted cell populations. Gene expression profiling by RNA-sequencing reveals that the NKX6.1-positive population closely resembles mature human beta cells and the functional evaluation of purified populations shows that the glucose-responsive beta-like cells are enriched within the NKX6.1-positive population. These reporter lines provide a valuable resource to the scientific community for the derivation of functional relevant pancreas and neuronal cell subtypes.

  Study EGAS00001002802

• WGS of high grade serous ovarian tumours and matched blood normals

  FASTQ reads for 81 matched tumour-normal WGS pairs for high grade serous ovarian cancer patients. Scottish HGSOC samples were collected via local Bioresource facilities at Edinburgh, Glasgow, Dundee and Aberdeen and stored in liquid Nitrogen until required. HGSOC patients were determined from pathology records and were included in the study where there was matched tumour and whole blood samples. Tumour samples were divided into two for DNA and RNA extraction and slivers of tissue were taken, fixed in formalin and embedded in paraffin wax (FFPE). Samples were only included if they were confirmed as HGSOC and there was greater than 40% tumour cellularity throughout the tumour, determined using H&E staining of the FFPE sections and pathology review. Somatic DNA was extracted from the tumour and germline DNA was extracted from whole blood. Somatic DNA was extracted using the Qiagen DNeasy Blood and tissue kit (cat no 69504). The tissue was initially homogenised using a Qiagen Bioruptor, followed by the manufacturers recommended protocol (including RNase digestion step). Germline DNA was extracted from 1-3ml whole blood using the Qiagen FlexiGene kit (cat no 51206) following the manufacturers recommended protocol. The resulting DNA underwent quality control as follows: firstly, A260 and A280nm were measured on a Denovix DS-11 Fx to qualitatively illustrate A260/280nm and A260/230nm ratios as surrogate measures of DNA purity. A260/280 had to be 1.8 or greater and A260/230 had to be 2.0 or greater. Then, DNA was quantified using LifeTechnologies Qubit dsDNA BR kit (cat no Q32850) and we required a minimum of 50ul at 25ng/ul for WGS. Thirdly, DNA was diluted to 25ng/ul and a representative sample was loaded onto a 0.8% TAE gel, ran at 100v for 60mins and then imaged using a BioRad ChemiDoc imaging system to visualise the DNA quality.

  Dataset EGAD00001009049

• WGS of high grade serous ovarian tumours and matched blood normals

  FASTQ reads for 34 matched tumour-normal WGS pairs for high grade serous ovarian cancer patients. Scottish HGSOC samples were collected via local Bioresource facilities at Edinburgh, Glasgow, Dundee and Aberdeen and stored in liquid Nitrogen until required. HGSOC patients were determined from pathology records and were included in the study where there was matched tumour and whole blood samples. Tumour samples were divided into two for DNA and RNA extraction and slivers of tissue were taken, fixed in formalin and embedded in paraffin wax (FFPE). Samples were only included if they were confirmed as HGSOC and there was greater than 40% tumour cellularity throughout the tumour, determined using H&sE staining of the FFPE sections and pathology review. Somatic DNA was extracted from the tumour and germline DNA was extracted from whole blood. Somatic DNA was extracted using the Qiagen DNeasy Blood and tissue kit (cat no 69504). The tissue was initially homogenised using a Qiagen Bioruptor, followed by the manufacturers recommended protocol (including RNase digestion step). Germline DNA was extracted from 1-3ml whole blood using the Qiagen FlexiGene kit (cat no 51206) following the manufacturers recommended protocol. The resulting DNA underwent quality control as follows: firstly, A260 and A280nm were measured on a Denovix DS-11 Fx to qualitatively illustrate A260/280nm and A260/230nm ratios as surrogate measures of DNA purity. A260/280 had to be 1.8 or greater and A260/230 had to be 2.0 or greater. Then, DNA was quantified using LifeTechnologies Qubit dsDNA BR kit (cat no Q32850) and we required a minimum of 50ul at 25ng/ul for WGS. Thirdly, DNA was diluted to 25ng/ul and a representative sample was loaded onto a 0.8% TAE gel, ran at 100v for 60mins and then imaged using a BioRad ChemiDoc imaging system to visualise the DNA quality.

  Dataset EGAD00001010135

• UK10K_OBESITY_SCOOP

  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 SCOOP samples are part of the Obesity group and will undergo exome sequencing. Severe Childhood Onset Obesity Project (SCOOP) is a sub-cohort of the Genetics Of Obesity Study (GOOS) cohort established by Sadaf Farooqi and Steve O’Rahilly at the University of Cambridge over the last 12 years. The GOOS cohort contains >4,000 patients of diverse geographic origin, many of whom have monogenic and syndromic forms of obesity, and includes patients that are offspring of consanguineous union. SCOOP is a subset of >1500 UK Caucasian patients with severe, early onset obesity (all patients have a BMI Standard Deviation Score (SDS) > 3 and obesity onset before the age of 10 years), in whom all known monogenic causes of obesity have been excluded. GWAS data on the SCOOP cohort will be available (WTCCC2 independent study) at the time of the start of this study. Data from this cohort has demonstrated that the prevalence of the common obesity risk alleles in FTO, MC4R and NEGR1 are amongst the highest within SCOOP, demonstrating its value also in the study of genetic variants with an impact on more common obesity. For further information with regard to this cohort please contact Sadaf Farooqi (isf20@medschl.cam.ac.uk).

  Study EGAS00001000124

• Assessment of the Toll-like receptor 3 response in hepatocytes

  Toll like receptor 3 (TLR3) is an endosomal pattern recognition receptor (PRR), recognizing double stranded RNA (dsRNA) as a distinct Pathogen-associated molecular pattern (PAMP) for viral infections. We aim to understand the contribution of TLR3 to the dsRNA response in hepatocytes. The innate immune response of cells of hepatic origin (Huh7, Huh7.5, PH5CH and primary human hepatocytes (PHH)) was analyzed by transcriptome analysis (RNAseq) upon supernatant delivery or transfection of synthetic dsRNA (poly(I:C)). Expression of TLR3 and RIG-I was reconstituted by lentiviral transduction in Huh7 and Huh7.5 cells. Huh7 and Huh7.5 cells, devoid of functional TLR3 expression, did not respond to supernatant delivery of poly(I:C), in contrast to PH5CH and PHH, confirming a high selectivity of this method towards activating TLR3 in all hepatocyte models.

  Study EGAS00001005147

• Large Scale Meta-analysis Characterizes Genetic Architecture for Common Psoriasis-associated Variants

  The genotypes obtained from the Affymetrix Biobank Plus array constituted one of eight Caucasian datasets used in a large genome-wide meta-analysis of the association of psoriasis cases versus unaffected controls. With a combined effective sample size of > 39,000 individuals, this study identified 16 new psoriasis loci achieving genome-wide significance, increasing the total number of identified psoriasis loci for European-ancestry populations to 63.

  Study phs001306