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• Assessing gene expression profiling from FFPE Patient Samples: A Comparison of Two Library Preparation Approaches and Recommendations

  Next-Generation Sequencing (NGS) has transformed cancer research and clinical practice, with Whole Exome Sequencing (WES) driving advances in mutational profiling and personalized oncology. Yet, transcriptomic signatures remain essential for understanding disease mechanisms, including therapy resistance pathways. RNA sequencing (RNA-seq), however, faces unique challenges when dealing with low-input or degraded RNA, as often found in archival formalin-fixed paraffin-embedded (FFPE) tissues. Although previous studies have compared library preparation protocols, rapidly evolving technologies call for updated evaluations. Here, we present a direct comparison of two FFPE-compatible stranded RNA-seq library preparation kits: TaKaRa™ SMARTer® Stranded Total RNA-Seq Kit v2 (Kit A) and Illumina Stranded Total RNA Prep Ligation with Ribo-Zero Plus (Kit B). Both kits generated high-quality RNA-seq data, yet important differences emerged. Notably, Kit A achieved comparable gene expression quantification to Kit B while requiring 20-fold less RNA input, a crucial advantage for limited samples, albeit with increased sequencing depth. We critically discuss these results in relation to RNA availability, technical performance, cost-effectiveness, processing time, and automation potential, offering practical guidance for selecting optimal RNA-seq strategies in clinical and translational research settings

  Study EGAS50000001066

• Efficacy of two different FGFR-inhibitors in a patient with extrahepatic cholangiocarcinoma harboring an FGFR2 mutation

  Cholangiocarcinomas (CCAs) is a type of cancer with few effective systemic therapies. Elucidation of the molecular landscape of the disease from genomic studies based on next generation sequencing (NGS) has contributed to the introduction of new targeted therapies. One of these treatments consists of a class of small molecules that target members of the FGFR family of receptor tyrosine kinases. These drugs are effective and have been approved for cholangiocarcinomas with fusions or rearrangements of FGFR genes. In contrast, the role of these inhibitors in cholangiocarcinomas with mutations in FGFR genes is less well defined. We report here a patient with a cholangiocarcinoma bearing a FGFR2 p.Ser252Trp mutation. The patient was treated with two different FGFR inhibitors, as the first caused ocular toxicity. She obtained clinical benefit from both. This case illustrates the efficacy of FGFR inhibitors on cholangiocarcinoma with specific point mutations. This is the first case to report the clinical benefit of these drugs in FGFR2 p.Ser252Trp mutation. Clinical benefit can be sustained, as seen in our patient. Our case also shows that FGFR inhibitors-induced adverse effects, such as ocular toxicities, may not recur after re-challenge with an alternative drug of the same class.

  Study EGAS50000000015

• Exome sequencing of a novel cervical cancer cell line

  Human cancer cell lines are largely used in the searching for new antineoplastic agents. However, due to the artifacts of a long-term in culture, cell lines do not always represent the realistic tumor cell behavior. This has motivated the development of models that better mimetics the tumor tissue, among them, the establishment of primary cell cultures. In this work, we establish and characterized a low-passage cervix cancer cell line from a Brazilian patient with squamous cell carcinoma. The phenotype confirms the epithelial and tumor origin, through cytokeratins, EpCAM, and p16 staining. Whole exome sequencing showed relevant somatic mutations in several genes including BRCA2, TGFBR1, and IRX2 genes. CNV analysis by nanostring and WGS revealed amplification in genes related mainly with kinases proteins, involved in proliferation, migration and cell differentiation, such as EGFR, PIK3CA, and MAPK7. Overexpression of EGFR was confirmed by phospho RTK-array and western blot analysis. Furthermore, the cell was sensitivity to cisplatin, with IC50 13 times lower than SiHa cell line. In conclusion, this cervical cancer cell line presents molecular alterations that are an important tool for leading pre-clinical studies of new drugs that target one or more of the altered pathways.

  Study EGAS00001003343

• Summary statistics from genome-wide association study in glioma of 12,488 cases and 18,169 controls.

  Genome-wide association studies (GWAS) have transformed our understanding of glioma susceptibility, but individual studies have had limited power to identify risk loci. We performed a meta-analysis of existing GWAS and two new GWAS, which totaled 12,488 cases and 18,169 controls. We identified five new loci for glioblastoma (GBM) at 1p31.3 (rs12752552; P = 2.04 × 10-9, odds ratio (OR) = 1.22), 11q14.1 (rs11233250; P = 9.95 × 10-10, OR = 1.24), 16p13.3 (rs2562152; P = 1.93 × 10-8, OR = 1.21), 16q12.1 (rs10852606; P = 1.29 × 10-11, OR = 1.18) and 22q13.1 (rs2235573; P = 1.76 × 10-10, OR = 1.15), as well as eight loci for non-GBM tumors at 1q32.1 (rs4252707; P = 3.34 × 10-9, OR = 1.19), 1q44 (rs12076373; P = 2.63 × 10-10, OR = 1.23), 2q33.3 (rs7572263; P = 2.18 × 10-10, OR = 1.20), 3p14.1 (rs11706832; P = 7.66 × 10-9, OR = 1.15), 10q24.33 (rs11598018; P = 3.39 × 10-8, OR = 1.14), 11q21 (rs7107785; P = 3.87 × 10-10, OR = 1.16), 14q12 (rs10131032; P = 5.07 × 10-11, OR = 1.33) and 16p13.3 (rs3751667; P = 2.61 × 10-9, OR = 1.18). These data substantiate that genetic susceptibility to GBM and non-GBM tumors are highly distinct, which likely reflects different etiology.

  Study EGAS00001003372

• Reconstituting the transcriptome and DNA methylome landscapes of human implantation at single-cell resolution

  Implantation is a milestone event during mammalian embryogenesis. Due to the extreme difficulty of obtaining in vivo human early post-implantation embryos, the gene regulatory network and epigenetics controlling human embryo implantation remains elusive. Here, combining an in vitro culture system for human post-implantation development and single-cell omics sequencing technologies, over 10,000 single cells at five representative stages of pre/post-implantation development were systematically analyzed. Unsupervised dimensionality reduction and clustering algorithm of the transcriptome data show stepwise implantation routes for the epiblast, primitive endoderm, and trophectoderm lineages, suggesting preparation for the establishment of a mother-to-offspring connection after implantation. Female embryos showed asynchronous progress of dosage loss of X chromosomes during implantation. Furthermore, using the single cell trio-Seq (scTrio-Seq) strategy, re-methylation of the genomes of all the three lineages was unambiguously revealed. Surprisingly, the genome re-methylation of PE lineage were much slower than both EPI and TE lineages during the implantation process, indicating distinct methylome features between EPI and PE although both of which were derived from ICM. Collectively, our work paves the way for understanding the complex molecular mechanisms that regulate human embryo implantation, informing new insights and future efforts in early embryonic development and reproductive medicine.

  Study EGAS00001003443

• METABRIC: Data from Batra et al (2021); DNA methylation landscapes of 1538 breast cancers reveal a replication-linked clock, epigenomic instability and cis-regulation

  DNA methylation is aberrant in cancer, but the dynamics, regulatory role and clinical implications of such epigenetic changes are still poorly understood. Here, RRBS-based profiles of 1538 breast tumors and 244 normal breast tissues from the METABRIC cohort are reported, facilitating detailed analysis of DNA methylation within a rich context of genomic, transcriptional, and clinical data. Tumor methylation from immune and stromal signatures are deconvoluted leading to the discovery of a tumor replication-linked clock with genome-wide methylation loss in non-CpG island sites. Unexpectedly, methylation in most tumor CpG islands follows two replication-independent processes of gain (MG) or loss (ML) that we term epigenomic instability. Epigenomic instability is correlated with tumor grade and stage, TP53 mutations and poorer prognosis. After controlling for these global trans-acting trends, as well as for X-linked dosage compensation effects, cis-specific methylation and expression correlations are uncovered at hundreds of promoters and over a thousand distal elements. Some of these targeted known tumor suppressors and oncogenes. In conclusion, the study demonstrates that global epigenetic instability can erode cancer methylomes and expose them to localized methylation aberrations in-cis resulting in transcriptional changes seen in tumors.

  Study EGAS00001004327

• Clonal selection and double hit events involving tumor suppressor genes underlie relapse from total therapy

  In order to gain insights into the mechanisms of disease progression in multiple myeloma (MM) patients treated with multi-agent and high-dose chemotherapy we have performed a longitudinal study of 33 patients entered into Total Therapy protocols using gene expression profiling, high resolution copy number analyses and whole exome sequencing. The results of this study show the importance of acquired mutations in MM driver genes, bi-allelic events affecting tumor suppressor genes and increased proliferation rates in driving relapse, which is enhanced by intra-clonal heterogeneity and Darwinian type clonal evolution. Branching was the most frequent evolution pattern, characterized by clonal loss and appearance of new clones. We found a higher number of lost and acquired copy number aberrations and an excess of bi-allelic inactivation of tumor suppressor genes in GEP70 high risk (HiR) cases, consistent with genomic instability being a key feature of HiR MM. In conclusion, our study further stresses the impact of known prognostic markers and driver genes on the fitness level of MM cells to survive multi-agent chemotherapy. The frequent bi-allelic loss of tumor suppressor genes in HiR cases and the potentially resulting failure of treatment with DNA damaging agents highlight the need for new therapies for HiR MM.

  Study EGAS00001001810

• The Prediction and Prevention of Preeclampsia

  The Prediction and Prevention of Preeclampsia (PREDO) is a prospective birth cohort study of Finnish women who were pregnant between 2005 and 2010 and their children. The PREDO study cohort was set up to identify novel risk factors and biomarkers in pregnant women associated with the development of preeclampsia and intrauterine growth restriction (IUGR). Two groups of pregnant women were enrolled during the first trimester of their pregnancy: first, pregnant women with a known clinical risk factor status for preeclampsia and IUGR (N=1079; 969 of these women had at least one and 110 had none of the known risk factors for preeclampsia and IUGR), and second, pregnant women who volunteered to participate regardless of their risk factor status for preeclampsia and IUGR (N=3698). Both samples were monitored during pregnancy with four clinical visits and bi-weekly self-reports. The post-delivery follow-up has taken place at approximately 2 weeks, 6 months, and 3.5 years after the delivery. The most recent follow-up started in 2016 and is ongoing. In the high-risk sample, cord blood samples were collected and genome-wide genotyping has been conducted with Illumina OmniExpressExome 1.2 and genome-wide methylation status has been analysed with Illumina 450k HM array.

  Study EGAS00001001898

• A recessive PRDM13 mutation results in congenital hypogonadotropic hypogonadism and cerebellar hypoplasia

  The PRDM13 (PR Domain containing 13) putative chromatin modifier and transcriptional regulator functions downstream of the transcription factor PTF1A, which controls GABAergic fate in the spinal cord and neurogenesis in the hypothalamus. Here, we report a novel, recessive syndrome associated with PRDM13 mutation. Patients exhibited intellectual disability, ataxia with cerebellar hypoplasia, scoliosis and delayed puberty with congenital hypogonadotropic hypogonadism (CHH). Expression studies revealed Prdm13/PRDM13 transcripts in the developing hypothalamus and cerebellum in mouse and human. An analysis of hypothalamus and cerebellum development in mice homozygous for a Prdm13 mutant allele revealed a significant reduction in the number of Kisspeptin (Kiss1) neurons in the hypothalamus and PAX2+ progenitors emerging from the cerebellar ventricular zone. The latter was accompanied by ectopic expression of the glutamatergic lineage marker TLX3. Prdm13-deficient mice displayed cerebellar hypoplasia, normal gonadal structure, but delayed pubertal onset. Together, these findings identify PRDM13 as a critical regulator of GABAergic cell fate in the cerebellum and of hypothalamic kisspeptin neuron development, providing a mechanistic explanation for the co-occurrence of CHH and cerebellar hypoplasia in this syndrome. To our knowledge, this is the first evidence linking disrupted PRDM13-mediated regulation of Kiss1 neurons to CHH in humans.

  Study EGAS00001005878

• Clinical genome sequencing uncovers potentially targetable truncations and fusions of MAP3K8 in spitzoid and other melanomas

  Spitzoid melanoma is a specific morphologic variant of melanoma that most commonly affects children and adolescents and ranges on the spectrum of malignancy from low-grade to overtly malignant. These tumors are generally driven by fusions of ALK, RET, NTRK1/3, MET, ROS1 and BRAF. However, in approximately 50% of cases, no genetic driver has been established. Clinical whole genome and transcriptome sequencing (RNA-Seq) of a spitzoid tumor from an adolescent revealed a novel gene fusion of MAP3K8, encoding a serine−threonine kinase that activates MEK. The patient who had exhausted all other therapeutic options was treated with a MEK inhibitor and underwent a transient clinical response. We subsequently analyzed spitzoid tumors from 49 patients by RNA-Seq and found in-frame fusions or C-terminal truncations in 33% of cases. The fusion transcripts and truncated genes all contained MAP3K8 exons 1-8 but lacked the autoinhibitory final exon. Data mining of RNA-Seq from the Cancer Genome Atlas (TCGA) uncovered analogous MAP3K8 rearrangements in 1.5% of adult melanomas. Thus, MAP3K8 rearrangements - uncovered by comprehensive clinical sequencing of a single case - are the most common genetic event in spitzoid melanoma, are present in adult melanomas, and could be amenable to MEK inhibition.

  Study EGAS00001003430

• WXS and RNA-seq for 22 patients treated with radiation + immunotherapy

  Over 500 active clinical trials are investigating combination radiation (RT) and immune checkpoint blockade (ICB) as a cancer treatment; however, the majority of trials have found no positive interaction. We performed a comprehensive molecular analysis of a randomized phase I clinical trial of patients with non-small cell lung cancer (NSCLC) treated with concurrent or sequential ablative RT and ICB. Concurrent treatment was superior to sequential treatment in augmenting local and distant tumor responses and improving overall survival in a subset of patients with highly aneuploid tumors, but not in those with less aneuploid tumors. Our analysis suggested that concurrent treatment eliminated immunologically cold aneuploid tumors better than sequential treatment and improved patient outcomes. In addition, we report that RT alone decreased intratumoral cytotoxic effector T cell and adaptive immune gene expression signatures, in contrast to upregulation of key immune pathways after concurrent administration of RT and ICB. Our findings distinguish the differential genomic and transcriptomic effects of RT versus RT and ICB and challenge the prevailing paradigm that local ablative RT beneficially stimulates the immune response. We propose the use of tumor aneuploidy as a novel biomarker and therapeutic target in personalizing treatment approaches for patients with NSCLC treated with RT and ICB.

  Study EGAS00001006212

• Whole genome sequencing delineates regulatory, structural, and cryptic splice variants in early onset cardiomyopathy

  Cardiomyopathy (CMP) is a heritable disorder. Over 50% cases are gene-elusive on clinical gene panel testing. The contribution of variants in non-coding DNA elements that result in cryptic splicing and regulate gene expression has not been explored. We analyzed whole genome sequencing (WGS) data in a discovery cohort of 209 pediatric CMP patients and 1,953 independent replication genomes and exomes. We searched for protein-coding variants, and non-coding variants predicted to affect the function or expression of genes. Thirty-nine % cases harbored pathogenic coding variants in known CMP genes, and 5% harbored high-risk loss-of-function (LoF) variants in additional candidate CMP genes. Fifteen % harbored high-risk regulatory variants in promoters and enhancers of CMP genes (Odds ratio 2.25, p=6.70×10-7 versus controls). Genes involved in α-dystroglycan glycosylation (FKTN, DTNA) and desmosomal signaling (DSC2, DSG2) were most highly enriched for regulatory variants (Odds ratio 6.7-58.1). Functional effects were confirmed in patient myocardium and reporter assays in human cardiomyocytes, and in zebrafish CRISPR knockouts. We provide strong evidence for the genomic contribution of functionally active variants in new genes and in regulatory elements of known CMP genes to early-onset CMP.

  Study EGAS00001004929

• Significant and pervasive effects of RNA degradation on Nanopore direct RNA sequencing

  Abstract Oxford Nanopore direct RNA sequencing (DRS) is capable of sequencing complete RNA molecules and accurately measuring gene and isoform expression. However, as DRS is designed to profile intact RNA, expression quantification may be more heavily dependent upon RNA integrity than alternate RNA-seq methodologies. It is currently unclear how RNA degradation impacts DRS or if it can be corrected for. To assess the impact of RNA integrity on DRS we performed a degradation time-series using SH-SY5Y neuroblastoma cells. Our results demonstrate that degradation is a significant and pervasive factor that can bias DRS measurements, including a reduction in library complexity resulting in an overrepresentation of short genes and isoforms. Degradation also biases differential expression analyses; however, we find explicit correction can almost fully recover meaningful biological signal. In addition, DRS provided less biased profiling of partially degraded samples than nanopore cDNA-PCR sequencing. Overall, we find samples with RIN > 9.5 can be treated as undegraded and samples with RIN > 7 can be utilised for DRS with appropriate correction. These results establish the suitability of DRS for a wide range of samples, including partially degraded in-vivo clinical and post-mortem samples, whilst limiting the confounding effect of degradation on expression quantification.

  Study EGAS00001006542

• Hexanucleotide repeat expansions in C9orf72 alter microglial responses and prevent a coordinated glial reaction in ALS

  Neuroinflammation is an important hallmark in amyotrophic lateral sclerosis (ALS). Experimental evidence has highlighted a role of microglia in the modulation of motor neuron degeneration. However, the exact contribution of microglia to both sporadic and genetic forms of ALS is still unclear. We generated single nuclei profiles of spinal cord and motor cortex from sporadic and C9orf72 ALS patients, as well as controls. We particularly focused on the transcriptomic responses of both microglia and astrocytes. We confirmed that C9orf72 is highly expressed in microglia and shows a diminished expression in carriers of the hexanucleotide repeat expansion (HRE). This resulted in an impaired response to disease, with specific deficits in phagocytic and lysosomal transcriptional pathways. Astrocytes also displayed a dysregulated response in C9orf72 ALS patients, remaining in a homeostatic state. This suggests that C9orf72 HRE alters a coordinated glial response, which ultimately would increase the risk for developing ALS. Our results indicate that C9orf72 HRE results in a selective microglial loss-of-function, likely impairing microglial-astrocyte communication and preventing a global glial response. This is relevant as it indicates that sporadic and familial forms of ALS may present a different cellular substrate, which is of great importance for patient stratification and treatment.

  Study EGAS00001006711

• T cell landscape definition by multi-omics identifies galectin-9 as novel immunotherapy target in chronic lymphocytic leukemia (CLL)

  Failure of immunotherapy after applying checkpoint inhibitors or CAR-T cells is linked to T cell exhaustion. Here, we explored the T cell landscape in chronic lymphocytic leukemia (CLL) by single-cell omics analyses of blood, bone marrow and lymph node samples of patients and spleen samples of a CLL mouse model. By single-cell RNA- sequencing, mass cytometry (CyTOF), and multiplex image analysis of tissue microarrays, we defined the spectrum of phenotypes and transcriptional programs of T cells and and their differentiation state trajectories. We identified disease-specific accumulation of distinct regulatory T cell subsets and T cells harboring an exhausted phenotype exclusively in the CLL lymph node tissue. Integration of TCR data revealed a clonal expansion of CD8+ precursor exhausted T cells, suggesting their reactivity for CLL cells. Interactome analyses identified the TIM3 ligand Galectin-9 as novel immunoregulatory molecule in CLL. Blocking of Galectin-9 in CLL-bearing mice slowed down disease development and reduced the number of TIM3 expressing T cells. Galectin-9 expression correlated with shorter survival of CLL patients. Thus, Galectin-9 contributes to immune escape in CLL and represents a novel target for immunotherapy.

  Study EGAS00001006864

• Dataset developed for use with EOSC4Cancer of synthetic colorectal cancer tumor/normal pairs.

  This dataset contains 10 tumor and normal pairs synthetic WGS data of colorectal cancer that were simulated in a standard format of Illumina paired-end reads. The NEAT read simulator (version 3.0, https://github.com/zstephens/neat-genreads) was utilized to synthetize these 10 pairs of tumor and normal WGS data. In the procedure of data generation, simulated parameters (i.e., sequencing error statistics, read fragment length distribution and GC% coverage bias) were learned from data models provided by NEAT. The average sequencing depth for tumor and normal samples aimed to reach around 110X and 60X, respectively. For generation of synthetic normal WGS data per each sample, a germline variant profile from a real patient was down-sampled randomly, representing 50% germline variants of a given patient. These were mixed with the other 50% in silico germline variants that were modelled randomly using an average mutation rate (0.001), finally constituting a full germline profile for normal synthetic WGS data. For generation of synthetic tumor WGS data per each sample, a pre-defined somatic short variant profile (SNVs+Indels) learnt from a real CRC patient was added to the germline variant profile used for creating the normal synthetic WGS data of the same patient, consisting of the variants for tumor sample. Neither copy number profile nor structural variation profile was introduced into the tumor synthetic WGS data. Tumor content and ploidy were assumed to be 100% and 2, respectively. For mapping/variant detection, the Sarek pipeline v3.1.2 (https://nf-co.re/sarek/3.1.2) was used, specifically: 1. BWA v0.7.17-r1188 for read mapping 2. GATK v4.3.0.0 for pre-processing BAM file (including markduplicates and recalibration). 2. Mutect2 (GATK v4.3.0.0) for somatic variant calling 3. Strelka2 v2.9.10 for germline and somatic variant calling Metadata information of 10 CRC patients used for the generation of synthetic normal and tumor WGS data: Patient_id Tumor_barcode Normal_barcode Age Sex Tissue Cancer SIM007 SIM007_T SIM007_N 71 F Rectal Primary CRC SIM008 SIM008_T SIM008_N 45 F Colon Neuroendocrine Metastasis CRC SIM010 SIM010_T SIM010_N 62 M Colon Metastasis CRC SIM011 SIM011_T SIM011_N 55 M Colon Neuroendocrine Metastasis CRC SIM012 SIM012_T SIM012_N 57 M Rectal Metastasis CRC SIM013 SIM013_T SIM013_N 69 M Colon Metastasis CRC SIM014 SIM014_T SIM014_N 68 M Colon Neuroendocrine primary CRC SIM015 SIM015_T SIM015_N 58 F Colon Primary CRC SIM016 SIM016_T SIM016_N 49 M Colon/Rectal Primary CRC SIM017 SIM017_T SIM017_N 78 M Colon Neuroendocrine primary CRC

  Dataset EGAD50000000564

• FASTQ files of total RNA-Seq data from the POPS SGA (Small for Gestational Age) samples

  All the samples were obtained from the Pregnancy Outcome Prediction–a prospective cohort study of nulliparous women attending the Rosie Hospital, Cambridge (UK) for their dating ultrasound scan between January 14, 2008, and July 31, 2012. Ethical approval for the study was given by the Cambridgeshire 2 Research Ethics Committee (reference number 07/H0308/163) and all participants provided written informed consent. Cases of preeclampsia (PET) were defined on the basis of the 2013 ACOG criteria and cases of small for gestational age (SGA)infants were confined to severe SGA, i.e. a customized birth weight <5th percentile. Chorionic villi from the corresponding placentas (free from decidua, visible infarction, calcification, hematoma, or damage) were collected and processed within 30 minutes of separation from the uterus. After repeated washes in chilled phosphate buffered saline, the samples were placed in RNA later (Applied Biosystems) and stored at -80°C. Total placental RNA was extracted using mirVana Isolation Kit (Ambion). For each placenta, approximately 5 mg of tissue were homogenized in the Lysis/Binding solution for 20 sec at 6 m/s using a bead beater (FastPrep24) and Lysing Matrix D Tubes (MP Biomedicals). The samples were then spun at 13,000 rpm for 5 min at 4°C and the supernatants recovered. Afterwards, the manufacturer's instructions were followed. Immediately after the RNA extraction, placental RNA samples were DNase-treated using DNA-free DNA Removal Kit (Ambion), aliquoted, and stored in -80°C. Quantity and quality of the RNA samples were assessed using the Agilent 2100 Bioanalyzer, the Agilent RNA 6000 Nano Kit (Agilent Technologies), and Qubit fluorometer. Libraries were prepared starting with 300-500 ng of good quality total RNA (RIN ≥7.5) using the TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero Human/Mouse/Rat (Illumina), according to the manufacturer's instructions. The kit contains 96 uniquely indexed adapter combinations in order to allow pooling of multiple samples prior to sequencing. After determining their size (with the Agilent 2100 Bioanalyzer and the Agilent High Sensitivity DNA Kit by Agilent Technologies) and concentration (by qPCR with the KAPA Illumina ABI Prism Library Quantification Kit, Kapa Biosystems), libraries have been pooled and sequenced (single-end, 125 bp) using a Single End V4 Cluster Kit and an Illumina HiSeq2500 or HiSeq4000 instrument.

  Dataset EGAD00001003507

• FASTQ files of total RNA-Seq data from the POPS PET (pre-eclamptic) samples

  All the samples were obtained from the Pregnancy Outcome Prediction–a prospective cohort study of nulliparous women attending the Rosie Hospital, Cambridge (UK) for their dating ultrasound scan between January 14, 2008, and July 31, 2012. Ethical approval for the study was given by the Cambridgeshire 2 Research Ethics Committee (reference number 07/H0308/163) and all participants provided written informed consent. Cases of preeclampsia (PET) were defined on the basis of the 2013 ACOG criteria and cases of small for gestational age (SGA)infants were confined to severe SGA, i.e. a customized birth weight <5th percentile. Chorionic villi from the corresponding placentas (free from decidua, visible infarction, calcification, hematoma, or damage) were collected and processed within 30 minutes of separation from the uterus. After repeated washes in chilled phosphate buffered saline, the samples were placed in RNA later (Applied Biosystems) and stored at -80°C. Total placental RNA was extracted using mirVana Isolation Kit (Ambion). For each placenta, approximately 5 mg of tissue were homogenized in the Lysis/Binding solution for 20 sec at 6 m/s using a bead beater (FastPrep24) and Lysing Matrix D Tubes (MP Biomedicals). The samples were then spun at 13,000 rpm for 5 min at 4°C and the supernatants recovered. Afterwards, the manufacturer's instructions were followed. Immediately after the RNA extraction, placental RNA samples were DNase-treated using DNA-free DNA Removal Kit (Ambion), aliquoted, and stored in -80°C. Quantity and quality of the RNA samples were assessed using the Agilent 2100 Bioanalyzer, the Agilent RNA 6000 Nano Kit (Agilent Technologies), and Qubit fluorometer. Libraries were prepared starting with 300-500 ng of good quality total RNA (RIN ≥7.5) using the TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero Human/Mouse/Rat (Illumina), according to the manufacturer's instructions. The kit contains 96 uniquely indexed adapter combinations in order to allow pooling of multiple samples prior to sequencing. After determining their size (with the Agilent 2100 Bioanalyzer and the Agilent High Sensitivity DNA Kit by Agilent Technologies) and concentration (by qPCR with the KAPA Illumina ABI Prism Library Quantification Kit, Kapa Biosystems), libraries have been pooled and sequenced (single-end, 125 bp) using a Single End V4 Cluster Kit and an Illumina HiSeq2500 or HiSeq4000 instrument.

  Dataset EGAD00001003508

• A novel recurrent mutation in MITF predisposes to familial and sporadic melanoma

  We conducted whole-genome sequencing of probands from several melanoma families, identifying one individual carrying a novel germline variant (c.G1075A, NM_000248.3; p.E318K, NP_000239.1; rs149617956) in the melanoma lineage-specific oncogene MITF. While the variant cosegregated with melanoma in some, but not all cases in the family, linkage analysis of 31 families subsequently identified to carry the variant generated a LOD score of 2.7 under a dominant model, suggesting E318K as a possible intermediate risk variant. Consistent with this, E318K was significantly associated with melanoma in a large Australian case-control sample, giving an odds ratio (OR) of 2.33, 95% CI 1.21-4.70 (case and control carrier frequency, 0.0165 and 0.0072, respectively; P=0.008). Likewise, it was similarly associated in an independent case-control sample from the United Kingdom (UK P=0.012; combined P=0.0003, OR 2.19, 95% CI 1.41-3.45). In the Australian sample, the variant allele was significantly over-represented in cases with a family history of melanoma (OR 2.95, 95% CI 1.23-6.92), multiple primary melanomas (OR 4.22, 95% CI 1.52-10.91), or both (OR 8.37, 95% CI 2.58-23.80). The variant allele was also associated with increased nevus count (combined P=0.002, OR 2.54, 95% CI 1.42-4.55) and non-blue eye color (combined P=0.008, OR 2.01, 95% CI 1.11-3.81). Functional analysis of E318K showed that MITF encoded by the variant allele had impaired sumoylation and differentially regulated several MITF targets. These data indicate that MITF is a melanoma predisposition gene and highlights the utility of whole-genome sequencing to identify novel rare variants associated with disease susceptibility.

  Study phs000419

• Mortality and risk of progression to adult T-cell leukemia/lymphoma in patients with HTLV-1-associated myelopathy/tropical spastic paraparesis

  Human T-cell leukemia virus (HTLV)-1 causes the functionally debilitating disease HTLV-1-associated myelopathy/ tropical spastic paraparesis (HAM/TSP) as well as adult T-cell leukemia lymphoma (ATLL). Although there were concerns that the mortality of HAM/TSP could be affected by the development of ATLL, prospective evidence was lacking in this area. In this 5-year prospective cohort study, we determined the mortality, prevalence, and incidence of ATLL in 527 HAM/TSP patients. The standard mortality ratio of HAM/TSP patients was 2.25, and ATLL was one of the major causes of death (5/33 deaths). ATLL prevalence and incidence in these patients were 3.0% and 3.81 per 1,000 person-years, respectively. To identify patients at a high risk of developing ATLL, flow cytometry, Southern blotting, and targeted sequencing data were analyzed in a separate cohort of 218 HAM/TSP patients. In 17% of the HAM/TSP patients, we identified an increase in T-cells positive for cell adhesion molecule 1 (CADM1), a marker for ATLL and HTLV-1 infected cells. Genomic analysis of these patients revealed that somatic mutations of HTLV-1 infected cells were seen in 90% of the cases and 11% of them had dominant clone and developed ATLL in the longitudinal observation. In this study, we were able to demonstrate for the first time that the increased mortality in patients with HAM/TSP and a significant effect of ATLL on their prognosis. Having dominant clonal expansion of HTLV-1- infected cells with ATLL-associated somatic mutations may be important characteristics of patients with HAM/TSP who are at an increased risk of developing ATLL.

  Study JGAS000226

• Single cell RNA sequencing of synovial B cells in early Rheumatoid Arthritis

  This dataset includes 3432 paired single cell sequencing fastq files derived from synovial B cells of 5 early Rheumatoid Arthritis patients. Libraries were prepared using the SmartSeq2 protocol. All wells contained ERCC spike-ins. Libraries were sequenced on an Illumina NovaSeq instrument with 2x100bp paired-end reads yielding a median of 2.5M reads/well. Sample aliases ending on 368 and 384 represent empty control wells. Sample aliases starting with P11417_4 and P13157_1 belong to ACPA- patient A7, sample aliases starting with P11417_5 and P11417_6 belong to ACPA+ patient A1, sample aliases starting with P13157_2 belong to ACPA- patient A3, sample aliases starting with P13157_3 and P13157_6 belong to ACPA+ patient A2, sample aliases starting with P13157_4 and P13157_5 belong to ACPA- patient A4.

  Dataset EGAD00001009076

• The long term effects of chemotherapy on normal blood - WGS dataset

  In developed countries, ~10% of individuals are exposed to systemic chemotherapy for cancer and other diseases. Many chemotherapeutic agents act by increasing DNA damage in cancer cells, hence triggering cell death. However, there is limited understanding of the extent and consequences of collateral DNA damage to normal tissues. To investigate the impact of chemotherapy on mutation burdens and cell population structure of a normal tissue we sequenced blood cell genomes from 23 individuals, aged 3-80 years, treated with a range of chemotherapy regimens. Substantial additional mutation loads with characteristic mutational signatures were imposed by some chemotherapeutic agents, but there were differences in burden between different classes of agent, different agents of the same class and different blood cell types. Chemotherapy also induced premature changes in the cell population structure of normal blood, similar to those of normal ageing. The results constitute an initial survey of the long-term biological consequences of cytotoxic agents to which a substantial fraction of the population is exposed during the course of their disease management, raising mechanistic questions and highlighting opportunities for mitigation of adverse effects.

  Dataset EGAD00001015339

• HGSC lines: ATACseq and RNAseq, pre- vs post-treatment with HKMTi-1-005

  Formation of a DAC to support the deposition of RNAseq and ATACseq data, from pre- vs post-treatment high-grade serous ovarian carcinoma cell lines. The lines include PEO1, PEO4, PEA2, OVCAR5, OVCAR8, and the treatment includes use of a novel epigenetic drug called HKMTi-1-005.

  Dac EGAC50000000034

• Enrichment Data

  Fresh fecal samples were donated from healthy individuals. We evaluated the responses of healthy adult fecal microbiota to distinct carbon source conditions, pH levels and redox potentials. Metabolite concentrations were measured by HPLC. The total genomic DNA was extracted, sequenced and analyzed. The research project was approved by the Ethic Committee of the Canton of Zurich (2017-01290).

  Dac EGAC50000000346

• BipEx_Landen_SWEBIC

  BipEx: Bipolar Exome Sequencing Broad & Karolinska Institutet. EGAS00001005860. "This research project was a collaboration between the Karolinska Institute and the Stanley Center at the Broad Institute. In this project we sequenced and analyzed the whole exomes of X SWEBIC Bipolar case samples from collaborators in Sweden. Genomic DNA from each samples was sequenced to a mean depth of 20x"

  Dac EGAC50000000142