Portability of Scientific Workflows in NGS Data Analysis: A Case Study

The analysis of next-generation sequencing (NGS) data requires complex computational workflows consisting of dozens of autonomously developed yet interdependent processing steps. Whenever large amounts of data need to be processed, these workflows must be executed on a parallel and/or distributed systems to ensure reasonable runtime. Porting a workflow developed for a particular system on a particular hardware infrastructure to another system or to another infrastructure is non-trivial, which poses a major impediment to the scientific necessities of workflow reproducibility and workflow reusability. In this work, we describe our efforts to port a state-of-the-art workflow for the detection of specific variants in whole-exome sequencing of mice. The workflow originally was developed in the scientific workflow system snakemake for execution on a high-performance cluster controlled by Sun Grid Engine. In the project, we ported it to the scientific workflow system SaasFee that can execute workflows on (multi-core) stand-alone servers or on clusters of arbitrary sizes using the Hadoop. The purpose of this port was that also owners of low-cost hardware infrastructures, for which Hadoop was made for, become able to use the workflow. Although both the source and the target system are called scientific workflow systems, they differ in numerous aspects, ranging from the workflow languages to the scheduling mechanisms and the file access interfaces. These differences resulted in various problems, some expected and more unexpected, that had to be resolved before the workflow could be run with equal semantics. As a side-effect, we also report cost/runtime ratios for a state-of-the-art NGS workflow on very different hardware platforms: A comparably cheap stand-alone server (80 threads), a mid-cost, mid-sized cluster (552 threads), and a high-end HPC system (3784 threads)

Generation and characterization of a mitotane-resistant adrenocortical cell line

Mitotane is the only drug approved for the therapy of adrenocortical carcinoma (ACC). Its clinical use is limited by the occurrence of relapse during therapy. To investigate the underlying mechanisms in vitro, we here generated mitotane-resistant cell lines. After long-term pulsed treatment of HAC-15 human adrenocortical carcinoma cells with 70 µM mitotane, we isolated monoclonal cell populations of treated cells and controls and assessed their respective mitotane sensitivities by MTT assay. We performed exome sequencing and electron microscopy, conducted gene expression microarray analysis and determined intracellular lipid concentrations in the presence and absence of mitotane. Clonal cell lines established after pulsed treatment were resistant to mitotane (IC50 of 102.2 ± 7.3 µM (n = 12) vs 39.4 ± 6.2 µM (n = 6) in controls (biological replicates, mean ± s.d., P = 0.0001)). Unlike nonresistant clones, resistant clones maintained normal mitochondrial and nucleolar morphology during mitotane treatment. Resistant clones largely shared structural and single nucleotide variants, suggesting a common cell of origin. Resistance depended, in part, on extracellular lipoproteins and was associated with alterations in intracellular lipid homeostasis, including levels of free cholesterol, as well as decreased steroid production. By gene expression analysis, resistant cells showed profound alterations in pathways including steroid metabolism and transport, apoptosis, cell growth and Wnt signaling. These studies establish an in vitro model of mitotane resistance in ACC and point to underlying molecular mechanisms. They may enable future studies to overcome resistance in vitro and improve ACC treatment in vivo

Investigating APOE, APP-Aβ metabolism genes and Alzheimer’s disease GWAS hits in brain small vessel ischemic disease

Alzheimer’s disease and small vessel ischemic disease frequently co-exist in the aging brain. However, pathogenic links between these 2 disorders are yet to be identified. Therefore we used Taqman genotyping, exome and RNA sequencing to investigate Alzheimer’s disease known pathogenic variants and pathways: APOE ε4 allele, APP-Aβ metabolism and late-onset Alzheimer’s disease main genome-wide association loci (APOE, BIN1, CD33, MS4A6A, CD2AP, PICALM, CLU, CR1, EPHA1, ABCA7) in 96 early-onset small vessel ischemic disease Caucasian patients and 368 elderly neuropathologically proven controls (HEX database) and in a mouse model of cerebral hypoperfusion. Only a minority of patients (29%) carried APOE ε4 allele. We did not detect any pathogenic mutation in APP, PSEN1 and PSEN2 and report a burden of truncating mutations in APP-Aß degradation genes. The single-variant association test identified 3 common variants with a likely protective effect on small vessel ischemic disease (0.54>OR > 0.32, adj. p-value  1, adj. p-val<0.05) together with Apoe, Ms4a cluster and Cd33 during brain hypoperfusion and their overexpression correlated with the ischemic lesion size. Finally, the detection of Aβ oligomers in the hypoperfused hippocampus supported the link between brain ischemia and Alzheimer’s disease pathology

Combining callers improves the detection of copy number variants from whole-genome sequencing

Copy Number Variants (CNVs) are deletions, duplications or insertions larger than 50 base pairs. They account for a large percentage of the normal genome variation and play major roles in human pathology. While array-based approaches have long been used to detect them in clinical practice, whole-genome sequencing (WGS) bears the promise to allow concomitant exploration of CNVs and smaller variants. However, accurately calling CNVs from WGS remains a difficult computational task, for which a consensus is still lacking. In this paper, we explore practical calling options to reach the best compromise between sensitivity and sensibility. We show that callers based on different signal (paired-end reads, split reads, coverage depth) yield complementary results. We suggest approaches combining four selected callers (Manta, Delly, ERDS, CNVnator) and a regenotyping tool (SV2), and show that this is applicable in everyday practice in terms of computation time and further interpretation. We demonstrate the superiority of these approaches over array-based Comparative Genomic Hybridization (aCGH), specifically regarding the lack of resolution in breakpoint definition and the detection of potentially relevant CNVs. Finally, we confirm our results on the NA12878 benchmark genome, as well as one clinically validated sample. In conclusion, we suggest that WGS constitutes a timely and economically valid alternative to the combination of aCGH and whole-exome sequencing

Analyses of circRNA Expression throughout the Light-Dark Cycle Reveal a Strong Regulation of Cdr1as, Associated with Light Entrainment in the SCN

Circular RNAs (circRNAs) are a large class of relatively stable RNA molecules that are highly expressed in animal brains. Many circRNAs have been associated with CNS disorders accompanied by an aberrant wake-sleep cycle. However, the regulation of circRNAs in brain homeostasis over daily light-dark (LD) cycles has not been characterized. Here, we aim to quantify the daily expression changes of circRNAs in physiological conditions in healthy adult animals. Using newly generated and public RNA-Seq data, we monitored circRNA expression throughout the 12:12 h LD cycle in various mouse brain regions. We identified that Cdr1as, a conserved circRNA that regulates synaptic transmission, is highly expressed in the suprachiasmatic nucleus (SCN), the master circadian pacemaker. Despite its high stability, Cdr1as has a very dynamic expression in the SCN throughout the LD cycle, as well as a significant regulation in the hippocampus following the entry into the dark phase. Computational integration of different public datasets predicted that Cdr1as is important for regulating light entrainment in the SCN. We hypothesize that the expression changes of Cdr1as in the SCN, particularly during the dark phase, are associated with light-induced phase shifts. Importantly, our work revises the current beliefs about natural circRNA stability and suggests that the time component must be considered when studying circRNA regulation

Dataset for: Modeling chemotherapy induced neurotoxicity with human induced pluripotent stem cell (iPSC)-derived sensory neurons

Chemotherapy-induced peripheral neuropathy (CIPN) is a frequent and potentially irreversible adverse event of cytotoxic chemotherapy. We evaluate whether sensory neurons derived from induced pluripotent stem cells (iPSC-DSN) can serve as human disease model system for chemotherapy induced neurotoxicity. Sensory neurons differentiated from two established induced pluripotent stem cell lines were used (s.c. BIHi005-A https://hpscreg.eu/cell- line/BIHi005-A and BIHi004-B https://hpscreg.eu/cell-line/BIHi004-B, Berlin Institute of Health Stem Cell Core Facility). Cell viability and cytotoxicity assays were performed, comparing susceptibility to four neurotoxic and two non-neurotoxic drugs. RNA sequencing analyses in paclitaxel vs. vehicle (DMSO)treated sensory neurons were performed. Treatment of iPSC-DSN for 24 h with the neurotoxic drugs paclitaxel, bortezomib, vincristine and cisplatin led to a dose dependent decline of cell viability in clinically relevant IC50 ranges, which was not the case for the non-neurotoxic compounds doxorubicin and 5-fluorouracil. RNA sequencing analyses at 24 h, i.e. before paclitaxel-induced cell death occurred, revealed the differential expression of genes of neuronal injury, cellular stress response, and sterol pathways in response to 1 mu M paclitaxel. Neuroprotective effects of lithium chloride co-incubation, which were previously shown in rodent dorsal root ganglia, could be replicated in human iPSC-DSN. Cell lines from the two different donors BIHi005-A and BIHi004-B showed different responses to the neurotoxic treatment in cell viability and cytotoxicity assays

Proteomic Changes of Tissue-Tolerable Plasma Treated Airway Epithelial Cells and Their Relation to Wound Healing

Background. The worldwide increasing number of patients suffering from nonhealing wounds requires the development of new safe strategies for wound repair. Recent studies suggest the possibility of nonthermal (cold) plasma application for the acceleration of wound closure. Methods. An in vitro wound healing model with upper airway S9 epithelial cells was established to determine the macroscopically optimal dosage of tissue-tolerable plasma (TTP) for wound regeneration, while a 2D-difference gel electrophoresis (2D-DIGE) approach was used to quantify the proteomic changes in a hypothesis-free manner and to evaluate the balance of beneficial and adverse effects due to TTP application. Results. Plasma doses from 30 s up to 360 s were tested in relation to wound closure after 24 h, 48 h, 72 h, 96 h, and 120 h, in which lower doses (30, 60, and 120 s) resulted in dose-dependent improved wound healing rate compared to untreated cells. Thereby, the 120 s dose caused significantly the best wound healing properties after 96 and 120 h. The proteome analysis combined with IPA revealed that a lot of affected stress adaptation responses are linked to oxidative stress response emphasizing oxidative stress as a possible key event in the regeneration process of epithelial cells as well as in the adaptation to plasma exposure. Further cellular and molecular functions like proliferation and apoptosis were significantly up- or downregulated by all TTP treatments but mostly by the 120 s dose. Conclusions. For the first time, we were able to show plasma effects on cellular adaptation of upper airway epithelial S9 cells improving wound healing. This is of particular interest for plasma application, for example, in the surgery field of otorhinolaryngology or internal medicine

VarFish: comprehensive DNA variant analysis for diagnostics and research

VarFish is a user-friendly web application for the quality control, filtering, prioritization, analysis, and user-based annotation of DNA variant data with a focus on rare disease genetics. It is capable of processing variant call files with single or multiple samples. The variants are automatically annotated with population frequencies, molecular impact, and presence in databases such as ClinVar. Further, it provides support for pathogenicity scores including CADD, MutationTaster, and phenotypic similarity scores. Users can filter variants based on these annotations and presumed inheritance pattern and sort the results by these scores. Variants passing the filter are listed with their annotations and many useful link-outs to genome browsers, other gene/variant data portals, and external tools for variant assessment. VarFish allows users to create their own annotations including support for variant assessment following ACMG-AMP guidelines. In close collaboration with medical practitioners, VarFish was designed for variant analysis and prioritization in diagnostic and research settings as described in the software's extensive manual. The user interface has been optimized for supporting these protocols. Users can install VarFish on their own in-house servers where it provides additional lab notebook features for collaborative analysis and allows re-analysis of cases, e.g. after update of genotype or phenotype databases

An exploratory investigation of brain collateral circulation plasticity after cerebral ischemia in two experimental C57BL/6 mouse models

Brain collateral circulation is an essential compensatory mechanism in response to acute brain ischemia. To study the temporal evolution of brain macro and microcollateral recruitment and their reciprocal interactions in response to different ischemic conditions, we applied a combination of complementary techniques (T2 weighted magnetic resonance imaging [MRI], time of flight [TOF] angiography [MRA], cerebral blood flow [CBF] imaging and histology) in two different mouse models. Hypoperfusion was either induced by permanent bilateral common carotid artery stenosis (BCCAS) or 60 minute transient unilateral middle cerebral artery occlusion (MCAO). In both models, collateralization is a very dynamic phenomenon with a global effect affecting both hemispheres. Patency of ipsilateral posterior communicating artery (PcomA) represents the main variable survival mechanism and the main determinant of stroke lesion volume and recovery in MCAO whereas the promptness of external carotid artery retrograde flow recruitment together with PcomA patency, critically influence survival, brain ischemic lesion volume and retinopathy in BCCAS mice. Finally, different ischemic gradients shape microcollateral density and size