Biology subjects

Stadler, P. F.

Publications and source records attributed to Stadler, P. F..

36 records · Page 2Linked to original sources

bioRxiv2021

Manipulating base quality scores enables variant calling from bisulfite sequencing alignments using conventional Bayesian approaches

Calling germline SNP variants from bisulfite-converted sequencing data poses a challenge for conventional software, which have no inherent capability to dissociate true polymorphisms from artificial mutations induced by the chemical treatment. Nevertheless, SNP data is desirable both for genotyping and to understand the DNA methylome in the context of the genetic background. The confounding effect of bisulfite conversion can be resolved by observing differences in allele counts on a per-strand basis, whereby artificial mutations are reflected by non-complementary base pairs. Herein, we present a computational pre-processing approach for adapting sequence alignment data, thus indirectly enabling downstream analysis in this manner using conventional variant calling software such as GATK or Freebayes. In comparison to specialised tools, the method represents a marked improvement in precision-sensitivity based on high-quality, published benchmark datasets for both human and model plant variants.

bioinformatics↗

bioRxiv2021

PTEN regulates adipocyte progenitor growth, differentiation and cellular aging

The tumor suppressor phosphatase and tensin homolog (PTEN) negatively regulates the insulin signaling pathway. Germline PTEN pathogenic variants cause PTEN Hamartoma Tumor Syndrome (PHTS), associated with lipoma development in children. It remains unclear which mechanisms trigger this aberrant adipose tissue growth. Adipocyte progenitor cells (APCs) lose their capacity to differentiate into adipocytes during continuous culture, while APCs from PHTS patients lipomas retain their adipogenic potential over a prolonged period. To investigate the role of PTEN in adipose tissue development we performed functional assays and RNA sequencing of control and PTEN knockdown APCs. Reduction of PTEN levels using siRNA or CRISPR lead to an enhanced proliferation and differentiation of APCs. FOXO1 was downregulated on the mRNA level while inactivation through phosphorylation increased. FOXO1 phosphorylation initiates the expression of the lipogenesis activating transcription factor SREBP1. SREBP1 levels were higher after PTEN knockdown and may account for the enhanced adipogenesis. To validate this we overexpressed constitutively active FOXO1 in PTEN CRISPR cells and found reduced adipogenesis, accompanied by a SREBP1 downregulation. We observed that PTEN levels were upregulated during long term culture of wild type APCs. PTEN CRISPR cells showed less senescence compared to controls and the senescence marker CDKN1A (p21) was downregulated in PTEN knockdown cells. Cellular senescence was the most significantly enriched pathway found in RNA sequencing of PTEN knockdown vs. control cells. These results provide evidence that PTEN is involved in the regulation of APCs proliferation, differentiation and senescence, thereby contributing to aberrant adipose tissue growth in PHTS patients.

cell biology↗

bioRxiv2020

Comprehensive benchmarking of software for mapping whole genome bisulfite data: from read alignment to DNA methylation analysis

Whole genome bisulfite sequencing is currently at the forefront of epigenetic analysis, facilitating the nucleotide-level resolution of 5-methylcytosine (5mC) on a genome-wide scale. Specialised software have been developed to accommodate the unique difficulties in aligning such sequencing reads to a given reference, building on the knowledge acquired from model organisms such as human, or Arabidopsis thaliana. As the field of epigenetics expands its purview to non-model plant species, new challenges arise which bring into question the suitability of previously established tools. Herein, nine short-read aligners are evaluated: Bismark, BS-Seeker2, BSMAP, BWA-meth, ERNE-BS5, GEM3, GSNAP, Last, and segemehl. Precision-recall of simulated alignments, in comparison to real sequencing data obtained from three natural accessions, reveals on-balance that BWA-meth and BSMAP are able to make the best use of the data during mapping. The influence of difficult-to-map regions, characterised by deviations in sequencing depth over repeat annotations, is evaluated in terms of the mean absolute deviation of the resulting methylation calls in comparison to a realistic methylome. Downstream methylation analysis is responsive to the handling of multi-mapping reads relative to mapping quality (MAPQ), and potentially susceptible to bias arising from the increased sequence complexity of densely-methylated reads.

bioinformatics↗

bioRxiv2020

Supervised and Unsupervised Classification of lncRNA Subtypes

Many small nucleolar RNAs and many of the hairpin precursors of miRNAs are processed from long non-protein-coding (lncRNA) host genes. In contrast to their highly conserved and heavily structured payload, the host genes feature poorly conserved sequences. Nevertheless there is mounting evidence that the host genes have biological functions. No obvious connections between the function of the host genes and the function of their payloads have been reported. Here we inverstigate whether there is an association of host gene function or mechanisms with the type of payload. To assess this hypothesis we test whether the miRNA host genes (MIRHGs), snoRNA host genes (SNHGs), and other lncRNAs host genes can be distinguished based on sequence and structure features. A positive answer would imply a correlation between host genes and their payload. While the three classes can be distinguished reliably when the classifier is allowed to extract features from the payloads, this is no longer the case when only sequence and structure of parts of the host gene distal from the snoRNAs or miRNA payload is used for classification. Our data indicate that the functions of MIRHGs and SNHGs are largely independent of the functions of their payloads. Furthermore, there is no evidence that the MIRHGs and SNHGs form coherent classes of long non-coding RNAs distinguished by features other than their payloads.

bioinformatics↗

bioRxiv2020

Beyond Plug and Pray: Context Sensitivity and in silico Design of Artificial Neomycin Riboswitches

Gene regulation in prokaryotes often depends on RNA elements such as riboswitches or RNA thermometers located in the 5’ untranslated region of mRNA. Rearrangements of the RNA structure in response, e. g., to the binding of small molecules or ions control translational initiation or premature termination of transcription and thus mRNA expression. Such structural responses are amenable to computational modeling, making it possible to rationally design synthetic riboswitches for a given aptamer. Starting from an artificial aptamer, we construct the first synthetic transcriptional riboswitches that respond to the antibiotic neomycin. We show that the switching behavior in vivo critically depends not only on the sequence of the riboswitch itself, but also on its sequence context. We therefore developed in silico methods to predict the impact of the context, making it possible to adapt the design and to rescue non-functional riboswitches. We furthermore analyze the influence of 5’ hairpins with varying stability on neomycin riboswitch activity. Our data highlight the limitations of a simple plug-and-play approach in the design of complex genetic circuits and demonstrate that detailed computational models significantly simplify, improve, and automate the design of transcriptional circuits. Our design software is available under a free license on Github.1Competing Interest StatementThe authors have declared no competing interest.List of Abbreviationsauarbitrary time unitsAURaptamer upstream regionbgaBβ-galactosidasebgaBthe β-galactosidase genedLdecoupling leaderE. coliEscherichia colieGFPthe enhanced green fluorescent proteinegfpthe enhanced green fluorescent protein geneGFPgreen fluorescent proteinLHleader hairpinLMmutated leaderMFEminimum free energymRNAmessenger RNAoLoriginal leaderPospositive controlRBSribosomal binding siteRFUrelative fluorescence unitRNAribonucleic acidRNAPRNA polymeraseRNase ERibonuclease ERppHRNA 5’ pyrophosphohydrolaserRNAribosomal RNASELEXsystematic evolution of ligands by exponential enrichmentSSCsaline sodium citrate bufferTBETris-borate-EDTATDRterminator downstream regionTSStranscription start siteUunstructured regionUTRuntranslated regionView Full Text

synthetic biology↗

bioRxiv2020

Economic Genome Assembly from Low Coverage Illumina and Nanopore Data

We describe a new approach to assemble genomes from a combination of low-coverage short and long reads. LazyBastard starts from a bipartite overlap graph between long reads and restrictively filtered short-read unitigs, which are then reduced to a long-read overlap graph G. Edges are removed from G to obtain first a consistent orientation and then a DAG. Using heuristics based on properties of proper interval graphs, contigs are extracted as maximum weight paths. These are translated into genomic sequence only in the final step. A prototype implementation of LazyBastard, entirely written in python, not only yields significantly more accurate assemblies of the yeast and fruit fly genomes compared to state-of-the-art pipelines but also requires much less computational effort. FundingRSF / Helmholtz Association 18-44-06201; Deutsche Academische Austauschdienst, DFG STA 850/19-2 within SPP 1738; German Federal Ministery of Education an Research 031A538A, de.NBI-RBC

bioinformatics↗

bioRxiv2020

Assessing the Quality of Cotranscriptional Folding Simulations

Structural changes in RNAs are an important contributor to controlling gene expression not only at the post-transcriptional stage but also during transcription. A subclass of riboswitches and RNA thermometers located in the 5 region of the primary transcript regulates the downstream functional unit - usually an ORF - through premature termination of transcription. Such elements not only occur naturally but they are also attractive devices in synthetic biology. The possibility to design such riboswitches or RNA thermometers is thus of considerable practical interest. Since these functional RNA elements act already during transcription, it is important to model and understand the dynamics of folding and, in particular, the formation of intermediate structures concurrently with transcription. Cotranscriptional folding simulations are therefore an important step to verify the functionality of design constructs before conducting expensive and labour-intensive wet lab experiments. For RNAs, full-fledged molecular dynamics simulations are far beyond practical reach both because of the size of the molecules and the time scales of interest. Even at the simplified level of secondary structures further approximations are necessary. The BarMap approach is based on representing the secondary structure landscape for each individual transcription step by a coarse-grained representation that only retains a small set of low-energy local minima and the energy barriers between them. The folding dynamics between two transcriptional elongation steps is modeled as a Markov process on this representation. Maps between pairs of consecutive coarse-grained landscapes make it possible to follow the folding process as it changes in response to transcription elongation. In its original implementation, the BarMap software provides a general framework to investigate RNA folding dynamics on temporally changing landscapes. It is, however, difficult to use in particular for specific scenarios such as cotranscriptional folding. To overcome this limitation, we developed the user-friendly BarMap-QA pipeline described in detail in this contribution. It is illustrated here by an elaborate example that emphasizes the careful monitoring of several quality measures. Using an iterative workflow, a reliable and complete kinetics simulation of a synthetic, transcription regulating riboswitch is obtained using minimal computational resources. All programs and scripts used in this contribution are free software and available for download as a source distribution for Linux(R), or as a platform-independent Docker(R) image including support for Apple macOS(R) and Microsoft Windows(R).

bioinformatics↗

bioRxiv2019

TerrestrialMetagenomeDB: a public repository of curated and standardized metadata for terrestrial metagenomes

Microbiome studies focused on the genetic potential of microbial communities (metagenomics) became standard within microbial ecology. MG-RAST and the Sequence Read Archive (SRA), the two main metagenome repositories, contain over 202 858 public available metagenomes and this number has increased exponentially. However, mining databases can be challenging due to misannotated, misleading and decentralized data. The main goal of TerrestrialMetagenomeDB is to make it easier for scientists to find terrestrial metagenomes of interest that could be compared with novel datasets in meta-analyses. We defined terrestrial metagenomes as those that do not belong to marine environments. Further, we curated the database using text mining to assign potential descriptive keywords that better contextualize environmental aspects of terrestrial metagenomes, such as biomes and materials. TerrestrialMetagenomeDB release 1.0 includes 15 194 terrestrial metagenomes from SRA and MG-RAST. Together, the downloadable data amounts to 68 Tbp. In total, 199 terrestrial terms were divided into 14 categories. These metagenomes span 84 countries, 31 biomes and 7 main source materials. The TerrestrialMetagenomeDB is publicly available at https://webapp.ufz.de/tmdb.

bioinformatics↗

bioRxiv2019

Unbiased Map of Transcription Termination Sites in Haloferax volcanii reveals manifold termination patterns

Archaeal genomes are densely packed; thus, correct transcription termination is an important factor for orchestrated gene expression. A systematic analysis of RNA 3 termini, to identify transcription termination sites (TTS) using RNAseq data has hitherto only been performed in two archaea. In this study, only part of the genome had been investigated. Here, we developed a novel algorithm that allows an unbiased, genome-wide identification of RNA 3 termini independent of annotation. In an RNA fraction enriched for primary transcripts by terminator exonuclease (TEX) treatment we identified 1,543 RNA 3 termini. A strong sequence signature consistent with known termination events at intergenic loci indicates a clear enrichment for native TTS among them. Using these data we determined distinct putative termination motifs for intergenic (a T stretch) and coding regions (AGATC). In vivo reporter gene tests of selected TTS confirmed termination at these sites, which exemplify the different motifs. For several genes, more than one termination site was detected, resulting in transcripts with different lengths of the 3 untranslated region.

molecular biology↗

bioRxiv2019

flowEMMi: An automated model-based clustering tool for microbial cytometric data

BackgroundFlow cytometry (FCM) is a powerful single-cell based measurement method to ascertain multidimensional optical properties of millions of cells. FCM is widely used in medical diagnostics and health research. There is also a broad range of applications in the analysis of complex microbial communities. The main concern in microbial community analyses is to track the dynamics of microbial subcommunities. So far, this can be achieved with the help of time-consuming manual clustering procedures that require extensive user-dependent input. In addition, several tools have recently been developed by using different approaches which, however, focus mainly on the clustering of medical FCM data or of microbial samples with a well-known background, while much less work has been done on high-throughput, online algorithms for two-channel FCM.\n\nResultsWe bridge this gap with flowEMMi, a model-based clustering tool based on multivariate Gaussian mixture models with subsampling and foreground/background separation. These extensions provide a fast and accurate identification of cell clusters in FCM data, in particular for microbial community FCM data that are often affected by irrelevant information like technical noise, beads or cell debris. flowEMMi outperforms other available tools with regard to running time and information content of the clustering results and provides near-online results and optional heuristics to reduce the running-time further.\n\nConclusionsflowEMMi is a useful tool for the automated cluster analysis of microbial FCM data. It overcomes the user-dependent and time-consuming manual clustering procedure and provides consistent results with ancillary information and statistical proof.

bioinformatics↗

bioRxiv2019

Bi-Alignments as Models of Incongruent Evolution of RNA Sequence and Structure

RNA molecules may experience independent selection pressures on their sequence and (secondary) structure. Structural features then may be preserved without maintaining their exact position along the sequence. In such cases, corresponding base pairs are no longer formed by homologous bases, leading to the incongruent evolutionary conservation of sequence and structure. In order to model this phenomenon, we introduce bi-alignments as a superposition of two alignments: one modeling sequence homology; the other, structural homology. We show that under natural assumptions on the scoring functions, bi-alignments form a special case of 4-way alignments, in which the incongruencies are measured as indels in the pairwise alignment of the two alignment copies. A preliminary survey of the Rfam database suggests that incongruent evolution of RNAs is not a very rare phenomenon.\n\nAvailabilityOur software is freely available at https://github.com/s-will/BiAlign

bioinformatics↗

bioRxiv2019

Splicing conservation signals in plant long non-coding RNAs

Long non-coding RNAs (lncRNAs) have recently emerged as prominent regulators of gene expression in eukaryotes. LncRNAs often drive the modification and maintenance of gene activation or gene silencing states via chromatin conformation rearrangements. In plants, lncRNAs have been shown to participate in gene regulation, and are essential to processes such as vernalization and photomorphogenesis. Despite their prominent functions only over a dozen lncRNAs have been experimentally and functionally characterized. Similar to its animal counterparts, the rates of sequence divergence are much higher in plant lncRNAs than in protein coding mRNAs, making it difficult to identify lncRNA conservation using traditional sequence comparison methods. Beyond this, little is known about the evolutionary patterns of lncRNAs in plants. Here, we characterized the splicing conservation of lncRNAs in Brassicaceae. We generated a whole-genome alignment of 16 Brassica species and used it to identify synthenic lncRNA orthologues. Using a scoring system trained on transcriptomes from A. thaliana and B. oleracea, we identified splice sites across the whole alignment and measured their conservation. Our analysis revealed that 17.9% (112/627) of all intergenic lncRNAs display splicing conservation in at least one exon, an estimate that is substantially higher to previous estimates of lncRNA conservation in this group. Our findings agree with similar studies in vertebrates, demonstrating that splicing conservation can be evidence of stabilizing selection. We provide conclusive evidence for the existence of evolutionary deeply conserved lncRNAs in plants and describe a generally applicable computational workflow to identify functional lncRNAs in plants.

genomics↗

bioRxiv2018

TERribly Difficult: Searching for Telomerase RNAs in Saccharomycetes

AbstractThe telomerase RNA in yeasts is large, usually > 1,000 nt, and contains functional elements that have been extensively studied experimentally in several disparate species. Nevertheless, they are very difficult to detect by homology-based methods and so far have escaped annotation in the majority of the genomes of Saccharomycotina. This is a consequence of sequences that evolve rapidly at nucleotide level, are subject to large variations in size, and are highly plastic with respect to their secondary structures. Here we report on a survey that was aimed at closing this gap in RNA annotation. Despite considerable efforts and the combination of a variety of different methods, it was only partially successful. While 27 new telomerase RNAs were identified, we had to restrict our efforts to the subgroup Saccharomycetacea because even this narrow subgroup was diverse enough to require different search models for different phylogenetic subgroups. More distant branches of the Saccharomycotina still remain without annotated telomerase RNA.

bioinformatics↗

bioRxiv2018

Genome-wide features of introns are evolutionary decoupled among themselves and from genome size throughout Eukarya

The impact of spliceosomal introns on genome and organismal evolution remains puzzling. Here, we investigated the correlative associations among genome-wide features of introns from protein-coding genes (e.g., size, density, genome-content, repeats), genome size and multicellular complexity on 461 eukaryotes. Thus, we formally distinguished simple from complex multicellular organisms (CMOs), and developed the program GenomeContent to systematically estimate genomic traits. We performed robust phylogenetic controlled analyses, by taking into account significant uncertainties in the tree of eukaryotes and variation in genome size estimates. We found that changes in the variation of some intron features (such as size and repeat composition) are only weakly, while other features measuring intron abundance (within and across genes) are not, scaling with changes in genome size at the broadest phylogenetic scale. Accordingly, the strength of these associations fluctuates at the lineage-specific level, and changes in the length and abundance of introns within a genome are found to be largely evolving independently throughout Eukarya. Thereby, our findings are in disagreement with previous estimations claiming a concerted evolution between genome size and introns across eukaryotes. We also observe that intron features vary homogeneously (with low repetitive composition) within fungi, plants and stramenophiles; but they vary dramatically (with higher repetitive composition) within holozoans, chlorophytes, alveolates and amoebozoans. We also found that CMOs and their closest ancestral relatives are characterized by high intron-richness, regardless their genome size. These patterns contrast the narrow distribution of exon features found across eukaryotes. Collectively, our findings unveil spliceosomal introns as a dynamically evolving non-coding DNA class and strongly argue against both, a particular intron feature as key determinant of eukaryotic gene architecture, as well as a major mechanism (adaptive or non-adaptive) behind the evolutionary dynamics of introns over a large phylogenetic scale. We hypothesize that intron-richness is a pre-condition to evolve complex multicellularity.

evolutionary biology↗

bioRxiv2017

Time-Consistent Reconciliation Maps and ForbiddenTime Travel

BackgroundIn the absence of horizontal gene transfer it is possible to reconstruct the history of gene families from empirically determined orthology relations, which are equivalent to event-labeled gene trees. Knowledge of the event labels considerably simplifies the problem of reconciling a gene tree T with a species trees S, relative to the reconciliation problem without prior knowledge of the event types. It is well-known that optimal reconciliations in the unlabeled case may violate time-consistency and thus are not biologically feasible. Here we investigate the mathematical structure of the event labeled reconciliation problem with horizontal transfer.\n\nResultsWe investigate the issue of time-consistency for the event-labeled version of the reconciliation problem, provide a convenient axiomatic framework, and derive a complete characterization of time-consistent reconciliations. This characterization depends on certain weak conditions on the event-labeled gene trees that reflect conditions under which evolutionary events are observable at least in principle. We give an [O](|V(T)|log(|V(S)|))-time algorithm to decide whether a time-consistent reconciliation map exists. It does not require the construction of explicit timing maps, but relies entirely on the comparably easy task of checking whether a small auxiliary graph is acyclic. The algorithms are implemented in C++ using the boost graph library and are freely available at https://github.com/Nojgaard/tc-recon.\n\nSignificanceThe combinatorial characterization of time consistency and thus biologically feasible reconciliation is an important step towards the inference of gene family histories with horizontal transfer from orthology data, i.e., without presupposed gene and species trees. The fast algorithm to decide time consistency is useful in a broader context because it constitutes an attractive component for all tools that address tree reconciliation problems.

evolutionary biology↗

bioRxiv2017

Large-Scale Evolutionary Patterns of Protein Domain Distributions in Eukaryotes

The genomic inventory of protein domains is an important indicator of an organisms regulatory and metabolic capabilities. Existing gene annotations, however, can be plagued by substantial ascertainment biases that make it difficult to obtain and compare quantitative domain data. We find that quantitative trends across the Eukarya can be investigated based on a combination of gene prediction and standard domain annotation pipelines. Species-specific training is required, however, to account for the genomic peculiarities in many lineages. In contrast to earlier studies we find wide-spread statistically significant avoidance of protein domains associated with distinct functional high-level gene-ontology terms.\n\n1998 ACM Subject Classification J.3 Life and Medical Sciences

bioinformatics↗

bioRxiv2017

Alzheimer related genes show accelerated evolution

Alzheimer's disease (AD) is a neurodegenerative disorder of unknown cause with complex genetic and environmental traits. Here, we show that gene structures of loci, that show AD-associated changes in their expression, evolve faster than the genome at large. This phylogenetic trait of AD suggests a critical pathogenetic role of recent adaptive evolution of human brain and might have far reaching consequences with respect to the appropriateness of model systems and the development of disease-modifying strategies.

evolutionary biology↗

bioRxiv2016

Design Specifications for Cellular Regulation

A critical feature of all cellular processes is the ability to control the rate of gene or protein expression and metabolic flux in changing environments through regulatory feedback. We review the many ways that regulation is represented through causal, logical and dynamical components. Formalizing the nature of these components promotes effective comparison among distinct regulatory networks and provides a common framework for the potential design and control of regulatory systems in synthetic biology.

systems biology↗