Biology subjects

Gawron, J.

Publications and source records attributed to Gawron, J..

4 recordsLinked to original sources

bioRxiv2024

DemoTape: Computational demultiplexing of targeted single-cell sequencing data

BackgroundSingle-cell sequencing can provide novel insights into the understanding and treatment of diseases. In cancer, for example, intratumor heterogeneity is a major cause of treatment resistance and relapse. Although technological progress has substantially increased the throughput of sequenced cells, single-cell sequencing remains cost and labor-intensive. Multiplexing, i.e., the pooling and subsequent joint preparation and sequencing of samples, followed by a demultiplexing step, is a common practice to reduce expenses and confounding batch effects, especially in single-cell RNA sequencing. ResultsHere, we introduce demoTape, a computational demultiplexing method for targeted single-cell DNA sequencing (scDNA-seq) data based on a distance metric between individual cells at single-nucleotide polymorphisms loci. To validate demoTape, we sequence three B-cell lymphoma patients separately and multiplexed on the Tapestri platform. We find similar genotypes, clones, and evolutionary histories in all three samples when comparing the individual with the demultiplexed samples. Using the three individually sequenced samples, we simulate multiplexed ground truth data and show that demoTape outperforms state-of-the-art demultiplexing methods designed for RNA sequencing data. Additionally, we demonstrate through downsampling that the inferred clonal composition remained largely stable for samples with fewer cells despite the inevitable loss in resolution of low-frequency clones. ConclusionsMultiplexing and subsequent genotype-based demultiplexing of scDNA-seq will reduce costs and workload, eventually allowing the sequencing of more samples. This will open new possibilities and accelerate the investigation of biological questions where cellular heterogeneity on the genomic level plays a crucial role.

bioinformatics↗

bioRxiv2024

Robust and cost-efficient single-cell sequencing through combinatorial pooling

Single-cell sequencing is widely used to study molecular cell-to-cell heterogeneity. Even though the cost of sequencing has dropped throughout the last decades, single-cell assays remain expensive, because they require strategies to index molecules by cells. The high costs of indexing can be mitigated by pooling samples prior to sequencing library preparation. Computational methods have been developed to leverage molecular features that are distinct between different samples to separate the pools into distinct datasets. However, since all multiplexed samples are processed in the same way, information on the origin of each demultiplexed dataset is lost. To map datasets to their sample of origin, additional information such as molecular indexing or additional genotyping is needed. Here, we propose a class of experimental designs that allows identifying the sample of origin of each demultiplexed dataset, only relying on the genetic profiles of the samples and the composition of pools. Our approach is based on splitting and pooling samples in specific combinations. We find a most cost-efficient experimental design in this class and prove its optimality. We present a dynamic programming algorithm to iteratively simplify an optimal experimental design by breaking it into several independent designs while maintaining optimality. Furthermore, we propose a subclass of experimental designs which allow robust sample identification even under partial failure of the experiment and present a provably optimal design in this subclass. We provide an implementation for automatic sample identification under these optimal combinatorial pooling strategies and demonstrate its functionality in a simulation study.

bioinformatics↗

bioRxiv2024

Human iPSC-derived myelinating organoids andgloboid cells to study Krabbe Disease

Krabbe disease (Kd) is a lysosomal storage disorder (LSD) caused by the deficiency of the lysosomal galactosylceramidase (GALC) which cleaves the myelin enriched lipid galactosylceramide (GalCer). Accumulated GalCer is catabolized into the cytotoxic lipid psychosine that causes myelinating cells death and demyelination which recruits microglia/macrophages that fail to digest myelin debris and become globoid cells. Here, to understand the pathological mechanisms of Kd, we used induced pluripotent stem cells (iPSCs) from Kd patients to produce myelinating organoids and microglia. We show that Kd organoids have no obvious defects in neurogenesis, astrogenesis, and oligodendrogenesis but manifest early myelination defects. Specifically, Kd organoids showed shorter but a similar number of myelin internodes than Controls at the peak of myelination and a reduced number and shorter internodes at a later time point. Interestingly, myelin is affected in the absence of autophagy and mTOR pathway dysregulation, suggesting lack of lysosomal dysfunction which makes this organoid model a very valuable tool to study the early events that drive demyelination in Kd. Kd iPSC-derived microglia show a marginal rate of globoid cell formation under normal culture conditions that is drastically increased upon GalCer feeding. Under normal culture conditions, Kd microglia show a minor LAMP1 content decrease and a slight increase in the autophagy protein LC3B. Upon GalCer feeding, Kd cells show accumulation of autophagy proteins and strong LAMP1 reduction that at a later time point are reverted showing the compensatory capabilities of globoid cells. Altogether, this supports the value of our cultures as tools to study the mechanisms that drive globoid cell formation and the compensatory mechanism in play to overcome GalCer accumulation in Kd.

neuroscience↗

bioRxiv2024

Loss of prohibitin 2 in Schwann cells dysregulates key transcription factors controlling developmental myelination

Schwann cells are critical for the proper development and function of the peripheral nervous system, where they form a mutually beneficial relationship with axons. Past studies have highlighted that a pair of proteins called the prohibitins play major roles in Schwann cell biology. Prohibitins are ubiquitously expressed and versatile proteins. We have previously shown that while prohibitins play a crucial role in Schwann cell mitochondria for long-term myelin maintenance and axon health, they may also be present at the Schwann cell-axon interface during development. Here, we expand on this work, showing that drug-mediated modulation of prohibitins in vitro disrupts myelination and confirming that Schwann cell-specific ablation of prohibitin 2 (Phb2) in vivo results in early and severe defects in peripheral nerve development. Using a proteomic approach in vitro, we identify a pool of candidate PHB2 interactors that change their interaction with PHB2 depending on the presence of axonal signals. Furthermore, we show in vivo that loss of Phb2 in mouse Schwann cells causes ineffective proliferation and dysregulation of transcription factors EGR2 (KROX20), POU3F1 (OCT6) and POU3F2 (BRN2) that are necessary for proper Schwann cell maturation. Schwann cell-specific deletion of Jun, a transcription factor associated with negative regulation of myelination, confers partial rescue of the development defect seen in mice lacking Schwann cell Phb2. This work develops our understanding of Schwann cell biology, revealing that Phb2 may directly or indirectly modulate the timely expression of transcription factors necessary for proper peripheral nervous system development, and proposing candidates that may play a role in PHB2-mediated integration of axon signals in the Schwann cell.

neuroscience↗