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To overcome the ethical and technical limitations of in vivo human disease models, the broader scientific community frequently employs model organism-derived cell lines to investigate disease mechanisms, pathways, and therapeutic strategies. Despite the widespread use of certain in vitro models, many still lack contemporary genomic analysis supporting their use as a proxy for the affected human cells and tissues. Consequently, it is imperative to determine how accurately and effectively any proposed biological surrogate may reflect the biological processes it is assumed to model. One such cellular surrogate of human disease is the established mouse neural precursor cell line, SN4741, which has been used to elucidate mechanisms of neurotoxicity in Parkinson disease for over 25 years. Here, we are using a combination of classic and contemporary genomic techniques – karyotyping, RT-qPCR, single cell RNA-seq, bulk RNA-seq, and ATAC-seq – to characterize the transcriptional landscape, chromatin landscape, and genomic architecture of this cell line, and evaluate its suitability as a proxy for midbrain dopaminergic neurons in the study of Parkinson disease. We find that SN4741 cells possess an unstable triploidy and consistently exhibits low expression of dopaminergic neuron markers across assays, even when the cell line is shifted to the non-permissive temperature that drives differentiation. The transcriptional signatures of SN4741 cells suggest that they are maintained in an undifferentiated state at the permissive temperature and differentiate into immature neurons at the non-permissive temperature; however, they may not be dopaminergic neuron precursors, as previously suggested. Additionally, the chromatin landscapes of SN4741 cells, in both the differentiated and undifferentiated states, are not concordant with the open chromatin profiles of ex vivo, mouse E15.5 forebrain- or midbrain-derived dopaminergic neurons. Overall, our data suggest that SN4741 cells may reflect early aspects of neuronal differentiation but are likely not a suitable proxy for dopaminergic neurons as previously thought. The implications of this study extend broadly, illuminating the need for robust biological and genomic rationale underpinning the use of in vitro models of molecular processes. [ABSTRACT FROM AUTHOR]

Haploid cell lines are a valuable research tool with broad applicability for genetic assays. As such the fully haploid human cell line, eHAP1, has been used in a wide array of studies. However, the absence of a corresponding reference genome sequence for this cell line has limited the potential for more widespread applications to experiments dependent on available sequence, like capture-clone methodologies. We generated ~15× coverage Nanopore long reads from ten GridION flowcells and utilized this data to assemble a de novo draft genome using minimap and miniasm and subsequently polished using Racon. This assembly was further polished using previously generated, low-coverage, Illumina short reads with Pilon and ntEdit. This resulted in a hybrid eHAP1 assembly with >90% complete BUSCO scores. We further assessed the eHAP1 long read data for structural variants using Sniffles and identify a variety of rearrangements, including a previously established Philadelphia translocation. Finally, we demonstrate how some of these variants overlap open chromatin regions, potentially impacting regulatory regions. By integrating both long and short reads, we generated a high-quality reference assembly for eHAP1 cells. The union of long and short reads demonstrates the utility in combining sequencing platforms to generate a high-quality reference genome de novo solely from low coverage data. We expect the resulting eHAP1 genome assembly to provide a useful resource to enable novel experimental applications in this important model cell line. • Generation of a reference genome for the human haploid cell line, eHAP1 • Long but noisy Nanopore sequencing combined with accurate but short Illumina sequencing yields a polished reference genome • Structural variants, including the Philadelphia translocation, can be identified using exclusively long reads [ABSTRACT FROM AUTHOR]