Biology subjects

Chovanec, P.

Publications and source records attributed to Chovanec, P..

7 recordsLinked to original sources

bioRxiv2026

Robust Chromosome Segmentation in Sparse Single-Cell Whole-Genome Sequencing Using a k-Segment Constrained Hidden Markov Model

Genome integrity is essential for cellular and organismal fitness. Loss of heterozygosity (LOH), which eliminates the buffering effect of heterozygosity and can expose deleterious recessive alleles, is an important form of somatic genome alteration associated with aging and tumorigenesis. To characterize LOH across cell populations, we use sci-L3, a high-throughput combinatorial-indexing approach for single-cell whole-genome sequencing (WGS). The throughput, however, comes at the cost of sparse coverage of individual genomes, complicating LOH inference from phased heterozygous sites. We therefore developed a k-segment constrained hidden Markov model (HMM) that explicitly limits the maximum number of state transitions and implicitly enforces learning a much lower transition probability than unconstrained HMM models, enabling robust segmentation with learned transition probabilities and optional SNP binning. Benchmarking against orthogonal sci-L3-Strand-seq segmentation, which resolves all seven mitotic crossover outcomes, the method achieved 86% LOH recall and 88% precision. Haplotype-aware analyses support genuine LOH in 68-78% of sampled apparent false-positive calls, suggesting that the reference-based precision of 88% may underestimate the actual precision, potentially approaching 96%. The k-segment HMM thus enables sensitive detection of LOH heterogeneity from sparse single-cell WGS and is applicable to systems with available haplotype phasing.

genomics↗

bioRxiv2026

Lesions initiating spontaneous mitotic crossover are minimally subject to non-homologous end joining

Homologous recombination (HR) between sister chromatids is the dominant outcome of replication-associated DNA repair, yet the lesions that initiate spontaneous mitotic crossovers remain poorly defined. Most mechanistic work on HR pathway choice uses enzymatically induced two-ended double-strand breaks (DSBs), where non-homologous end joining (NHEJ) is a major competing pathway. Whether NHEJ also competes for spontaneous, replication-associated lesions has not been directly tested. Here we use sci-L3-Strand-seq, a single-cell replication-template strand-specific sequencing method that maps sister chromatid exchange (SCE) genome-wide, to profile heterozygous and homozygous knockouts of NHEJ factors (LIG4, XRCC4) and the single-strand break (SSB) repair scaffold XRCC1 in HAP1 and BJ cell lines. NHEJ disruption produced only a modest (30%) increase in SCE, whereas XRCC1 loss caused a pronounced, 5-fold elevation. The dense, widespread SCE pattern in XRCC1-deficient cells is consistent with unrepaired SSBs being converted at replication forks into one-ended DSBs that lack a second end for ligation and therefore cannot engage NHEJ. In parallel, structural variation (SV) mapping in the same single cells revealed a strongly non-random landscape dominated by recurrent chromosome losses and clonal expansion, indicating selective pressure and stepwise genome evolution. SCE frequency did not correlate with SV burden or SV-defined subclones, demonstrating that error-free recombination and mutational rearrangement represent separable axes of genome maintenance. Recovery of reciprocal daughter-cell pairs with matching SCE breakpoints directly confirms that these events arose by inter-sister exchange in the preceding division. Together, these results show that spontaneous mitotic crossovers are driven by lesions largely incompatible with NHEJ and instead engage HR through replication-coupled SSB-to-DSB conversion, and that elevated error-free recombination is decoupled from the mutational SV landscape.

genomics↗

bioRxiv2026

Genome-wide mapping of Cas9-induced sister chromatid exchange across single and over 200 genomic targets

CRISPR/Cas9-induced DNA double-strand breaks (DSBs) are widely used for genome engineering, yet their capacity to provoke sister chromatid exchange (SCE) and associated genome instability remains incompletely understood, in part because exchanges between identical sister chromatids leave no sequence change and are invisible to conventional whole-genome sequencing. Using sci-L3-Strand-seq, a scalable single-cell template-strand sequencing platform for genome-wide SCE mapping, we quantified strand-switch events following targeted Cas9 cleavage. Cas9 targeting of a unique genomic locus led to strong local enrichment of SCE at the break site, reaching up to 41% in the same cell cycle and 17% in the subsequent division, indicating that DSB repair frequently engages non-local inter-sister repair. Extending the assay to 237 repetitive Alu targets using one Alu-specific sgRNA revealed mild and marginally statistically significant enrichment of on-target SCE across wild-type Cas9 and nickase variants (D10A and H840A). However, restricting analysis to a subset of cells with elevated SCE burden (>8 SCEs per cell) uncovered significant enrichment at programmed cut sites, suggesting that recombination at repetitive loci is conditionally engaged in highly recombinogenic cells. Reciprocal daughter-cell pair analysis further revealed large-scale structural alterations on chromosomes with induced SCEs and at SCE junctions, indicating that Cas9-induced SCEs do not universally reflect error-free homologous recombination. Disruption of DNA repair genes at the cut site, including LIG3, LIG4, XRCC1, and XRCC4, did not measurably alter SCE frequency per cell, consistent with delayed functional loss following editing and, in the case of essential genes such as LIG3, selection against disruptive alleles. Together, these findings demonstrate that Cas9-induced DSBs are potent local triggers of SCE at unique loci, while at repetitive regions SCE is more context-dependent and can be associated with structural alterations, highlighting the influence of lesion type and genomic context on recombination outcomes during genome editing.

genomics↗

bioRxiv2025

High-throughput linear amplification for single-cell template strand sequencing with sci-L3-Strand-seq

Despite the many advances in single cell genomics, detecting structural rearrangements in single cells, particularly error-free sister-chromatid exchanges, remains challenging. Here we describe sci-L3-Strand-seq, a combinatorial indexing method with linear amplification for DNA template strand sequencing that cost-effectively scales to millions of single cells, as a platform for mapping mitotic crossover and resulting genome instability events. We provide a computational framework to fully leverage the throughput, as well as the relatively sparse but multifaceted genotype information within each cell that includes strandedness, digital counting of copy numbers, and haplotype-aware chromosome segmentation, to systematically distinguish seven possible types of mitotic crossover outcomes. We showcase the power of sci-L3-Strand-seq by quantifying the rates of error-free and mutational crossovers in thousands of cells, enabling us to explore enrichment patterns of genomic and epigenomic features. The throughput of sci-L3-Strand-seq also gave us the ability to measure subtle phenotypes, opening the door for future large mutational screens. Furthermore, mapping clonal lineages provided insights into the temporal order of certain genome instability events, showcasing the potential to dissect cancer evolution. Altogether, we show the wide applicability of sci-L3-Strand-seq to the study of DNA repair and structural variations.

genomics↗

bioRxiv2025

VH-replacement shapes the antibody repertoire by targeting non-pairing heavy-chains

The diversity of antibodies underpins robust immune responses. During the formation of the antibody repertoire in early bone marrow B-cells, random antibody heavy-chain proteins are generated from recombined V, D and J gene-segments. Many are non- functional and are negatively selected. Critically, this phase of development is impacted by ageing and inflammation. To understand this process in normal mice we have undertaken the first in-depth analysis of heavy-chain selection at this pre-B cell transition. We find independent selection acting on three regions of the CDR3 antigen binding site, with particularly heavy counter-selection of certain productive VH/JH combinations. This led us to hypothesise that VH-replacement is targeting productive VDJ rearrangements that cannot pair with the surrogate light-chain (SLC). We detect VH-replacement recombination products that closely follow the pattern of counter- selection of productive VDJ. This reveals a physiological role for VH-replacement: the developmental release of B-cells that are stalled, with a productive but not SLC-pairing VDJ, leading to re-modelling of the early VDJ repertoire toward locus-distal VH that we show are more tolerant of CDR3 composition.

immunology↗

bioRxiv2020

RNA promotes the formation of spatial compartments in the nucleus

The nucleus is a highly organized arrangement of RNA, DNA, and protein molecules that are compartmentalized within three-dimensional (3D) structures involved in shared functional and regulatory processes. Although RNA has long been proposed to play a global role in organizing nuclear structure, exploring the role of RNA in shaping nuclear structure has remained a challenge because no existing methods can simultaneously measure RNA-RNA, RNA-DNA, and DNA-DNA contacts within 3D structures. To address this, we developed RNA & DNA SPRITE (RD-SPRITE) to comprehensively map the location of all RNAs relative to DNA and other RNAs. Using this approach, we identify many RNAs that are localized near their transcriptional loci (RNA-DNA) together with other diffusible ncRNAs (RNA-RNA) within higher-order DNA structures (DNA-DNA). These RNA-chromatin compartments span three major classes of nuclear functions: RNA processing (including ribosome biogenesis, mRNA splicing, snRNA biogenesis, and histone mRNA processing), heterochromatin assembly, and gene regulation. More generally, we identify hundreds of ncRNAs that form stable nuclear compartments in spatial proximity to their transcriptional loci. We find that dozens of nuclear compartments require RNA to guide protein regulators into these 3D structures, and focusing on several ncRNAs, we show that these ncRNAs specifically regulate heterochromatin assembly and the expression of genes contained within these compartments. Together, our results demonstrate a unique mechanism by which RNA acts to shape nuclear structure by forming high concentration territories immediately upon transcription, binding to diffusible regulators, and guiding them into spatial compartments to regulate a wide range of essential nuclear functions.

genomics↗

bioRxiv2019

Network analysis of promoter interactions reveals the hierarchical differences in genome organisation between human pluripotent states

SO_SCPCAPUMMARYC_SCPCAPA complex and poorly understood interplay between 3D genome organisation, transcription factors and chromatin state underpins cell identity. To gain a systems-level understanding of this interplay, we generated a high-resolution atlas of annotated chromatin interactions in naive and primed human pluripotent stem cells and developed a network-graph approach to examine the atlas at multiple spatial scales. Investigating chromatin interactions as a network uncovered highly connected hubs that changed substantially in interaction frequency and in transcriptional co-regulation between pluripotent states. Small hubs frequently merged to form larger networks in primed cells, often linked by newly-formed Polycomb-associated interactions. Importantly, we identified state-specific differences in enhancer activity and interactivity that corresponded with widespread reconfiguration of transcription factor binding and target gene expression. These findings provide multilayered insights into the gene regulatory control of human pluripotency and our systems-based network approach could be applied broadly to uncover new principles of 3D genome organisation.

genomics↗