Biology subjects

Cechova, M.

Publications and source records attributed to Cechova, M..

13 recordsLinked to original sources

bioRxiv2026

G-quadruplex structures act as a novel recognition motif for the meiosis-specific histone methyltransferase PRDM9

The histone methyltransferase PR domain containing protein 9 (PRDM9) is a key determinant of meiotic recombination in humans. It deposits activating histone marks thereby promoting recruitment of the meiotic recombination machinery. It recognizes DNA through a repetitive zinc-finger array that binds specific sequence motifs whose complementary G-rich strands can form DNA secondary structures, particularly G-quadruplexes (G4s). These may present an additional binding platform for PRDM9 and contribute to the formation of a chromatin environment permissive for meiotic recombination. We investigated the relationship between PRDM9 binding sites and G4 motifs using computational analyses of predicted and experimentally validated G4s and found that G4 motifs are among the most prevalent features at PRDM9 binding sites, with the strongest enrichment observed for highly stable G4s, largely independent of loop length. Using electrophoretic mobility shift assays, we further examined whether PRDM9 can bind short, single-stranded G4-forming oligonucleotides in addition to its canonical double-stranded DNA targets. PRDM9 directly bound folded G4 structures, and binding increased with G4 stability. This relationship was observed across different G4 motifs and following stabilization of the same G4 by increasing the potassium concentration or adding a G4-stabilizing ligand. PRDM9 also bound an artificial G4-forming sequence absent from the human genome, which was abolished when mutating the G4 motif to avoid structure formation. Together, these results support a model in which stable G4 structures facilitate PRDM9 recruitment by creating and discrete increased local chromatin accessibility, thereby contributing to the initiation of meiotic recombination. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/742039v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@428bdorg.highwire.dtl.DTLVardef@10ead06org.highwire.dtl.DTLVardef@8171c4org.highwire.dtl.DTLVardef@540459_HPS_FORMAT_FIGEXP M_FIG C_FIG

evolutionary biology↗

bioRxiv2026

Haplotype-resolved centromeric chromatin organization from a complete diploid human genome

Centromeres ensure proper chromosome segregation during cell division, yet the organization and regulation of centromeric chromatin within satellite DNA arrays remain incompletely understood. Here, we leverage the complete diploid human genome benchmark (T2T-HG002) to provide a detailed study of centromeric sequence and chromatin architecture on individual haplotypes. Using adaptive-sampling-enriched, ultra-long-read DiMeLo-seq, we achieve single-molecule chromatin profiling across all centromeres, revealing that along single chromatin fibers, CENP-A, the histone variant specifying centromere identity, forms multiple discrete subdomains within hypomethylated centromere dip regions (CDRs) that are flanked by H3K9me3-enriched heterochromatin. Despite underlying sequence variation, CDRs localize to sequence-homogeneous domains and maintain relatively balanced CENP-A dosage and aggregate length across all chromosomes and between haplotypes. Further, we show that bidirectional changes to centromeric and pericentromeric DNA methylation are accompanied by changes to centromeric chromatin architecture. In passaged cells with centromeric hypomethylation, subdomain boundaries are eroded, and adjacent CENP-A domains tend to merge and expand. Conversely, in pluripotent stem cells with centromeric hypermethylation, CDRs are fundamentally reorganized, such that discrete hypomethylated domains are frequently consolidated into broader contiguous tracts. These methylation-associated CDR restructuring events suggest that DNA methylation acts as a principal regulator of human centromere organization, with implications for understanding centromere plasticity, epigenetic inheritance, and chromosomal instability in development and disease.

genomics↗

bioRxiv2026

Fully T2T pedigree assemblies reveal genetic stability and epigenetic plasticity of human centromeres across inheritance and cell-fate transitions

Centromeres are essential chromosome components yet remain poorly understood due to their highly repetitive sequence architecture. Using fully-phased telomere-to-telomere diploid assemblies from a three-generation pedigree integrated with long-read epigenomes from matched peripheral blood mononuclear cells, induced pluripotent stem cells, and neural progenitor cells, we generate allele-resolved single basepair resolution maps of centromere genetic and epigenetic dynamics across inheritance, reprogramming, and differentiation. We show that centromeric dip regions (CDRs), which define the functional core of centromeres, are positionally stable across generations and cell-fate transitions. In contrast, CDR epigenetic architecture is highly dynamic. Reprogramming markedly attenuates CDR hypomethylation, which is partially restored during differentiation in parallel with global hypomethylation of active alpha-satellite arrays and coordinated changes in nucleosome organization and protein occupancy. Centromeric remodeling is insulated from X-chromosome status, including Xa, Xi, and erosion. Finally, de novo mutations arising during reprogramming are enriched in centromeric regions but depleted within functional centromeric cores.

genomics↗

bioRxiv2025

Complete genomes of a multi-generational pedigree to expand studies of genetic and epigenetic inheritance

Pedigree analysis remains the gold standard for rare disease diagnostics, yet whole genome sequencing studies typically omit critical regions like centromeres, telomeres, and acrocentric chromosome p-arms. Here, we present telomere-to-telomere (T2T) reference genomes for four self-identified African American individuals of admixed ancestry spanning three generations. Our parent-of-origin assigned, chromosome-level assemblies revealed precise meiotic recombination breakpoints in previously inaccessible regions, including recombination events across acrocentric and subtelomeric sequences. Centromeric regions were highly stable, with multi-megabase arrays inherited intact across three generations, while the position of kinetochore assembly sites remained consistent and predominantly associated with the p-arm proximal region. The relative lengths of telomeres on individual chromosomes were maintained across generations. Using a targeted rDNA assembly approach, we reconstructed a complete megabase-scale ribosomal DNA (rDNA) array corresponding to the paternal chromosome 14. This openly available pedigree provides a benchmark dataset for studying recombination and genetic and epigenetic variation across the complete genome.

genomics↗

bioRxiv2023

The Complete Sequence and Comparative Analysis of Ape Sex Chromosomes

Apes possess two sex chromosomes--the male-specific Y and the X shared by males and females. The Y chromosome is crucial for male reproduction, with deletions linked to infertility1. The X chromosome carries genes vital for reproduction and cognition2. Variation in mating patterns and brain function among great apes suggests corresponding differences in their sex chromosomes. However, due to their highly repetitive nature and incomplete reference assemblies, ape sex chromosomes have been challenging to study. Here, using the methodology developed for the telomere-to-telomere (T2T) human genome, we produced gapless assemblies of the X and Y chromosomes for five great apes (chimpanzee, bonobo, gorilla, Bornean and Sumatran orangutans) and a lesser ape, the siamang gibbon. These assemblies allowed us to untangle the intricacies of ape sex chromosome evolution. We found that, compared to the Xs, the ape Ys vary greatly in size and have low alignability and high levels of structural rearrangements. This divergence on the Y arises from the accumulation of lineage-specific ampliconic regions, palindromes, transposable elements, and satellites. Our analysis of Y chromosome genes revealed expansions of multi-copy gene families and signatures of purifying selection. Thus, the Y exhibits dynamic evolution, while the X is more stable. Mapping short-read sequencing data to these assemblies revealed diversity and selection patterns on sex chromosomes of >100 great ape individuals. These reference assemblies are expected to inform human evolution and conservation genetics of nonhuman apes, all of which are endangered species.

genomics↗

bioRxiv2022

The complete sequence of a human Y chromosome

The human Y chromosome has been notoriously difficult to sequence and assemble because of its complex repeat structure including long palindromes, tandem repeats, and segmental duplications1-3. As a result, more than half of the Y chromosome is missing from the GRCh38 reference sequence and it remains the last human chromosome to be finished4, 5. Here, the Telomere-to-Telomere (T2T) consortium presents the complete 62,460,029 base pair sequence of a human Y chromosome from the HG002 genome (T2T-Y) that corrects multiple errors in GRCh38-Y and adds over 30 million base pairs of sequence to the reference, revealing the complete ampliconic structures of TSPY, DAZ, and RBMY gene families; 41 additional protein-coding genes, mostly from the TSPY family; and an alternating pattern of human satellite 1 and 3 blocks in the heterochromatic Yq12 region. We have combined T2T-Y with a prior assembly of the CHM13 genome4 and mapped available population variation, clinical variants, and functional genomics data to produce a complete and comprehensive reference sequence for all 24 human chromosomes.

genomics↗

bioRxiv2022

Altered sequencing success at non-B-DNA motifs

Modern sequencing technologies are not error-free, and might have elevated error rates at some locations of the genome. A potential cause for such elevated error rates is the formation of alternative DNA structures (non-B DNA), such as G-quadruplexes (G4s), Z-DNA, or cruciform structures, during sequencing. Approximately 13% of the human genome has the potential to form such structures, which have been previously shown to affect the activity of DNA polymerases and helicases. Here we tested whether motifs with the potential to form non-B DNA (non-B motifs) influence the sequencing success of three major sequencing technologies--Illumina, Pacific Biosciences (PacBio) HiFi, and Oxford Nanopore Technologies (ONT). We estimated sequencing success by computing the rates of single-nucleotide, insertion, and deletion errors, as well as by evaluating mean read depth and mean base quality. Overall, all technologies exhibited altered sequencing success for most non-B motif types. Single-nucleotide error rates were generally increased for G-quadruplexes (G4s) and Z-DNA motifs in all three technologies. Illumina and PacBio HiFi deletion error rates were also increased for all non-B types except for Z-DNA motifs, while in ONT they were increased substantially only for G4 motifs. Insertion error rates for non-B motifs were highly elevated in Illumina, moderately elevated in PacBio HiFi, and only slightly elevated in ONT. Using Poisson regression modeling, we evaluated how non-B DNA motifs and other factors influence sequencing error profiles. Using the error rates at non-B motifs, we developed a probabilistic approach to determine the number of false-positive single-nucleotide variants (SNVs) in different sample size and variant frequency cutoff scenarios, as well as in previously generated sequencing data sets (1000Genomes, Simons Genome Diversity Project, and gnomAD). Overall, the effect of non-B DNA on sequencing should be considered in downstream analyses, particularly in studies with limited read depth--e.g., single-cell and ancient DNA sequencing, as well as sequencing of pooled population samples--and when scoring variants with low frequency (e.g., singletons). Because each sequencing technology analyzed has a unique error profile at non-B motifs, a combination of different technologies should be considered in future sequencing studies of such motifs, to maximize accuracy.

genomics↗

bioRxiv2021

HiC-TE: a computational pipeline for Hi-C data analysis shows a possible role of repeat family interactions in the genome 3D organization

The role of repetitive DNA in the 3D organization of the interphase nucleus in plant cells is a subject of intensive study. High-throughput chromosome conformation capture (Hi-C) is a sequencing-based method detecting the proximity of DNA segments in nuclei. We combined Hi-C data, plant reference genome data and tools for the characterization of genomic repeats to build a Nextflow pipeline identifying and quantifying the contacts of specific repeats revealing the preferential homotypic interactions of ribosomal DNA, DNA transposons and some LTR retrotransposon families. We provide a novel way to analyze the organization of repetitive elements in the 3D nucleus.

bioinformatics↗

bioRxiv2020

Dynamic evolution of great ape Y chromosomes

The mammalian male-specific Y chromosome plays a critical role in sex determination and male fertility. However, because of its repetitive and haploid nature, it is frequently absent from genome assemblies and remains enigmatic. The Y chromosomes of great apes represent a particular puzzle: their gene content is more similar between human and gorilla than between human and chimpanzee, even though human and chimpanzee shared a more recent common ancestor. To solve this puzzle, here we constructed a dataset including Ys from all extant great ape genera. We generated assemblies of bonobo and orangutan Ys, from short and long sequencing reads, and aligned them with the publicly available human, chimpanzee and gorilla Y assemblies. Analyzing this dataset, we found that the genus Pan, including chimpanzee and bonobo, experienced accelerated substitution rates. Additionally, Pan also exhibited elevated gene death rates. These observations are consistent with high levels of sperm competition in Pan. Furthermore, we inferred that the great ape common ancestor already possessed multi-copy sequences homologous to most human and chimpanzee palindromes. Nonetheless, each species also acquired distinct ampliconic sequences. We also detected increased chromatin contacts between and within palindromes (from Hi-C data), likely facilitating gene conversion and structural rearrangements. Moreover, our ENCODE data analysis suggested that Y palindromes exist to promote gene conversion preventing degradation of not only genes, as is commonly believed, but also gene regulatory sites. Our results highlight the dynamic mode of Y chromosome evolution, and open avenues for studies of male-specific dispersal in endangered great ape species.

evolutionary biology↗

bioRxiv2018

Noise-Cancelling Repeat Finder: Uncovering tandem repeats in error-prone long-read sequencing data

SummaryTandem DNA repeats can be sequenced with long-read technologies, but cannot be accurately deciphered due to the lack of computational tools taking high error rates of these technologies into account. Here we introduce Noise-Cancelling Repeat Finder (NCRF) to uncover putative tandem repeats of specified motifs in noisy long reads produced by Pacific Biosciences and Oxford Nanopore sequencers. Using simulations, we validated the use of NCRF to locate tandem repeats with motifs of various lengths and demonstrated its superior performance as compared to two alternative tools. Using real human whole-genome sequencing data, NCRF identified long arrays of the (AATGG)n repeat involved in heat shock stress response.\n\nAvailability and implementationNCRF is implemented in C, supported by several python scripts. Source code, under the MIT open source license, and simulation data are available at https://github.com/makovalab-psu/NoiseCancellingRepeatFinder, and also in bioconda.

bioinformatics↗

bioRxiv2018

High inter- and intraspecific turnover of satellite repeats in great apes

BackgroundSatellite repeats are a structural component of centromeres and telomeres, and in some instances their divergence is known to drive speciation. Due to their highly repetitive nature, satellite sequences have been understudied and underrepresented in genome assemblies. To investigate their turnover in great apes, we studied satellite repeats in human, chimpanzee, bonobo, gorilla, and Sumatran and Bornean orangutans, using unassembled short and long sequencing reads.\n\nResultsThe density of satellite repeats, as identified from accurate short reads (Illumina), varied greatly among great ape genomes. These were dominated by a handful of abundant repeated motifs, frequently shared among species, which formed two groups: (1) the (AATGG)n repeat (critical for heat shock response) and its derivatives; and (2) subtelomeric 32-mers. Using the densities of abundant repeats, individuals could be classified into species. However clustering did not reproduce the accepted species phylogeny, suggesting rapid repeat evolution. Several abundant repeats were enriched in males vs. females; using existing Y chromosome assemblies or FIuorescent In Situ Hybridization, we validated location for some of them on the Y. Finally, applying a novel computational tool, we identified many satellite repeats completely embedded within long Oxford Nanopore and Pacific Biosciences reads. Such repeats were up to 59 kb in length and consisted of perfect repeats interspersed with other similar sequences.\n\nConclusionsOur results based on sequencing reads generated with three different technologies provide the first detailed characterization of great ape satellite repeats, and open new avenues for exploring their functions.

evolutionary biology↗

bioRxiv2017

Non-B DNA affects polymerization speed and error rate in sequencers and living cells

DNA conformation may deviate from the classical B-form in ~13% of the human genome. Non-B DNA regulates many cellular processes; however, its effects on DNA polymerization speed and accuracy have not been investigated genome-wide. Such an inquiry is critical for understanding neurological diseases and cancer genome instability. Here we present the first simultaneous examination of DNA polymerization kinetics and errors in the human genome sequenced with Single-Molecule-Real-Time technology. We show that polymerization speed differs between non-B and B-DNA: it decelerates at G-quadruplexes and fluctuates periodically at disease-causing tandem repeats. Analyzing polymerization kinetics profiles, we predict and validate experimentally non-B DNA formation for a novel motif. We demonstrate that several non-B motifs affect sequencing errors (e.g., G-quadruplexes increase error rates) and that sequencing errors are positively associated with polymerase slowdown. Finally, we show that highly divergent G4 motifs have pronounced polymerization slowdown and high sequencing error rates, suggesting similar mechanisms for sequencing errors and germline mutations.

genomics↗

bioRxiv2017

RecoverY : K-mer based read classification for Y-chromosome specific sequencing and assembly

MotivationThe haploid mammalian Y chromosome is usually under-represented in genome assemblies due to high repeat content and low depth due to its haploid nature. One strategy to ameliorate the low coverage of Y sequences is to experimentally enrich Y-specific material before assembly. Since the enrichment process is imperfect, algorithms are needed to identify putative Y-specific reads prior to downstream assembly. A strategy that uses k-mer abundances to identify such reads was used to assemble the gorilla Y (Tomaszkiewicz et al 2016). However, the strategy required the manual setting of key parameters, a time-consuming process leading to sub-optimal assemblies.\n\nResultsWe develop a method, RecoverY, that selects Y-specific reads by automatically choosing the abundance level at which a k-mer is deemed to originate from the Y. This algorithm uses prior knowledge about the Y chromosome of a related species or known Y transcript sequences. We evaluate RecoverY on both simulated and real data, for human and gorilla, and investigate its robustness to important parameters. We show that RecoverY leads to a vastly superior assembly compared to alternate strategies of filtering the reads or contigs. Compared to the preliminary strategy used in Tomaszkiewicz et al (2016), we achieve a 33% improvement in assembly size and a 20% improvement in the NG50, demonstrating the power of automatic parameter selection.\n\nAvailabilityOur tool RecoverY is freely available at https://github.com/makovalab-psu/RecoverY\n\nContactkmakova@bx.psu.edu, pashadag@cse.psu.edu\n\nSupplementary informationAttached as an additional file.

bioinformatics↗