Biology subjects

Logsdon, G.

Publications and source records attributed to Logsdon, G..

5 recordsLinked to original sources

bioRxiv2026

Protein-only centromeric chromatin assembly streamlines human artificial chromosome formation

Human artificial chromosomes (HACs) are inherited through cell divisions alongside natural chromosomes, serving as tools for interrogating chromosomal elements and as vectors for large genetic cargoes (1-5). Despite recent progress (6-8), large (i.e., multiple Mb) HACs have not been reported. Further, HAC formation via epigenetic seeding of centromeric chromatin currently requires prior engineering of recipient human cells (6-8), hampering their potential deployment in many useful cell types. Here, we designed, built, and delivered to human cells a 2 Mb HAC construct that is [~]3 times larger than the prior generation. We also report a robust epigenetic centromere seeding approach that initiates immediately upon delivery to the human cell cytoplasm and bypasses genetic engineering of target cells. The HACs are then faithfully inherited in the absence of selection. Thus, formation of functional centromeric chromatin in the same cell cycle of HAC delivery drives high efficiency HAC formation. TeaserA system for efficient protein-only centromere seeding permits visualizing the first step of HAC formation

synthetic biology↗

bioRxiv2023

RAmbler: de novo genome assembly of complex repetitive regions

Complex repetitive regions (also called segmental duplications) in eukaryotic genomes often contain essential functional and regulatory information. Despite remarkable algorithmic progress in genome assembly in the last twenty years, modern de novo assemblers still struggle to accurately reconstruct these highly repetitive regions. When sequenced reads will be long enough to span all repetitive regions, the problem will be solved trivially. However, even the third generation of sequencing technologies on the market cannot yet produce reads that are sufficiently long (and accurate) to span every repetitive region in large eukaryotic genomes. In this work, we introduce a novel algorithm called RAmbler to resolve complex repetitive regions based on high-quality long reads (i.e., PacBio HiFi). We first identify repetitive regions by mapping the HiFi reads to the draft genome assembly and by detecting un-usually high mapping coverage. Then, (i) we compute the k-mers that are expected to occur only once in the genome (i.e., single copy k-mers, which we call unikmers), (ii) we barcode the HiFi reads based on the presence and the location of their unikmers, (iii) we compute an overlap graph solely based on shared barcodes, (iv) we reconstruct the sequence of the repetitive region by traversing the overlap graph. We present an extensive set of experiments comparing the performance of RAmbler against Hifiasm, HiCANU and Verkko on synthetic HiFi reads generated over a wide range of repeat lengths, number of repeats, heterozygosity rates and depth of sequencing (over 140 data sets). Our experimental results indicate that RAmbler outperforms Hifiasm, HiCANU and Verkko on the large majority of the inputs. We also show that RAmbler can resolve several long tandem repeats in Arabidopsis thaliana using real HiFi reads. The code for RAmbler is available at https://github.com/sakshar/rambler. CCS CONCEPTSApplied computing [->] Bioinformatics; Computational genomics; Molecular sequence analysis; * Theory of computation [->] Graph algorithms analysis.

bioinformatics↗

bioRxiv2023

Conservation of chromatin organization within human and primate centromeres

The focal attachment of the kinetochore to the centromere is essential for genome maintenance, yet the highly repetitive nature of satellite regional centromeres, such as those in humans, limits our understanding of their chromatin organization. We demonstrate that single-molecule chromatin fiber sequencing (Fiber-seq) can uniquely co-resolve kinetochore and surrounding chromatin architectures along point centromeres, revealing largely homogeneous single-molecule kinetochore occupancy along each chromosome. In contrast, extension of Fiber-seq to regional satellite centromeres exposed marked per-molecule heterogeneity in their chromatin organization. Regional CENP-A-marked centromere cores uniquely contain a dichotomous chromatin organization (dichromatin) composed of compacted nucleosome arrays punctuated with highly accessible chromatin patches. CENP-B occupancy phases dichromatin to the underlying alpha-satellite repeat within centromere cores, but is not necessary for dichromatin formation. Centromere core dichromatin is a conserved feature between humans despite the marked divergence of their underlying alpha-satellite organization and is similarly a conserved feature along regional centromeres that lack satellite repeats in gibbon. Overall, the chromatin organization of regional centromeres is defined by marked per-molecule heterogeneity, likely buffering kinetochore attachment against sequence and structural variability within regional centromeres. HighlightsO_LIDichotomous accessible and compacted chromatin (dichromatin) marks centromere cores C_LIO_LIHighly accessible chromatin patches punctuate sites of kinetochore attachment C_LIO_LIDichromatin can form irrespective of CENP-B occupancy C_LIO_LIConservation within centromeres is mediated at the level of chromatin, not DNA C_LI

genomics↗

bioRxiv2021

Complete genomic and epigenetic maps of human centromeres

Existing human genome assemblies have almost entirely excluded highly repetitive sequences within and near centromeres, limiting our understanding of their sequence, evolution, and essential role in chromosome segregation. Here, we present an extensive study of newly assembled peri/centromeric sequences representing 6.2% (189.9 Mb) of the first complete, telomere-to-telomere human genome assembly (T2T-CHM13). We discovered novel patterns of peri/centromeric repeat organization, variation, and evolution at both large and small length scales. We also found that inner kinetochore proteins tend to overlap the most recently duplicated subregions within centromeres. Finally, we compared chromosome X centromeres across a diverse panel of individuals and uncovered structural, epigenetic, and sequence variation at single-base resolution across these regions. In total, this work provides an unprecedented atlas of human centromeres to guide future studies of their complex and critical functions as well as their unique evolutionary dynamics. One-sentence summaryDeep characterization of fully assembled human centromeres reveals their architecture and fine-scale organization, variation, and evolution.

genomics↗

bioRxiv2021

Epigenetic Patterns in a Complete Human Genome

The completion of the first telomere-to-telomere human genome, T2T-CHM13, enables exploration of the full epigenome, removing limitations previously imposed by the missing reference sequence. Existing epigenetic studies omit unassembled and unmappable genomic regions (e.g. centromeres, pericentromeres, acrocentric chromosome arms, subtelomeres, segmental duplications, tandem repeats). Leveraging the new assembly, we were able to measure enrichment of epigenetic marks with short reads using k-mer assisted mapping methods. This granted array-level enrichment information to characterize the epigenetic regulation of these satellite repeats. Using nanopore sequencing data, we generated base level maps of the most complete human methylome ever produced. We examined methylation patterns in satellite DNA and revealed organized patterns of methylation along individual molecules. When exploring the centromeric epigenome, we discovered a distinctive dip in centromere methylation consistent with active sites of kinetochore assembly. Through long-read chromatin accessibility measurements (nanoNOMe) paired to CUT&RUN data, we found the hypomethylated region was extremely inaccessible and paired to CENP-A/B binding. With long-reads we interrogated allele-specific, longrange epigenetic patterns in complex macro-satellite arrays such as those involved in X chromosome inactivation. Using the single molecule measurements we can clustered reads based on methylation status alone distinguishing epigenetically heterogeneous and homogeneous areas. The analysis provides a framework to investigate the most elusive regions of the human genome, applying both long and short-read technology to grant new insights into epigenetic regulation.

genomics↗