Biology subjects

McDonald, T. L.

Publications and source records attributed to McDonald, T. L..

8 recordsLinked to original sources

bioRxiv2026

Fiber-TEnCATS reveals haplotype-specific chromatin accessibility and DNA methylation at human L1HS loci

Human-specific long interspersed nuclear element-1 (L1HS) is an active and autonomous retrotransposon in the human genome. Changes in its transcription and transposition are known to affect cellular processes involved in development and aging, and diseases such as neurological disorders and cancer. To better understand natural variability in epigenetic patterns that affect L1HS regulation, we developed a targeted long-read method to simultaneously profile individual haplotypes for DNA methylation and chromatin accessibility across L1HS loci in a healthy human cell line trio. We show that the intronic L1HS in the ZNF638 gene consistently displays high chromatin accessibility and DNA hypomethylation with bidirectional transcription. Our approach also reveals additional intronic and intergenic L1HS copies with allele-specific chromatin accessibility and methylation, and instances of reduced promoter DNA methylation that does not correspond with increased chromatin accessibility. We also identify potential cases of non-Mendelian inheritance of DNA methylation patterns over a subset of L1HS promoters. Our methods high coverage over L1HS loci enables detection and profiling of loci that are missed even by long-read-based assemblies and enables more accurate inheritance tracing of L1HS insertions. Overall, our results offer new insights into the locus-specific regulation of both reference and non-reference L1HS within the human genome.

genomics↗

bioRxiv2025

Multi-platform framework for mapping somatic retrotransposition in human tissues

Mobile element insertions (MEI) shape the human genome in both germline and somatic tissues. While inherited MEIs are well characterized, mapping somatic MEIs (sMEI) in non-cancer tissues remains challenging due to their low allelic fraction and repetitive nature. We established an integrative framework for sMEI analysis leveraging modern sequencing technologies and analytical innovations. We first benchmarked sMEI detection and demonstrated advantages of long-read and MEI-targeted sequencing for ultra-low-frequency events using a mixture of well-established cell lines. We then showed that haplotype phasing and donor-specific assemblies refine sMEI detection, effectively distinguishing from germline and false signals in in-silico tumor-normal mixtures. We further developed a source-tracing strategy based on internal sequence variation, expanding the catalogue of active source elements beyond traditional transduction-based methods. Applying this framework to donor tissues, we identified 18 rare somatic L1 insertions, revealing structural and source diversity. Our work provides a foundational framework and biological insight into sMEIs.

genomics↗

bioRxiv2024

A personalized multi-platform assessment of somatic mosaicism in the human frontal cortex

Somatic mutations in individual cells create genomic mosaicism, influencing genetic disorders and cancers. While clonal mutations in cancers are well-studied, rarer somatic variants in normal tissues remain poorly characterized. This study systematically evaluates detection methods using a personalized donor-specific assembly (DSA) from a neurotypical individuals dorsolateral prefrontal cortex assessed with Oxford Nanopore, NovaSeq, linked-read sequencing, Cas9-targeted long-read sequencing (TEnCATS), and single-neuron MALBAC amplification. The haplotype-resolved DSA improved cross-platform analysis, dramatically increasing phasing rates. Germline SNVs, structural variations (SVs), and transposable elements (TEs) were recalled with 99.4%-99.7% accuracy in bulk tissue, and phased haplotype analysis reduced false positives by 15.4%-75.1% for putative somatic candidates. Long-read single-neuron sequencing detected nine somatic SV candidates, demonstrating enhanced sensitivity for rare variants, while TEnCATS identified eight low-frequency somatic TE candidates. These findings highlight advanced methodologies for precise somatic variant detection, critical for understanding mosaicisms role in health and disease.

genomics↗

bioRxiv2023

Relating polar bears killed, human presence, and ice conditions in Svalbard 1987-2019

Conflicts between humans and polar bears have been predicted to increase as polar bear prime habitat, sea ice, is decreasing. In Svalbard, a strong protection and strict control schemes have secured near complete records of bears killed and found dead since 1987. We analyzed the trend in the number of kills and related this to human visitation to the island. We found a slight decrease in the number of kills in the period 1987-2019, and a decrease in per capita number of kills when monthly kills were compared to the monthly number of visitors disembarking in the main settlement. We then used a discrete choice resource selection model to assess whether polar bear kill events are related to attributes of the kill sites and environmental conditions at the time. We divided Svalbard in four sectors, North, East, South, and West, and monthly average ice cover was calculated in 25-km rings around Svalbard, rings that were further delineated by the four sectors. We found that the odds of a kill was greater along the shoreline, and that the odds would be reduced by 50% when moving only 900 m from the shoreline when all sectors were included. Distance from other covariates like settlements, trappers cabins, and landing sites for tourists did for the most part not have a significant impact on the odds of a kill. Sectorwise, ice cover had no significant impact on the odds for a kill. The decreasing trend in kills of polar bears might partly be explained by the success of strict protection and management regimes of Svalbard wilderness.

ecology↗

bioRxiv2022

On-Ramp: a tool for rapid, multiplexed validation of plasmids using nanopore sequencing

Recombinant plasmid vectors are versatile tools which have facilitated discoveries in molecular biology, genetics, proteomics, and many other fields. As the enzymatic and bacterial processes used to create recombinant DNA can introduce errors, sequence validation is an essential step in plasmid assembly. Sanger sequencing is the current standard for plasmid validation, however this method is limited by an inability to sequence through complex secondary structure and lacks scalability when applied to full-plasmid sequencing of multiple plasmids due to read-length limits. While next-generation sequencing (NGS) does provide full-plasmid sequencing at scale, it is impractical and costly when utilized outside of library-scale validation. Here we present OnRamp (Oxford nanopore-based Rapid Analysis of Multiplexed Plasmids), an alternative method for routine plasmid validation which combines the advantages of NGSs full plasmid coverage and scalability with Sangers affordability and accessibility by leveraging nanopores novel long-read sequencing technology. We include customized wet-lab protocols for plasmid preparation along with a pipeline designed for analysis of read data obtained using these protocols. This analysis pipeline is built into the OnRamp webapp (http://OnRamp.BoyleLab.org), which generates alignments between actual and predicted plasmid sequences, quality scores, and read-level views in a user-friendly manner, precluding the need for programming experience in analyzing nanopore results. Here we describe the OnRamp protocols and pipeline, and demonstrate our ability to obtain full sequences from pooled plasmids while detecting sequence variation even in regions of high secondary structure, at less than half the cost of equivalent Sanger sequencing.

molecular biology↗

bioRxiv2021

Cas9 targeted enrichment of mobile elements using nanopore sequencing

Mobile element insertions (MEIs) are highly repetitive genomic sequences that contribute to inter- and intra-individual genetic variation and can lead to genetic disorders. Targeted and whole-genome approaches using short-read sequencing have been developed to identify reference and non-reference MEIs; however, the read length hampers detection of these elements in complex genomic regions. Here, we pair Cas9 targeted nanopore sequencing with computational methodologies to capture active MEIs in human genomes. We demonstrate parallel enrichment for distinct classes of MEIs, averaging 44% of reads on targeted signals. We show an individual flow cell can recover a remarkable fraction of MEIs (97% L1Hs, 93% AluYb, 51% AluYa, 99% SVA_F, and 65% SVA_E). We identify twenty-one non-reference MEIs in GM12878 overlooked by modern, long-read analysis pipelines, primarily in repetitive genomic regions. This work introduces the utility of nanopore sequencing for MEI enrichment and lays the foundation for rapid discovery of elusive, repetitive genetic elements.

genetics↗

bioRxiv2020

The inducible lac operator-repressor system is functional in zebrafish cells

BackgroundZebrafish are a foundational model organism for studying the spatio-temporal activity of genes and their regulatory sequences. A variety of approaches are currently available for editing genes and modifying gene expression in zebrafish, including RNAi, Cre/lox, and CRISPR-Cas9. However, the lac operator-repressor system, a component of the E. coli lac operon which has been adapted for use in many other species and is a valuable, flexible tool for studying the inducible modulation of gene expression, has not previously been tested in zebrafish. ResultsHere we demonstrate that the lac operator-repressor system robustly decreases expression of firefly luciferase in cultured zebrafish fibroblast cells. Our work establishes the lac operator-repressor system as a promising tool for the manipulation of gene expression in whole zebrafish. ConclusionsOur results lay the groundwork for the development of lac-based reporter assays in zebrafish, and adds to the tools available for investigating dynamic gene expression in embryogenesis. We believe that this work will catalyze the development of new reporter assay systems to investigate uncharacterized regulatory elements and their cell-type specific activities.

molecular biology↗

bioRxiv2019

finFindR: Computer-assisted Recognition and Identification of Bottlenose Dolphin Photos in R

O_LIPhotographic identification is an essential research and management tool for studying population size and dynamics of common bottlenose dolphins (Tursiops truncatus). Photographic identification involves recognizing individuals based on unique dorsal fin markings. Manual identification of dolphins, while successful, is labor-intensive and time-consuming. To shorten processing times, we developed a series of neural networks that finds fins, assesses their unique characteristics, and matches them to an existing catalog.\nC_LIO_LIOur software, finFindR, shortens photo-ID processing times by autonomously finding and isolating (i.e., \"cropping\") dolphin fins in raw field photographs, tracing the trailing edge of fins in cropped images, and producing a sorted list of likely identities from a catalog of known individuals. The program then presents users with the top 50 most likely matching identities, allowing users to view side-by-side image pairs and make final identity determinations.\nC_LIO_LIDuring testing on two sets of novel images, finFindR placed the correct individual in the first position of its ordered list in 88% (238/272 and 354/400) of test cases. finFindR placed the correct identity among the top 10 ranked images in 94% of test cases, and among the top 50 ranked images in 97% of test cases. Hence, if a match does not exist in the first 50 images of finFindRs ordered list, researchers can be almost certain (~97%) that a match does not exist in the entire catalog.\nC_LIO_LIDuring a head-to-head blind test of the human-only and finFindR-assisted matching methods, two experienced photo-ID technicians both achieved 97% correct identification of identities when matched against a catalog containing over 2,000 known individuals. However, the manual-only technician examined 124 images on average before making a match, while the technician using finFindR examined only 10 images on average before finding a match.\nC_LIO_LIWe conclude that finFindR will facilitate equal or improved match accuracy while greatly reducing the number of examined photos. The faster matches, automated detection, and automated cropping afforded by finFindR will greatly reduce typical photo-ID processing times.\nC_LI

bioinformatics↗