Biology subjects

Parmalee, N. L.

Publications and source records attributed to Parmalee, N. L..

4 recordsLinked to original sources

bioRxiv2025

Comprehensive benchmarking of somatic single-nucleotide variant and indel detection at ultra-low allele fractions using short- and long-read data

Mosaic mutations in normal tissues occur at low variant allele fractions (VAFs), complicating detection. To benchmark strategies, the SMaHT Network created a cell-line mixture (1:49) and produced ultra-deep whole-genome sequencing using short and long reads (five centers, 180-500x each). We assembled a reference of 44,008 mosaic SNVs and 2,059 Indels, cross-validation between platforms to expose limits of short-read analysis. We also partitioned the genome by mappability to examine the impact of genomic context, added a negative reference set, and accounted for culture-derived mutations. When seven institutions applied eleven algorithms to mixture data, call sets were largely discordant across tools and replicates, partly reflecting stochastic presence of low-VAF mutations in biological replicants. For >2% VAF SNVs, sensitivity and precision approached [~]80% at [≥]300x, with little gain from additional sequencing. This work provides a comprehensive framework for reliable detection of low-VAF mutations in non-cancer tissues and a valuable resource for the community.

bioinformatics↗

bioRxiv2025

A telomere-to-telomere map of somatic mutation burden and functional impact in cancer

Oncogenesis involves widespread genetic and epigenetic alterations, yet the full spectrum of somatic variation genome-wide remains unresolved. We generated a near-telomere-to-telomere (T2T) diploid assembly of a donor paired with deep short- and long-read sequencing of their melanoma. This revealed that 16% of somatic variants occur in sequences absent from GRCh38, with satellite repeats acting as hotspots for UV-induced damage due to sequence-intrinsic mutability and inefficient repair. Centromere kinetochore domains emerged as focal sites of structural, genetic, and epigenetic variation, leading to remodeling of centromere kinetochore binding domains during tumor evolution. Single-molecule telomere reconstructions uncovered cycles of attrition, deletion, and telomerase-mediated extension that shape cancer telomeres. Finally, diploid chromatin maps exposed that copy number alterations and epimutations, rather than point mutations, predominate in rewiring cancer regulatory programs. These findings define the full landscape of a cancers somatic variation and their functional impact, establishing a blueprint for T2T studies of mosaicism.

genomics↗

bioRxiv2024

Deaminase-assisted single-molecule and single-cell chromatin fiber sequencing

Gene regulation is mediated by the co-occupancy of numerous proteins along individual chromatin fibers. However, our tools for deeply profiling how proteins co-occupy individual fibers, especially at the single-cell level, remain limited. We present Deaminase-Assisted single-molecule chromatin Fiber sequencing (DAF-seq), which leverages a non-specific double-stranded DNA deaminase toxin A (SsDddA) to efficiently stencil protein occupancy along DNA molecules via selective deamination of accessible cytidines, which are preserved via C-to-T transitions upon DNA amplification. We demonstrate that DAF-seq enables [~]200,000-fold enrichment of target loci for single-molecule footprinting at near single-nucleotide resolution, enabling the precise delineation of the regulatory logic guiding neighboring proteins to cooperatively occupy chromatin fibers. Furthermore, DAF-seq enables the synchronous identification of single-molecule chromatin and genetic architectures - resolving the functional impact of rare somatic variants, as well as transitional chromatin states guiding haplotype-selective promoter actuation. Finally, we demonstrate that single-cell DAF-seq enables the accurate reconstruction of the diploid genome and epigenome from a single cell, revealing that a cells accessible regulatory landscape can diverge by as much as 63% while still retaining the cells identity. Overall, DAF-seq enables the comprehensive characterization of protein occupancy and chromatin accessibility across entire chromosomes with single-nucleotide, single-molecule, single-haplotype, and single-cell precision.

genomics↗

bioRxiv2024

A haplotype-resolved view of human gene regulation

Diploid human cells contain two non-identical genomes, and differences in their regulation underlie human development and disease. We present Fiber-seq Inferred Regulatory Elements (FIRE) and show that FIRE provides a more comprehensive and quantitative snapshot of the accessible chromatin landscape across the 6 Gbp diploid human genome, overcoming previously unrecognized biases in existing regulatory element catalogs. FIRE enables comprehensive detection of haplotype-selective chromatin accessibility (HSCA), exposing novel imprinted elements lacking underlying parent-of-origin CpG methylation differences, and gene regulatory modules that permit genes to escape X chromosome inactivation. We uncover that the human leukocyte antigen (HLA) locus harbors the most HSCA in immune cells, where we resolve specific transcription factor (TF) binding events disrupted by disease-associated variants. Finally, we demonstrate that the regulatory landscape of a cell is littered with autosomal somatic chromatin epimutations that are propagated by clonal expansions to create mitotically stable and non-genetically deterministic chromatin alterations.

genomics↗