Biology subjects

Gresham, D.

Publications and source records attributed to Gresham, D..

26 records · Page 2Linked to original sources

bioRxiv2021

Fluctuating environments maintain genetic diversity through neutral fitness effects and balancing selection

Genetic variation is the raw material upon which selection acts. The majority of environmental conditions change over time and therefore may result in variable selective effects. How temporally fluctuating environments impact the distribution of fitness effects and in turn population diversity is an unresolved question in evolutionary biology. Here, we employed continuous culturing using chemostats to establish environments that switch periodically between different nutrient limitations and compared the dynamics of selection to static conditions. We used the pooled Saccharomyces cerevisiae haploid gene deletion collection as a synthetic model for populations comprising thousands of unique genotypes. Using barcode sequencing (barseq), we find that static environments are uniquely characterized by a small number of high fitness genotypes that rapidly dominate the population leading to dramatic decreases in genetic diversity. By contrast, fluctuating environments are enriched in genotypes with neutral fitness effects and an absence of extreme fitness genotypes contributing to the maintenance of genetic diversity. We also identified a unique class of genotypes whose frequencies oscillate sinusoidally with a period matching the environmental fluctuation. Oscillatory behavior corresponds to large differences in short term fitness that are not observed across long timescales pointing to the importance of balancing selection in maintaining genetic diversity in fluctuating environments. Our results are consistent with a high degree of environmental specificity in the distribution of fitness effects and the combined effects of reduced and balancing selection in maintaining genetic diversity in the presence of variable selection.

evolutionary biology↗

bioRxiv2019

Nanopore sequencing undergoes catastrophic sequence failure at inverted duplicated DNA sequences

Inverted duplicated sequences are a common feature of structural variants (SVs) and copy number variants (CNVs). Analysis of CNVs containing inverted duplicated sequences using nanopore sequencing identified recurrent aberrant behavior characterized by incorrect and low confidence base calls that result from a systematic elevation in the current recorded by the sequencing pore. The coincidence of inverted duplicated sequences with catastrophic sequence failure suggests that secondary DNA structures may impair transit through the nanopore.

genomics↗

bioRxiv2019

Microbial community structure and ecology in sediments of a pristine mangrove forest

Mangrove forests are coastal intertidal ecosystems, characterized by mangrove trees growing in slow moving saline waters, that constitute a large portion of the coastline in the tropical and subtropical regions. The dynamic water regime created by the tides results in different microhabitats in which microbial communities play an essential role in the functioning and maintenance of the mangrove ecosystem. However, little is known about the diversity of taxa within these micro-habitats and their functional roles, as only a small fraction of these organisms can be cultured in the laboratory. In this study, we characterized the microbial community present in three distinct regions of mangrove sediments from the Serinhaem estuary, part of the Atlantic Forest biome within the Environmental Protection Area of Pratigi. We sampled sediments from regions below the tidal waterline (submerged), intertidal regions (intertidal), and regions above the tidal waterline (seco). More than 85% of all the sequences in the samples belonged to 6 of 42 identified phyla: Proteobacteria (30.6%), Firmicutes (30%), Chloroflexi (8.7%), Planctomycetes (5.7%), Crenarchaeota (5.4%) and Actinobacteria (5.3%). Diversity indices show that the submerged regions of the mangrove forest exhibit the greatest diversity and richness relative to the other regions. Notably, the intertidal region has the least diversity, suggesting that the dynamics of environmental variables in this region has an important influence on microbial diversity. Furthermore, distance metrics indicate that submerged sediments are more homogeneous while the seco region exhibits greater variability between locations. Finally, we found that the most abundant microbial families in the sediments are associated with nutrient cycling consistent with the essential role of the microbiome in maintaining the health of the mangrove ecology.

microbiology↗

bioRxiv2019

Gene regulatory network reconstruction using single-cell RNA sequencing of barcoded genotypes in diverse environments

Understanding how gene expression programs are controlled requires identifying regulatory relationships between transcription factors and target genes. Gene regulatory networks are typically constructed from gene expression data acquired following genetic perturbation or environmental stimulus. Single-cell RNA sequencing (scRNAseq) captures the gene expression state of thousands of individual cells in a single experiment, offering advantages in combinatorial experimental design, large numbers of independent measurements, and accessing the interaction between the cell cycle and environmental responses that is hidden by population-level analysis of gene expression. To leverage these advantages, we developed a method for transcriptionally barcoding gene deletion mutants and performing scRNAseq in budding yeast (Saccharomyces cerevisiae). We pooled diverse genotypes in 11 different environmental conditions and determined their expression state by sequencing 38,285 individual cells. We developed, and benchmarked, a framework for learning gene regulatory networks from scRNAseq data that incorporates multitask learning and constructed a global gene regulatory network comprising 12,018 interactions. Our study establishes a general approach to gene regulatory network reconstruction from scRNAseq data that can be employed in any organism.

genomics↗

bioRxiv2018

Single-cell copy number variant detection reveals the dynamics and diversity of adaptation

Copy number variants (CNVs) are a pervasive, but understudied source of genetic variation and evolutionary potential. Long-term evolution experiments in chemostats provide an ideal system for studying the molecular processes underlying CNV formation and the temporal dynamics of de novo CNVs. Here, we developed a fluorescent reporter to monitor gene amplifications and deletions at a specific locus with single-cell resolution. Using a CNV reporter in nitrogen-limited chemostats, we find that GAP1 CNVs are repeatedly generated and selected during the early stages of adaptive evolution resulting in predictable dynamics of CNV selection. However, subsequent diversification of populations defines a second phase of evolutionary dynamics that cannot be predicted. Using whole genome sequencing, we identified a variety of GAP1 CNVs that vary in size and copy number. Despite GAP1s proximity to tandem repeats that facilitate intrachromosomal recombination, we find that non-allelic homologous recombination (NAHR) between flanking tandem repeats occurs infrequently. Rather, breakpoint characterization revealed that for at least 50% of GAP1 CNVs, origin-dependent inverted-repeat amplification (ODIRA), a DNA replication mediated process, is the likely mechanism. We also find evidence that ODIRA generates DUR3 CNVs, indicating that it may be a common mechanism of gene amplification. We combined the CNV reporter with barcode lineage tracking and found that 103-104 independent CNV-containing lineages initially compete within populations, which results in extreme clonal interference. Our study introduces a novel means of studying CNVs in heterogeneous cell populations and provides insight into the underlying dynamics of CNVs in evolution.

evolutionary biology↗

bioRxiv2018

Global analysis of gene expression dynamics identifies factors required for accelerated mRNA degradation

Cellular responses to changing environments frequently involve rapid reprogramming of the transcriptome. Regulated changes in mRNA degradation rates can accelerate reprogramming by clearing or stabilizing extant transcripts. Here, we measured mRNA stability using 4-thiouracil labeling in the budding yeast Saccharomyces cerevisiae during a nitrogen upshift and found that 78 mRNAs are subject to destabilization. These transcripts include Nitrogen Catabolite Repression (NCR) and carbon metabolism mRNAs, suggesting that mRNA destabilization is a mechanism for targeted reprogramming. To explore the molecular basis of destabilization we implemented a SortSeq approach to screen using the pooled deletion collection library for trans factors that mediate rapid GAP1 mRNA repression. We combined low-input multiplexed Barcode sequencing with branched-DNA single-molecule mRNA FISH and Fluorescence-activated cell sorting (BFF) to identify that the Lsm1-7p/Pat1p complex and general mRNA decay machinery are important for GAP1 mRNA clearance. We also find that the decapping modulator SCD6, translation factor eIF4G2, and the 5 UTR of GAP1 are important for this repression, suggesting that translational control may impact the post-transcriptional fate of mRNAs in response to environmental changes.

genetics↗

bioRxiv2017

Adaptive Evolution Of Transcription Factor Binding Affinities

Understanding the molecular basis of gene expression evolution is a central problem in evolutionary biology. However, connecting changes in gene expression to increased fitness, and identifying the functional basis of those changes, remains challenging. To study adaptive evolution of gene expression in real time, we performed long term experimental evolution (LTEE) of Saccharomyces cerevisiae (budding yeast) in ammonium-limited chemostats. Following several hundred generations of continuous selection we found significant divergence of nitrogen-responsive gene expression in lineages with increased fitness. In multiple independent lineages we found repeated selection for non-synonymous mutations in the zinc finger DNA binding domain of the activating transcription factor (TF), GAT1, that operates within incoherent feedforward loops to control expression of the nitrogen catabolite repression (NCR) regulon. Missense mutations in the DNA binding domain of GAT1 reduce its binding affinity for the GATAA consensus sequence in a promoter-specific manner, resulting in increased expression of ammonium permease genes via both direct and indirect effects, thereby conferring increased fitness. We find that altered transcriptional output of the NCR regulon results in antagonistic pleiotropy in alternate environments and that the DNA binding domain of GAT1 is subject to purifying selection in natural populations. Our study shows that adaptive evolution of gene expression can entail tuning expression output by quantitative changes in TF binding affinities while maintaining the overall topology of a gene regulatory network.

evolutionary biology↗

bioRxiv2017

Incorporation of unique molecular identifiers in TruSeq adapters improves the accuracy of quantitative sequencing

Quantitative analysis of next-generation sequencing data requires discriminating duplicate reads generated by PCR from identical molecules that are of unique origin. Typically, PCR duplicates are defined as sequence reads that align to the same genomic coordinates using reference-based alignment. However, identical molecules can be independently generated during library preparation. The false positive rate of coordinate-based deduplication has not been well characterized and may introduce unforeseen biases during analyses. We developed a cost-effective sequencing adapter design by modifying Illumina TruSeq adapters to incorporate a unique molecular identifier (UMI) while maintaining the capacity to undertake multiplexed sequencing. Incorporation of UMIs enables identification of bona fide PCR duplicates as identically mapped reads with identical UMIs. Using TruSeq adapters containing UMIs (TrUMIseq adapters), we find that accurate removal of PCR duplicates results in enhanced data quality for quantitative analysis of allele frequencies in heterogeneous populations and gene expression.\n\nMethod SummaryTrUMIseq adapters incorporate unique molecular identifiers in TruSeq adapters while maintaining the capacity to multiplex sequencing libraries using existing workflows. The use of UMIs increases the accuracy of quantitative sequencing assays, including RNAseq and allele frequency estimation, by enabling accurate detection of PCR duplicates.

genomics↗