Best Practices in Designing, Sequencing, and Identifying Random DNA Barcodes

Journal of Molecular Evolution, Journal Year: 2023, Volume and Issue: 91(3), P. 263 - 280

Published: Jan. 18, 2023

Random DNA barcodes are a versatile tool for tracking cell lineages, with applications ranging from development to cancer evolution. Here, we review and critically evaluate barcode designs as well methods of sequencing initial processing data. We first demonstrate how various design decisions affect data quality propose new that balances all considerations currently aware of. then discuss options the preparation libraries, including inline indices Unique Molecular Identifiers (UMIs). Finally, test performance several established bioinformatic pipelines extraction raw reads error correction. find both alignment regular expression-based approaches work extraction, error-correction designed specifically superior generic ones. Overall, this will help researchers approach their barcoding experiments in deliberate systematic way.

Language: Английский

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Distinguishing mutants that resist drugs via different mechanisms by examining fitness tradeoffs

eLife, Journal Year: 2024, Volume and Issue: 13

Published: Sept. 10, 2024

There is growing interest in designing multidrug therapies that leverage tradeoffs to combat resistance. Tradeoffs are common evolution and occur when, for example, resistance one drug results sensitivity another. Major questions remain about the extent which reliable, specifically, whether mutants provide a given all suffer similar tradeoffs. This question difficult because drug-resistant observed clinic, even those evolved controlled laboratory settings, often biased towards large fitness benefits. Thus, mutations (and mechanisms) may be more diverse than current data suggests. Here, we perform experiments utilizing lineage-tracking capture fuller spectrum of give yeast cells advantage fluconazole, antifungal drug. We then quantify each 774 across 12 environments, finding these group into classes with characteristically different Their unique imply affects through underlying mechanisms. Some groupings find surprising. For some resist single drugs do not their combination, while others do. And same gene have others. These findings, on hand, demonstrate difficulty relying consistent or intuitive when treatments. On other by demonstrating hundreds adaptive can reduced few groups characteristic tradeoffs, our findings yet empower strategies More generally speaking, grouping likely affect mechanisms, work guides efforts map phenotypic effects mutation.

Language: Английский

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Identifying Targets of Selection in Laboratory Evolution Experiments

Journal of Molecular Evolution, Journal Year: 2023, Volume and Issue: 91(3), P. 345 - 355

Published: Feb. 21, 2023

Language: Английский

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Distinguishing mutants that resist drugs via different mechanisms by examining fitness tradeoffs

eLife, Journal Year: 2024, Volume and Issue: 13

Published: March 18, 2024

There is growing interest in designing multidrug therapies that leverage tradeoffs to combat resistance. Tradeoffs are common evolution and occur when, for example, resistance one drug results sensitivity another. Major questions remain about the extent which reliable, specifically, whether mutants provide a given all suffer similar tradeoffs. This question difficult because drug-resistant observed clinic, even those evolved controlled laboratory settings, often biased towards large fitness benefits. Thus, mutations (and mechanisms) may be more diverse than current data suggests. Here, we perform experiments utilizing lineage-tracking capture fuller spectrum of give yeast cells advantage fluconazole, antifungal drug. We then quantify each 774 across 12 environments, finding these group into classes with characteristically different Their unique imply affects through underlying mechanisms. Some groupings find surprising. For some resist single drugs do not their combination, while others do. And same gene have others. These findings, on hand, demonstrate difficulty relying consistent or intuitive when treatments. On other by demonstrating hundreds adaptive can reduced few groups characteristic tradeoffs, our findings yet empower strategies More generally speaking, grouping likely affect mechanisms, work guides efforts map phenotypic effects mutation.

Language: Английский

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Distinguishing mutants that resist drugs via different mechanisms by examining fitness tradeoffs across hundreds of fluconazole-resistant yeast strains

Published: March 15, 2024

There is growing interest in designing multidrug therapies that leverage tradeoffs to combat resistance. Tradeoffs are common evolution and occur when, for example, resistance one drug results sensitivity another. Major questions remain about the extent which reliable, specifically, whether mutants provide a given all suffer similar tradeoffs. This question difficult because drug-resistant observed clinic, even those evolved controlled laboratory settings, often biased towards large fitness benefits. Thus, mutations (and mechanisms) may be more diverse than current data suggests. Here, we perform experiments utilizing lineage-tracking capture fuller spectrum of give yeast cells advantage fluconazole, antifungal drug. We then quantify each 774 across 12 environments, finding these group into 6 classes with characteristically different Their unique imply affects through underlying mechanisms. Some groupings find surprising. For some resist single drugs do not their combination, same gene have others. These findings, on hand, demonstrate difficulty relying consistent or intuitive when treatments. On other by demonstrating hundreds adaptive can reduced few groups characteristic tradeoffs, our findings empower strategies Finally, grouping likely affect mechanisms, work guides efforts map phenotypic effects mutation.

Language: Английский

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A shift from pleiotropic to modular adaptation revealed by a high-resolution two-step adaptive walk

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: April 18, 2024

ABSTRACT Evolution by natural selection is expected to be a slow and gradual process. In particular, the mutations that drive evolution are predicted small modular, incrementally improving number of traits. However, adaptive identified early in microbial experiments, cancer, other systems often provide substantial fitness gains pleiotropically improve multiple traits at once. We asked whether such common throughout adaptation or instead rare feature steps tend target key signaling pathways. To do so, we conducted barcoded second-step experiments initiated from five first-step prior yeast experiment. then isolated hundreds these measured their performance several growth phases, whole-genome sequencing clones. Here, found while vast majority mutants this condition show patterns pleiotropic - both fermentation respiration phases shift towards modular adaptation, mostly only rarely performance. also molecular basis genes cellular pathways involved mitochondrial function. Our results suggest particularly capable providing large, adaptively benefits organism due ability coherently affect many phenotypes As such, may serve as source stages evolution, once become exhausted, organisms adapt more gradually, acquiring smaller, mutations.

Language: Английский

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Single-cell heterogeneity in ribosome content and the consequences for the growth laws

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: April 23, 2024

Across species and environments, the ribosome content of cell populations correlates with population growth rate. The robustness universality this correlation have led to its classification as a "growth law." This law has fueled theories about how evolution selects for microbial organisms that maximize their rate based on nutrient availability, it informed models individual cells regulate rates ribosomal content. However, due methodological limitations, rarely been studied at level cells. While

Language: Английский

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A high-resolution two-step evolution experiment in yeast reveals a shift from pleiotropic to modular adaptation

PLoS Biology, Journal Year: 2024, Volume and Issue: 22(12), P. e3002848 - e3002848

Published: Dec. 5, 2024

Evolution by natural selection is expected to be a slow and gradual process. In particular, the mutations that drive evolution are predicted small modular, incrementally improving number of traits. However, adaptive identified early in microbial experiments, cancer, other systems often provide substantial fitness gains pleiotropically improve multiple traits at once. We asked whether such common throughout adaptation or instead rare feature steps tend target key signaling pathways. To do so, we conducted barcoded second-step experiments initiated from 5 first-step prior yeast experiment. then isolated hundreds these measured their performance several growth phases, whole genome sequencing clones. Here, found while vast majority mutants this condition show patterns pleiotropic adaptation—improving both fermentation respiration phases—second-step shift towards modular adaptation, mostly only rarely performance. also molecular basis genes cellular pathways involved mitochondrial function. Our results suggest may more likely large, adaptively benefits organism due ability coherently affect many phenotypes As such, serve as source stages evolution, once become exhausted, organisms adapt gradually, acquiring smaller, mutations.

Language: Английский

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Environmental memory alters the fitness effects of adaptive mutations in fluctuating environments

Nature Ecology & Evolution, Journal Year: 2024, Volume and Issue: 8(9), P. 1760 - 1775

Published: July 17, 2024

Language: Английский

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Quantifying microbial fitness in high-throughput experiments

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Aug. 20, 2024

Few concepts are as central to evolution is fitness, and yet the quantification of fitness often ambiguous. In particular, high-throughput experiments measure mutant in microbes increasingly common but vary widely their definitions which makes results difficult compare. What consequences these different statistics, there a best way quantify given context? Here we systematize set possible statistics according following three choices: 1) encoding relative abundance (e.g., transforming by log or logit function), 2) time scale over change abundance, 3) choice reference subpopulation for calculating bulk competition experiments, such those using DNA-barcoded mutants. We show that choices can lead significantly interpretations affecting magnitude effects, presence epistasis, even ranking across This confound predictions evolutionary dynamics gene functions. Altogether our demonstrate importance consistent reproducible experiments.

Language: Английский

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