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

/pdf/best-practices-in-designing-sequencing-and-identifying-1ewhz5i8.pdf

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

Identifying Targets of Selection in Laboratory Evolution Experiments

Adaptation is driven by the selection for beneficial mutations that provide a fitness advantage in the specific environment in which a population is evolving. However, environments are rarely constant or predictable. When an organism well adapted to one environment finds itself in another, pleiotropic effects of mutations that made it well adapted to its former environment will affect its success. To better understand such pleiotropic effects, we evolved both haploid and diploid barcoded budding yeast populations in multiple environments, isolated adaptive clones, and then determined the fitness effects of adaptive mutations in "non-home" environments in which they were not selected. We find that pleiotropy is common, with most adaptive evolved lineages showing fitness effects in non-home environments. Consistent with other studies, we find that these pleiotropic effects are unpredictable: they are beneficial in some environments and deleterious in others. However, we do find that lineages with adaptive mutations in the same genes tend to show similar pleiotropic effects. We also find that ploidy influences the observed adaptive mutational spectra in a condition-specific fashion. In some conditions, haploids and diploids are selected with adaptive mutations in identical genes, while in others they accumulate mutations in almost completely disjoint sets of genes.

Evolution of haploid and diploid populations reveals common, strong, and variable pleiotropic effects in non-home environments.

Role of genetic architecture in phenotypic plasticity.

Climate change and the increasing frequency and severity of drought events pose significant challenges for sustainable agriculture worldwide. Soil microorganisms, both beneficial and pathogenic, play a crucial role in mediating plant–environment interactions and shaping the overall functioning of agroecosystems. This review summarizes current knowledge on the adaptive mechanisms used by different groups of plant-beneficial soil microorganisms—rhizobacteria and arbuscular mycorrhizal fungi (AMF)—as well as phytopathogenic bacteria and fungi, in response to drought. The review focuses on identifying the commonalities and differences in the survival strategies of these groups of beneficial and pathogenic soil microorganisms under drought conditions. Additionally, it reviews and compares the plant defence mechanisms under drought conditions facilitated by rhizobacteria and AMF. Special attention is given to the genetic exchange between beneficial and pathogenic soil microorganisms through horizontal gene transfer (HGT), which allows them to exchange traits. It is observed that drought may favor enhanced genetic exchange and the spread of pathogenic traits in the soil microbiome. This review will be useful for a wide range of readers to better understand the dynamics of the soil microbiome under climate change and to apply this knowledge to sustainable agricultural practices. 

Plant–Soil Microbial Interaction: Differential Adaptations of Beneficial vs. Pathogenic Bacterial and Fungal Communities to Climate-Induced Drought

The phrase "survival of the fittest" has become an iconic descriptor of how natural selection works. And yet, precisely measuring fitness, even for single-celled microbial populations growing in controlled laboratory conditions, remains a challenge. While numerous methods exist to perform these measurements, including recently developed methods utilizing DNA barcodes, all methods are limited in their precision to differentiate strains with small fitness differences. In this study, we rule out some major sources of imprecision, but still find that fitness measurements vary substantially from replicate to replicate. Our data suggest that very subtle and difficult to avoid environmental differences between replicates create systematic variation across fitness measurements. We conclude by discussing how fitness measurements should be interpreted given their extreme environment dependence. This work was inspired by the scientific community who followed us and gave us tips as we live tweeted a high-replicate fitness measurement experiment at #1BigBatch. 

/pdf/extreme-sensitivity-of-fitness-to-environmental-conditions-bmvryu1z.pdf

Extreme Sensitivity of Fitness to Environmental Conditions: Lessons from #1BigBatch

Challenges and potential solutions for studying the genetic and phenotypic architecture of adaptation in microbes.

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

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

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Extreme Sensitivity of Fitness to Environmental Conditions: Lessons from #1BigBatch

Challenges and potential solutions for studying the genetic and phenotypic architecture of adaptation in microbes.

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