Gawron, R. | Search bioRxiv

bioRxiv2026

Development of a Microdroplet-Based Functional Genomic Screening pipeline by combination of DNA Nanoflowers and PURExpress Cell-Free Expression

We present a microfluidic workflow that couples reconstituted in vitro transcription-translation (IVTT) with ultra-high-throughput droplet screening to directly link genotype and phenotype within complex, heterogeneous DNA pools. The approach employs DNA nanoflowers as clonal, high-copy templates, enabling robust protein expression from single DNA molecules encapsulated in picoliter droplets. When integrated with fluorescence-assisted microdroplet sorting (FADS) and a DNA recovery pipeline that reconstituted selected libraries for subsequent iterative rounds, the platform achieves approximately 400-500-fold enrichment per selection cycle and supports functional discovery and directed evolution entirely independent of host cell expression. As a proof of principle, we demonstrate recovery of the recombinase RecA from an E. coli genomic library screened for single-stranded DNA binders, highlighting the platforms capability to identify DNA-interacting and DNA-modifying enzymes. By eliminating host-derived background activity and toxicity constraints that often complicate lysate- or cell-based metagenomic screens, this method expands access to enzyme classes that have historically been difficult to assay.

bioengineering↗

bioRxiv2024

Rethinking large scale phylogenomics with PhyloToL 6, a flexible toolkit to enable phylogeny-informed data curation and analysis

Eukaryotic diversity is largely microbial, with macroscopic lineages (plant, animals and fungi) nesting among a plethora of diverse protists. Understanding the evolutionary relationships among eukaryotes is rapidly advancing through omics analyses, but phylogenomics are challenging for microeukaryotes, particularly uncultivable lineages, as single-cell sequencing approaches generate a mixture of sequences from hosts, associated microbiomes, and contaminants. Moreover, many analyses of eukaryotic gene families and phylogenies rely on boutique datasets and methods that are challenging for other research groups to replicate. To address these challenges, we present EukPhylo v1.0, a modular, user-friendly pipeline that enables effective data curation through phylogeny-informed contamination removal, estimation of homologous gene families (GFs), and generation of both multisequence alignments and gene trees. Analyses can use a hook database of [~]15k ancient GFs or users can easily replace this hook with a set of gene families of interest. We demonstrate the power of EukPhylo, including a suite of stand-alone utilities, through analyses of 500 conserved GFs sampled from 1,000 diverse species of eukaryotes, bacteria and archaea. We show improvements in estimates of the eukaryotic tree of life, recovering clades that are well established in the literature, through successive rounds of curation using the EukPhylo contamination loop. The final trees corroborate numerous hypotheses in the literature (e.g. Opisthokonta, Rhizaria, Amoebozoa) while challenging others (e.g. CRuMs, Obazoa, Diaphoretickes). We believe that the flexibility and transparency of EukPhylo sets standards for curation of omics data for future studies. AUTHOR SUMMARYThe majority of eukaryotic lineages are microbial, with plants and animals nesting among diverse amoeba, flagellates, and other predominantly-microbial clades. Yet analyses of the evolution of microbial eukaryotes is hampered by the lack of tools for efficient analysis of genome-scaled data, especially in light of the genome complexity and high levels of contamination associated with these microorganism. Furthermore, many existing analyses rely on boutique datasets and decision making that lacks transparency and are thus difficult for others to repeat. To address these challenges, we present EukPhylo as an easy-to-use toolkit that fills a gap in phylogenomic methods, and we analyze of 500 gene families from 1,000 species to demonstrate the power of this approach in estimating the eukaryotic tree of life. EukPhylo enables exploration of eukaryotic evolution in a manner that is both transparent and easily repeatable, and hence can be used to illuminate the origin and diversification of eukaryotic life on Earth.

evolutionary biology↗