Seifert, C. | bioRxiv Science

bioRxiv2026

Automated refinement of metagenomic bins and estimation of binning success using itBins

Most prokaryotic genomes in public databases are genomes reconstructed from metagenomes, forming a compendium of multiple contiguous sequences (contigs) assembled from shotgun sequencing data. Binning algorithms for assigning contigs to metagenome assembled genomes (MAGs) are manifold and continuously improving in accuracy. However, binning errors, i.e. the incorrect assignment of a contig and coding sequence to a MAG, often propagate through various databases and confound taxonomic, metabolic and/or evolutionary analyses. Here we present itBins, a fully automated python-based software that enables ultra-fast refinement of metagenomic bins using a rule-based approach harnessing information from %GC content (%GC for brevity), coverage, and taxonomy of individual contigs. When applied to the low, medium, and high complexity data of the Critical Assessment of Metagenome Interpretation (CAMI I) challenge [1], itBins produced higher F1 scores (the harmonic mean of precision and recall) for all levels compared to other automated refinement tools, i.e., MDMcleaner and Rosella. Compared to manual refinement via uBin, itBins performed similarly well across all three complexity levels of the CAMI I dataset. With an average speed of 61 ms per bin, itBins is faster than all other refinement tools by at least three orders of magnitude when input data is accordingly available (%GC, coverage, and taxonomy), and was similarly fast when input data preparation was included in the processing time. Application to 64 real-world metagenomes from highly complex river mesocosms resulted in 259 medium-quality and 19 high-quality MAGs refined by itBins, while the other automated refinement tools failed in generating output at all or within 5000 hours of runtime. Finally, itBins also utilizes marker genes to determine the overall binning success for individual metagenomes, providing a crucial benchmark for the user to estimate the ecological relevance of their binned data. The herein introduced software itBins is broadly applicable to any type of metagenome data, integrates well with other software like DASTool, and enables swift and reliable refinement of genomes from metagenomes along with estimation of the overall binning success. itBins is distributed via EUPL 1.2 license and available at Codeberg (codeberg.org/JMK/itBins), GitHub (github.com/ProbstLab/itBins) and through Bioconda [2](bioconda.github.io/recipes/itbins/README.html).

bioinformatics↗

bioRxiv2021

Phosphorylation dynamics in a flg22 induced, heterotrimeric G protein dependent signaling network in Arabidopsis thaliana reveals a candidate PP2A phosphatase involved in AtRGS1 trafficking

flg22 is a 22-amino peptide released from bacterial flagellin, a Microbe-Associated Molecular Pattern (MAMP) that is recognized by the plant cell as a signal indicating that bacteria are present. On its own, flg22 initiates a rapid increase in cytoplasmic calcium, extracellular reactive oxygen species, and activation of a Mitogen Activated Protein Kinase (MAPK) cascade, all of which are activated within 15 minutes after the cell perceives flg22. Here we show a massive change in protein abundance and phosphorylation state of the Arabidopsis root cell proteome within this 15-minute duration in wildtype and a mutant deficient in G-protein coupled signaling. Integration of phosphoproteome with protein-protein interactome data followed by network topology analyses discovered that many of the flg22-induced phosphoproteome changes fall on proteins that comprise the G protein interactome and on the most highly populated hubs of the immunity network. Approximately 95% of the phosphorylation changes in the G-protein interactome depend on a functional heterotrimeric G protein complex, some occur on proteins that interact directly with components of G-coupled signal transduction. One of these is ATB, a substrate-recognition sub-unit of the PP2A Ser/Thr phosphatase and an interactor to Arabidopsis thaliana REGULATOR OF G SIGNALING 1 protein (AtRGS1), a 7-transmembrane spanning modulator of the nucleotide-binding state of the core G protein complex. AtRGS1 is phosphorylated by BAK1, a component of the flg22 receptor, to initiate AtRGS1 endocytosis. A null mutation of ATB confers high basal endocytosis of AtRGS1, suggesting sustained phosphorylated status. Loss of ATB confers traits associated with loss of AtRGS1. Because the basal level of AtRGS1 is lower in the atb null mutant in a proteasome-dependent manner, we propose that phosphorylation-dependent endocytosis of AtRGS1 is part of a mechanism to degrade AtRGS1 which then sustains activation of the G protein complex. Thus, the role of ATB is now established as a central component of phosphorylation-dependent regulation of system dynamics in innate immunity.

systems biology↗