We were able to achieve almost all of the envisioned tools and applications of the proposal. Regarding tool developments, we have published a single nucleotide analyser, metaSNV (Costea et al., PlosOne 2017, Van Rossum, Bioinformatics 2022), improved quality control for metagenomic assembled genomes to distinguish strains (Orakov et al., Genome Biol. 2021), devised methods for gene content analyses of conspecific strains as well as subspecies delineation protocols (Costea et al., Mol. Sys. Biol. 2017, Van Rossum, Bioinformatics 2022) and developed many other analysis tools and resources that are required to zoom into strain resolution (e.g. the bacterial genome resource proGenomes: Mende et al., NAR 2017 and NAR 2019; an update of our eggnog (meta)genome annotation resource: Huerta-Cepas et al., Mol Biol Evol. 2017 and NAR 2019; and a tool to annotate newly sequenced (meta)genomes: Huerta-Cepas et al., Mol Biol Evol. 2017 and Cantalapiedra et al., Mol Biol Evol. 2021). Regarding the identification of known strains in metagenomics data, we have developed respective pipelines that led, as proposed, to various biological discoveries. For example, we have, as intended, identified and quantified subspecies in the vast majority of abundant and prevalent human gut microbes, which have a distinct biogeography and which seem to be exclusive and stay with the host for a long time (Costea et al., Mol. Sys. Biol. 2017, Van Rossum et al., Bioinformatics 2022, Van Rossum et al., Nature Rev. Microbiol. 2020). We pointed to the need and power of strain level resolution in metagenomics (Schmidt et al., Cell 2018) and illustrated it with various findings, e.g. basic ones like extensive strain transmission along the gastrointestinal tract (Schmidt et al., elife 2019) or in relation to medication (Forslund et al., Nature 2021, Hildebrand et al., Gut 2019), or diseases in general (Parkinson’s, Bedarf et al., Genome Med. 2017 and pancreatic cancer, Kartal et al., Gut 2022). Furthermore, as intended, we analysed longitudinal data using SNV markers in medically relevant areas such as FMT (Li et al., Science 2016; Schmidt et al., bioRxiv 2021, Nat.Med. in revision) or in medically important species such as C.difficile (Ferretti et al., bioRxiv 2022, Nature Med., submitted) and explored, using strains, family strain exchange over time in a biogeographic context (Korpela et al., Genome Res. 2018, Hildebrand et al. Cell Host Microbe 2021). The latter enabled us to also to progress on the population genetics part in that we discovered different resistance and dispersal strategies (Hildebrand et al., Cell Host Microbe, 2021).
