The Special Issue brought together papers that highlighted the power of high-throughput sequencing (HTS) data to address classic questions in ecology and evolution, and/or use models/theory to infer key ecological and evolutionary processes, and make predictions, particularly focused on metabarcoding (amplicon) datasets in conjunction with complementary -omics data types. We highlight key papers that show the power of the new technology to address questions related to: (1) community assembly, and the interplay between competition, environmental filtering, and neutral processes, that can be inferred from the data, and how these change according to environmental conditions, and across successional and extended evolutionary time. (2) Interaction networks, and how these can show predictable changes over similar spatial and temporal gradients, providing insights into questions of biotic resilience. Studies also examined (3) cross scale interactions and those involving hosts and their microbiomes, with the critical development being the ease of comparison and integration across scales of organismic complexity, allowing insights at one scale to inform the other. The approach is also amenable to (4) studies of invasive species and biotic homogenization, providing insights of shifts in alpha and beta diversity across a wide range of spatial scales.