The Framework for Autogenerated Signature Technology for Nucleic Acids (FAST-NA) is a signature extraction method and accompanying software for detection of small nucleic acid sequences of concern in orders submitted for DNA synthesis. Building on prior success in developing FAST- NA for detection of viral pathogens, in this project we extended the application of FAST-NA to bacterial, eukaryotic, and viroid threats. We have also demonstrated that the FAST-NA method can be effective beyond its original intended application area, for both pathogen screening in next generation sequencing data and for region of interest detection in novel pathogens. Following the conclusion of government-supported development, FAST-NA has been further developed to produce a commercially licensed software product that is now being deployed within the DNA synthesis industry.