Sequencing is accepted as the “gold” standard for genetic analysis and continues to be used as a validation and reference tool. The idea of using sequence analysis directly for sample characterization has been met with skepticism. However, herein, utility of direct use of sequencing to identify multiple genomes present in samples is presented and reviewed. All samples and “pure” isolates are populations of genomes. Population-Sequencing is the use of probabilistic matching tools in combination with large volumes of sequence information to identify genomes present, based on DNA analysis across entire genomes to determine genome assignments, to calculate confidence scores of major and minor genome content. Accurate genome identification from mixtures without culture purification steps can achieve phylogenetic classification by direct analysis of millions of DNA fragments. Genome sequencing data of mixtures can function as biomarkers for use to interrogate genetic content of samples and to establish a sample profile, inclusive of major and minor genome components, drill down to identify rare SNP and mutation events, compare relatedness of genetic content between samples, profile-to-profile, and provide a probabilistic or statistical scoring confidence for sample characterization and attribution. The application of Population-Sequencing will facilitate sample characterization and genome identification strategies. 1. Background A paradigm shift in microbial-forensics is emerging due to the potential of interrogating comprehensive genome content of samples via direct application of Next-Generation sequencing. The innovative process of sequencing populations of entire pathogenic mixtures as opposed to sequencing isolates, from diverse samples, and comparing to a standard reference is described. The approach greatly accelerates and vastly shortens the interval from time/point of contact, to specific pathogen and strain identification. This abbreviated timeline is critical to respond to and treat exposure victims. Moreover, tracing the origins of strains is accentuated, which facilitates rapid investigation. Population-Sequencing will be more productive to provide genome information than to singly clone one genome from the sample population and then analyze that genome individually cultured from it. Herein, direct and practical solutions as well as some current limiting dogmas in this area are presented. Whole genome sequencing (WGS) analysis has become feasible for molecular biology and comparative genetics due in large part to available tools, such as BLAST
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