Abstract
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.
Highlights
Introduction to PopulationSequencing nucleotide and aminoacid sequence-based approaches have been used in the past for inferring microbial evolutionary relationships, over the last 10 years, these methods have been increasingly used for typing and characterizing their populations [37,38,39]
The benefit of whole genome analysis over a selected panel of signature markers was made evident in human forensics and analysis of mitochondrial DNA [10, 11]
With the advent of next-generation sequencing approaches allowing for metagenomic sampling of microbes from outbreak scenes [44], data are available to both characterize novel genomic elements of pathogens and trace those novel elements through evolutionary history for both identification and tracking purposes
Summary
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. Development of bioinformatics tools to process volumes of sequence data provides solutions to many challenges, including bacterial forensics where Population-Sequencing identifies genome content of samples and differentiates between samples by creating genetic diversity profiles unique to each sample. The benefit of whole genome analysis over a selected panel of signature markers was made evident in human forensics and analysis of mitochondrial DNA [10, 11] These pioneering applications of analysis of entire genomes used sequence information to measure dynamics of entire sequences of interest. These examples point us in the direction of looking at comprehensive sequence information to characterize samples, rather than analysis of discreet regions of genomes, intended to describe one genome in the mixture: the product of consensus sequencing. Instead of classical methods and instead of DNA methods that consider small percentages of the genome (PCR, etc.), whole genome sequencing can be directly applied to microbe identification [13,14,15]
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