![]() ![]() The relatively low cost of amplicon sequencing in comparison to other techniques (e.g., shotgun sequencing that sequences fragments of all present genetic material) has made it an increasingly popular technique 9, 10. The use of NGS technology and techniques such as amplicon sequencing (sequencing of amplified genes of interest) have allowed for analysis of large quantities of diverse environmental DNA 7 and have largely eliminated challenges associated with culturing and microscopic identification 8 in this context. In addition to the limitations of culturing, microscopic evaluation of environmental samples remains of limited utility because of challenges in high-resolution taxonomic identification and the inability to infer function from morphology 2. It is well known that fewer than 1% of species in the environment can be isolated and cultured, limiting the ability to identify rare and difficult-to-cultivate members of the community 4, 5, 6. Next-generation sequencing (NGS) has revolutionized the understanding of environmental systems by enabling characterization of microbial communities and their function through examination of DNA collected from samples that contain mixed assemblages of organisms 1, 2, 3. Specifically, it evaluates which data might have been obtained if a particular sample’s library size had been smaller and allows graphical representation of the effects of this library size normalization process upon diversity analysis results. While many deterministic data transformations are not tailored to produce equal library sizes, repeatedly rarefying reflects the probabilistic process by which amplicon sequencing data are obtained as a representation of the amplified source microbial community. This enables (i) proportionate representation of all observed sequences and (ii) characterization of the random variation introduced to diversity analyses by rarefying to a smaller library size shared by all samples. Here, repeated rarefying is proposed as a tool to normalize library sizes for diversity analyses. This process is often dismissed as statistically invalid because subsampling effectively discards a portion of the observed sequences, yet it remains prevalent in practice and the suitability of rarefying, relative to many other normalization approaches, for diversity analysis has been argued. Rarefaction is a widely used normalization technique that involves the random subsampling of sequences from the initial sample library to a selected normalized library size. Groups of samples typically have different library sizes that are not representative of biological variation library size normalization is required to meaningfully compare diversity between them. Amplicon sequencing data consist of discrete counts of sequence reads, the sum of which is the library size. In water resources management, it can be especially useful to evaluate ecosystem shifts in response to natural and anthropogenic landscape disturbances to signal potential water quality concerns, such as the detection of toxic cyanobacteria or pathogenic bacteria. Amplicon sequencing has revolutionized our ability to study DNA collected from environmental samples by providing a rapid and sensitive technique for microbial community analysis that eliminates the challenges associated with lab cultivation and taxonomic identification through microscopy. ![]()
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