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Rarify qiime4/5/2023 The amplification and sequencing of specific genes (i.e., taxonomic marker genes) enables characterization of microbial community composition ( Hodkinson and Grice, 2015) as a result, it has been successfully applied in many area of water research. ![]() In contrast, the relatively low cost of amplicon sequencing has made it an increasingly popular technique ( Clooney et al., 2016 Langille et al., 2013). ![]() While shotgun sequencing allows characterization of the entire community, including both taxonomic composition and functional gene profiles, it is not widely accessible due to high sequencing costs and computational requirements for analysis ( Bartram et al., 2011 Clooney et al., 2016 Langille et al., 2013). Metagenomics includes a conglomerate of different sequencing experimental designs including amplicon sequencing (sequencing of amplified genes of interest) and shotgun sequencing (the sequencing of fragments of present genetic material). Metagenomic studies employ next-generation sequencing technology to analyze environmental DNA ( Thomas et al., 2012) and have largely eliminated these challenges ( McMurdie and Holmes, 2014). 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 ( Hugerth and Andersson, 2017). 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 ( Bodor et al., 2020 Cho and Giovannoni, 2004 Ferguson et al., 1984). Next-generation sequencing has revolutionized the analysis of environmental systems through the characterization of microbial communities and their function by the study of DNA collected from samples that contain mixed assemblages of organisms ( Bartram et al., 2011 Hugerth and Andersson, 2017 Shokralla et al., 2012). The impact of normalized library size selection and rarefying with or without replacement in diversity analyses were evaluated herein. Rarefying may be a statistically valid normalization technique, but researchers should evaluate their data to make appropriate decisions regarding library size selection and subsampling type. Although it has been suggested that rarefying should be avoided altogether, we propose that repeatedly rarefying enables (i) characterization of the variation introduced to diversity analyses by this random subsampling and (ii) selection of smaller library sizes where necessary to incorporate all samples in the analysis. However, rarefying has been criticized as a normalization technique because data can be omitted through the exclusion of either excess sequences or entire samples, depending on the rarefied library size selected. The process of randomly subsampling sequences to a selected normalized library size from the sample library-rarefying-is one such normalization technique. Samples may have different library sizes and thus, a normalization technique is required to meaningfully compare them. ![]() DNA sequencing data consist of discrete counts of sequence reads, the total number of which is the library size. It has revolutionized our ability to study DNA collected from environmental samples by eliminating the challenges associated with lab cultivation and taxonomic identification. The application of amplicon sequencing in water research provides a rapid and sensitive technique for microbial community analysis in a variety of environments ranging from freshwater lakes to water and wastewater treatment plants.
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