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Updated: Jun 16, 2025

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing
Published on: October 15, 2019
A rapid phylogeny-based method for accurate community profiling of large-scale metabarcoding datasets
Lenore Pipes1, Rasmus Nielsen1,2
1Department of Integrative Biology, University of California, Berkeley, Berkeley, United States.
Abstract:
Environmental DNA (eDNA) is becoming an increasingly important tool in diverse scientific fields from ecological biomonitoring to wastewater surveillance of viruses. The fundamental challenge in eDNA analyses has been the bioinformatical assignment of reads to taxonomic groups. It has long been known that full probabilistic methods for phylogenetic assignment are preferable, but unfortunately, such methods are computationally intensive and are typically inapplicable to modern next-generation sequencing data. We present a fast approximate likelihood method for phylogenetic assignment of DNA sequences. Applying the new method to several mock communities and simulated datasets, we show that it identifies more reads at both high and low taxonomic levels more accurately than other leading methods. The advantage of the method is particularly apparent in the presence of polymorphisms and/or sequencing errors and when the true species is not represented in the reference database.
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