Abstract
Recent algorithmic advances in amplicon-based microbiome studies enable the inference of exact amplicon sequence fragments. These new methods enable the investigation of sub-operational taxonomic units (sOTU) by removing erroneous sequences. However, short (e.g., 150-nucleotide [nt]) DNA sequence fragments do not contain sufficient phylogenetic signal to reproduce a reasonable tree, introducing a barrier in the utilization of critical phylogenetically aware metrics such as Faith's PD or UniFrac. Although fragment insertion methods do exist, those methods have not been tested for sOTUs from high-throughput amplicon studies in insertions against a broad reference phylogeny. We benchmarked the SATé-enabled phylogenetic placement (SEPP) technique explicitly against 16S V4 sequence fragments and showed that it outperforms the conceptually problematic but often-used practice of reconstructing de novo phylogenies. In addition, we provide a BSD-licensed QIIME2 plugin (https://github.com/biocore/q2-fragment-insertion) for SEPP and integration into the microbial study management platform QIITA. IMPORTANCE The move from OTU-based to sOTU-based analysis, while providing additional resolution, also introduces computational challenges. We demonstrate that one popular method of dealing with sOTUs (building a de novo tree from the short sequences) can provide incorrect results in human gut metagenomic studies and show that phylogenetic placement of the new sequences with SEPP resolves this problem while also yielding other benefits over existing methods.
Highlights
Recent algorithmic advances in amplicon-based microbiome studies enable the inference of exact amplicon sequence fragments
A de novo phylogeny was constructed from Deblur sub-operational taxonomic units (sOTU) following the steps illustrated in the QIIME 2 Moving Pictures tutorial version 2017.12, i.e., using multiple-sequence alignment via MAFFT [13] and phylogenetic reconstruction via FastTree (QIIME2 uses a FastTree version with double precision) [14]
The only identifiable factor was the presence of a single archaeon that was composed of just three low-abundance sOTUs
Summary
Recent algorithmic advances in amplicon-based microbiome studies enable the inference of exact amplicon sequence fragments. These new methods enable the investigation of sub-operational taxonomic units (sOTU) by removing erroneous sequences. The latter approach works well for OTUs but is hindered for sOTUs by the absence of some taxa in the reference database. New studies and meta-analyses will be processed significantly faster as placements for the majority of affected sOTUs are already available in this common resource
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