An in-depth, updated benchmark for 16S amplicon sequencing
Amplicon-based techniques provide a rapid and cost-effective approach for profiling microbial communities. However, the observed microbial diversity is influenced by a wide range of factors, encompassing pre-analytical steps such as the choice of primers and target regions, as well as the bioinformatic pipeline, including the selection of tools, reference databases, and parameter settings.…
A meticulous, updated benchmark for 16S amplicon sequencing has been conducted, aiming to provide a more accurate representation of microbial communities. Numerous factors influence the observed microbial diversity, including the choice of primers and target regions, as well as the bioinformatic pipeline, which includes tool selection, reference databases, and parameter settings.
Previous studies have already produced benchmarks; however, recent updates to key tools and databases, specifically LotuS3, the Ribosomal Database Project, and GreenGenes2, necessitated a fresh investigation.
The researchers employed seven regions across three mock communities of varying complexity, assessing 38 different combinations of sequence resolution algorithms, taxonomic classifiers, search tools, and databases. V1-V3 region, combined with DADA2 and MMseqs2 paired with SILVA, DADA2 and Metaxa2, or LotuS3 (DADA2) with RDP, demonstrated the most precise estimates of true diversity based on the chosen metrics.
Despite optimal tool and database combinations, the benchmark revealed that even dominant genera can prove challenging to detect. Furthermore, the quantification of all genera can be significantly over- or under-estimated, highlighting the need for a more standardized and reliable approach in 16S amplicon sequencing.
Written by urgent.news from bioRxiv's reporting — not their text. Machine-written — may contain errors; check the original before relying on it.
