16S rRNA Gene Copy Number Normalization Does Not Provide More Reliable Conclusions in Metataxonomic Surveys
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16S rRNA Gene Copy Number Normalization Does Not Provide More Reliable Conclusions in Metataxonomic Surveys Robert Starke 1 & Victor Satler Pylro 2 & Daniel Kumazawa Morais 1,3 Received: 6 July 2020 / Accepted: 24 August 2020 # The Author(s) 2020
Abstract Sequencing 16S rRNA gene amplicons is the gold standard to uncover the composition of prokaryotic communities. The presence of multiple copies of this gene makes the community abundance data distorted and gene copy normalization (GCN) necessary for correction. Even though GCN of 16S data provided a picture closer to the metagenome before, it should also be compared with communities of known composition due to the fact that library preparation is prone to methodological biases. Here, we process 16S rRNA gene amplicon data from eleven simple mock communities with DADA2 and estimate the impact of GCN. In all cases, the mock community composition derived from the 16S sequencing differs from those expected, and GCN fails to improve the classification for most of the analysed communities. Our approach provides empirical evidence that GCN does not improve the 16S target sequencing analyses in real scenarios. We therefore question the use of GCN for metataxonomic surveys until a more comprehensive catalogue of copy numbers becomes available. Keywords 16S rRNA . Metataxonomic surveys . Gene
Amplicon sequencing of 16S rRNA gene is considered a gold standard to evaluate the composition of prokaryotic communities due to (i) low cost, (ii) easy availability, (iii) easy practicality of extraction and preparation kits, (iv) high taxonomic resolution as deep as the level of genera (or sometimes species) and (v) extensive databases. The concept of gold standards implies a level of perfection never attained by any biological test [1] which is why those are constantly challenged and replaced when appropriate [2]. Still, amplicon sequencing outcompetes (88,889 papers with “16S rRNA” as of June 9, 2020) Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00248-020-01586-7) contains supplementary material, which is available to authorized users. * Robert Starke [email protected] 1
Laboratory of Environmental Microbiology, Institute of Microbiology of the Czech Academy of Sciences, Praha, Czech Republic
2
Department of Biology, Federal University of Lavras—UFLA, Lavras, Minas Gerais, Brazil
3
Bioinformatics Core Facility, Institute of Microbiology of the Czech Academy of Sciences, Praha, Czech Republic
other possible techniques to describe the community structure such as metagenomics (22,106), metaproteomics (1717) or metatranscriptomics (2639) with many thousand publications in recent years. The general practice as shown by the myriads of publications does not comprise the correction of the obtained raw counts by 16S rRNA gene copy numbers per bacterial genome even though it is known that bacteria can have multiple copy numbers of the 16S rRNA gene and the normalization of 16S rRNA amplicon data gave a picture closer to
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