Comparative analysis of full-length 16s ribosomal RNA genome sequencing in human fecal samples using primer sets with different degrees of degeneracy

Next-generation sequencing has revolutionized the field of microbiology research and greatly expanded our knowledge of complex bacterial communities. Nanopore sequencing provides distinct advantages, combining cost-effectiveness, ease of use, high throughput, and high taxonomic resolution through it...

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Published in:	Frontiers in genetics Vol. 14; p. 1213829
Main Authors:	Waechter, Christian, Fehse, Leon, Welzel, Marius, Heider, Dominik, Babalija, Lek, Cheko, Juan, Mueller, Julian, Pöling, Jochen, Braun, Thomas, Pankuweit, Sabine, Weihe, Eberhard, Kinscherf, Ralf, Schieffer, Bernhard, Luesebrink, Ulrich, Soufi, Muhidien, Ruppert, Volker
Format:	Journal Article
Language:	English
Published:	Switzerland Frontiers Media S.A 26-07-2023
Subjects:	16S rRNA Genetics gut microbiome human fecal microbiome nanopore sequencing next-generation sequencing (NGS) oxford nanopore technologies (ONT) 16S rRNA human fecal microbiome nanopore sequencing gut microbiome next-generation sequencing (NGS) primer degeneracy oxford nanopore technologies (ONT) MinION Mk1C
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Summary:	Next-generation sequencing has revolutionized the field of microbiology research and greatly expanded our knowledge of complex bacterial communities. Nanopore sequencing provides distinct advantages, combining cost-effectiveness, ease of use, high throughput, and high taxonomic resolution through its ability to process long amplicons, such as the entire 16s rRNA genome. We examine the performance of the conventional 27F primer (27F-I) included in the 16S Barcoding Kit distributed by Oxford Nanopore Technologies (ONT) and that of a more degenerate 27F primer (27F-II) in the context of highly complex bacterial communities in 73 human fecal samples. The results show striking differences in both taxonomic diversity and relative abundance of a substantial number of taxa between the two primer sets. Primer 27F-I reveals a significantly lower biodiversity and, for example, at the taxonomic level of the phyla, a dominance of and as determined by relative abundances, as well as an unusually high ratio of / when compared to the more degenerate primer set (27F-II). Considering the findings in the context of the gut microbiomes common in Western industrial societies, as reported in the American Gut Project, the more degenerate primer set (27F-II) reflects the composition and diversity of the fecal microbiome significantly better than the 27F-I primer. This study provides a fundamentally relevant comparative analysis of the performance of two primer sets designed for sequencing of the entire 16s rRNA genome and suggests that the more degenerate primer set (27F-II) should be preferred for nanopore sequencing-based analyses of the human fecal microbiome.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Edited by: Nathan Olson, National Institute of Standards and Technology (NIST), United States Reviewed by: Dieter Maurice Tourlousse, National Institute of Advanced Industrial Science and Technology (AIST), Japan Hassan Ghazal, National Center for Scientific and Technical Research (CNRST), Morocco
ISSN:	1664-8021 1664-8021
DOI:	10.3389/fgene.2023.1213829