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

Frontiers in Genetics

Genetics

Sabine Pankuweit¹  Juan Cheko¹  Lek Babalija¹  Bernhard Schieffer¹  Ulrich Luesebrink¹  Julian Mueller¹  Volker Ruppert²  Muhidien Soufi²  Christian Waechter³  Dominik Heider⁴  Marius Welzel⁴  Leon Fehse⁴  Eberhard Weihe⁵  Ralf Kinscherf⁵  Jochen Pöling⁶  Thomas Braun⁶ 

[1] Department of Cardiology, University Hospital Marburg, Philipps University Marburg, Marburg, Germany;Department of Cardiology, University Hospital Marburg, Philipps University Marburg, Marburg, Germany;Center for Undiagnosed and Rare Diseases, University Hospital Marburg, Philipps University Marburg, Marburg, Germany;Department of Cardiology, University Hospital Marburg, Philipps University Marburg, Marburg, Germany;Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany;Department of Mathematics and Computer Science, Philipps University Marburg, Marburg, Germany;Institute of Anatomy and Cell Biology, Medical Faculty, Philipps University Marburg, Marburg, Germany;Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany;

关键词: 16S rRNA;    gut microbiome;    human fecal microbiome;    next-generation sequencing (NGS);    nanopore sequencing;    oxford nanopore technologies (ONT);    MinION Mk1C;    primer degeneracy;   

DOI  :  10.3389/fgene.2023.1213829

 received in 2023-04-28, accepted in 2023-07-10,  发布年份 2023

来源: Frontiers

【 摘 要 】

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 Firmicutes and Proteobacteria as determined by relative abundances, as well as an unusually high ratio of Firmicutes/Bacteriodetes 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 in situ 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.

【 授权许可】

Unknown   
Copyright © 2023 Waechter, Fehse, Welzel, Heider, Babalija, Cheko, Mueller, Pöling, Braun, Pankuweit, Weihe, Kinscherf, Schieffer, Luesebrink, Soufi and Ruppert.