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A multi-amplicon 16S rRNA sequencing and analysis method for improved taxonomic profiling of bacterial communities.

ABSTRACT: Metagenomic sequencing of bacterial samples has become the gold standard for profiling microbial populations, but 16S rRNA profiling remains widely used due to advantages in sample throughput, cost, and sensitivity even though the approach is hampered by primer bias and lack of specificity. We hypothesized that a hybrid approach, that combined targeted PCR amplification with high-throughput sequencing of multiple regions of the genome, would capture many of the advantages of both approaches. We developed a method that identifies and quantifies members of bacterial communities through simultaneous analysis of multiple variable regions of the bacterial 16S rRNA gene. The method combines high-throughput microfluidics for PCR amplification, short read DNA sequencing, and a custom algorithm named MVRSION (Multiple 16S Variable Region Species-Level IdentificatiON) for optimizing taxonomic assignment. MVRSION performance was compared to single variable region analyses (V3 or V4) of five synthetic mixtures of human gut bacterial strains using existing software (QIIME), and the results of community profiling by shotgun sequencing (COPRO-Seq) of fecal DNA samples collected from gnotobiotic mice colonized with a defined, phylogenetically diverse consortium of human gut bacterial strains. Positive predictive values for MVRSION ranged from 65%-91% versus 44%-61% for single region QIIME analyses (p?

SUBMITTER: Schriefer AE

PROVIDER: S-EPMC6375304 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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A multi-amplicon 16S rRNA sequencing and analysis method for improved taxonomic profiling of bacterial communities.

Schriefer Andrew E AE Cliften Paul F PF Hibberd Matthew C MC Sawyer Christopher C Brown-Kennerly Victoria V Burcea Lauren L Klotz Elliott E Crosby Seth D SD Gordon Jeffrey I JI Head Richard D RD

Journal of microbiological methods 20180929

Metagenomic sequencing of bacterial samples has become the gold standard for profiling microbial populations, but 16S rRNA profiling remains widely used due to advantages in sample throughput, cost, and sensitivity even though the approach is hampered by primer bias and lack of specificity. We hypothesized that a hybrid approach, that combined targeted PCR amplification with high-throughput sequencing of multiple regions of the genome, would capture many of the advantages of both approaches. We ...[more]

PMID: 30273610

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Project description:Sourdoughs (SDs) are spontaneously formed microbial ecosystems composed of various species of lactic acid bacteria (LAB) and acid-tolerant yeasts in food matrices of cereal flours mixed with water. To date, more than 90 LAB species have been isolated, significantly impacting the organoleptic characteristics, shelf life, and health properties of bakery products. To learn more about the unique bacterial communities involved in creating regional Bulgarian sourdoughs, we examined the metacommunities of five sourdoughs produced by spontaneous fermentation and maintained by backslopping in bakeries from three geographic locations. The 16S rRNA gene amplicon sequencing showed that the former genus Lactobacillus was predominant in the studied sourdoughs (51.0-78.9%). Weissella (0.9-42.8%), Herbaspirillum (1.6-3.8%), Serratia (0.1-11.7%), Pediococcus (0.2-7.5%), Bacteroides (0.1-1.3%), and Sphingomonas (0.1-0.5%) were also found in all 5 samples. Genera Leuconostoc, Enterococcus, Bacillus, and Asaia were sample-specific. It is interesting to note that the genus Weissella was more abundant in wholegrain samples. The greatest diversity at the species level was found in the former genus Lactobacillus, presented in the sourdough samples with 13 species. The UPGMA cluster analysis clearly demonstrated similarity in species' relative abundance between samples from the same location. In addition, we can conclude that the presence of two main clusters-one including samples from mountainous places (the cities of Smolyan and Bansko) and the other including samples from the city of Ruse (the banks of the Danube River)-may indicate the impact of climate and geographic location (e.g., terrain, elevation, land use, and nearby water bodies and their streams) on the abundance of microbiome taxa. As the bacterial population is crucial for bread standardization, we expect the local bakery sector to be interested in the relationship between process variables and their effect on bacterial dynamics described in this research study.

Dataset Information

A multi-amplicon 16S rRNA sequencing and analysis method for improved taxonomic profiling of bacterial communities.

Publications

A multi-amplicon 16S rRNA sequencing and analysis method for improved taxonomic profiling of bacterial communities.

Similar Datasets

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