Abstract
Background: The human intestinal microbiome plays a central role in overall health status, especially in early life stages. 16S rRNA amplicon sequencing is used to profile its taxonomic composition; however, multiomic approaches have been proposed as the most accurate methods for study of the complexity of the gut microbiota. In this study, we propose an optimized method for bacterial diversity analysis that we validated and complemented with metabolomics by analyzing fecal samples. Methods: Forty-eight different analytical combinations regarding (1) 16S rRNA variable region sequencing, (2) a feature selection approach, and (3) taxonomy assignment methods were tested. A total of 18 infant fecal samples grouped depending on the type of feeding were analyzed by the proposed 16S rRNA workflow and by metabolomic analysis. Results: The results showed that the sole use of V4 region sequencing with ASV identification and VSEARCH for taxonomy assignment produced the most accurate results. The application of this workflow showed clear differences between fecal samples according to the type of feeding, which correlated with changes in the fecal metabolic profile. Conclusion: A multiomic approach using real fecal samples from 18 infants with different types of feeding demonstrated the effectiveness of the proposed 16S rRNA-amplicon sequencing workflow.
Highlights
IntroductionThe gut microbiota is the population of microorganisms (mainly bacteria and viruses, protozoa, and fungi) and their collective genetic material present in the gastrointestinal tract
The gut microbiota is the population of microorganisms and their collective genetic material present in the gastrointestinal tract
Analyzing the dissimilarity data in detail, they clearly showed that use of the V4 region applying the amplicon sequence variants (ASVs) approach for feature selection and VSEARCH for taxonomy assignment produces the most accurate results at the three studied taxonomic levels (Table 2)
Summary
The gut microbiota is the population of microorganisms (mainly bacteria and viruses, protozoa, and fungi) and their collective genetic material present in the gastrointestinal tract. The intestinal microbiota plays an important role in human health, and the disruption of its composition, named dysbiosis, has been proposed as one of the most important factors involved in gastrointestinal diseases and other illnesses [1]. 16S rRNA amplicon sequencing is used to profile its taxonomic composition; multiomic approaches have been proposed as the most accurate methods for study of the complexity of the gut microbiota. Results: The results showed that the sole use of V4 region sequencing with ASV identification and VSEARCH for taxonomy assignment produced the most accurate results The application of this workflow showed clear differences between fecal samples according to the type of feeding, which correlated with changes in the fecal metabolic profile. Conclusion: A multiomic approach using real fecal samples from 18 infants with different types of feeding demonstrated the effectiveness of the proposed 16S rRNA-amplicon sequencing workflow
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