Difficulties Of Enterobacteriaceae Genome Annotation In Deciphering Gastrointestinal Microbiome Datasets Obtained By 16S RRNA Gene Amplicon Sequencing

Abstract

Sequencing of the 16S rRNA gene amplicon is the cornerstone of the method for studying diverse bacteria in complex microbial communities. However, its use is complicated by an error rate of 10–17% when annotating 16S rRNA gene sequences. In our study, we examined the degree of accuracy of the taxonomic database of Enterobacteriaceae, compiled using the SILVA 132 reference database and a previously obtained dataset, viz. the microbiome of the gastrointestinal tract in adolescents with normal body weight and obesity. Material and Methods — In this study, previously obtained 16S rRNA gene amplicon sequencing data were used, and the deciphering was carried out using the QIIME2 2019.4 platform. Phylogenetic analysis was performed using MEGA X software. Results — Phylogenetic analysis of this family based on the studied V3–V4 fragment was hampered by polyphyly among some genera, and for half of the variants of the amplicon sequences it was not possible to clarify their genus. Statistical analysis did not reveal significant differences between the samples. Conclusion — Although the average values of bacterial genera in the studied groups intuitively differed from each other, statistical analysis did not reveal significant differences between the samples. However, it can be assumed that a more detailed study of taxonomic diversity, taking into account factors, such as enterotype, duration of breastfeeding and family history, may reveal differences in the frequency distribution.