Abstract
intestinal microbiota is becoming a significant marker that reflects differences between health and disease status also in terms of gut-brain axis communication. Studies show that children with autism spectrum disorder (ASD) often have a mix of gut microbes that is distinct from the neurotypical children. Various assays are being used for microbiota investigation and were considered to be universal. However, newer studies showed that protocol for preparing DNA sequencing libraries is a key factor influencing results of microbiota investigation. The choice of DNA amplification primers seems to be the crucial for the outcome of analysis. In our study, we have tested 3 primer sets to investigate differences in outcome of sequencing analysis of microbiota in children with ASD. We found out that primers detected different portion of bacteria in samples especially at phylum level; significantly higher abundance of Bacteroides and lower Firmicutes were detected using 515f/806r compared to 27f/1492r and 27f*/1495f primers. So, the question is whether a gold standard of Firmicutes/Bacteroidetes ratio is a valuable and reliable universal marker, since two primer sets towards 16S rRNA can provide opposite information. Moreover, significantly higher relative abundance of Proteobacteria was detected using 27f/1492r. The beta diversity of sample groups differed remarkably and so the number of observed bacterial genera.
Highlights
Abstract intestinal microbiota is becoming a significant marker that reflects differences between health and disease status in terms of gut-brain axis communication
2371 different bacterial genera were detected in stool samples of 10 paediatric patients suffering from autism spectrum disorder (ASD)
Significant difference in number of bacterial genera was detected in comparison of primer set#2/set#1 (p = 6,89095E-05), set#2/set#3 (p = 2,63,482-E04), but not set#1/set#3 (p = 0.07)
Summary
Abstract intestinal microbiota is becoming a significant marker that reflects differences between health and disease status in terms of gut-brain axis communication. We have tested 3 primer sets to investigate differences in outcome of sequencing analysis of microbiota in children with ASD. Despite the fact that MPS technologies allow analysis of whole bacterial genomes, sequencing of 16S rRNA has become a gold standard in microbiota studies[10] These genes are of suitable length and structure for phylogenetic analysis[11]. New studies showed that amplification of different variable regions of 16S rRNA genes may result in different outcome and that results may be highly variable depending on primer set used It is well-known that full length 16S rRNA gene sequence can provide the most specific phylogenetic analysis. We tested 3 different primer sets for investigating microbiota of ASD patients For this purpose, stool was chosen as a suitable biological sample, as accessible in the children with ASD. The quality and the quantity of the detected bacterial genera differed between three primer sets
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