Evaluation of 16S rRNA primer sets for characterisation of microbiota in paediatric patients with autism spectrum disorder

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.

Multi-omic analysis along the gut-brain axis points to a functional architecture of autism

“…In total, we analyzed 597 ASD-control pairs. To reduce the batch effects and noise associated with primer choice in the 16S datasets, a major confounder in microbiome analyses, we restricted the 16S datasets to include only those targeting the variable region V4 of the bacterial ribosomal RNA, a region exhibiting higher heterogeneity and lower evolution rates than other variable regions[97, 98, 99]. Our analysis included 16S datasets obtained targeting the V4 region exclusively, the V3-V4 region, or the V4-V5 region.…”

A Journey on the Skin Microbiome: Pitfalls and Opportunities

“…This approach reduces diversity and simplifies the computational analyses, but it is not free from criticism [ 149 ]. For example, either the choice of the regions or the choice of the primers is known to affect the outcome deeply, introducing biases in the taxonomic picture, with artifactual under- or over-representation of some taxa [ 150 , 151 ]. Remarkably, it has also been demonstrated that few base pair mismatches can result in poor amplification of taxa otherwise abundant on the skin, such as Propionibacterium , especially when considering the V4 region [ 20 ].…”

Next-generation sequencing: insights to advance clinical investigations of the microbiome

“…As discussed, there are nine hypervariable regions in the 16S rRNA gene, and available primer sets typically amplify only a subset of these regions. Thus, the performance characteristics of the primer set chosen will influence the number of the analyzable reads ( 60 ) as well as the results of the analysis ( 61 ). For example, one study reports that the V4 primer set yields significantly more Bacteroides and lower Firmicutes reads than other primer sets tested; this is particularly notable given that the Bacteroidetes/Firmicutes ratio is a commonly reported metric ( 60 ).…”