Exploring the impact of bacterial DNA variation in fish microbiome studies

Next generation sequencing (NGS) has become a popular tool to investigate the bacterial microflora colonising fish. Despite the significant advances made in the field of NGS, technical issues still present challenges in studies employing this technology as they have been reported to introduce bias and distort the community compositions profiled in microbiome studies, ultimately reducing the accuracy and reproducibility of these meta-analyses. The following study applied 16S rRNA amplicon-sequencing to evaluate the impact of bacterial DNA (bDNA) variation on the analysis of intestinal microbiome communities in rainbow trout. In this study, authors optimised a 16S rRNA library preparation protocol which in addition to traditional steps, also included quantification bDNA yield from gastrointestinal digesta and titrating 16S rRNA libraries to standard bDNA concentration. This approach generated 16S rRNA libraries with improved sequencing read yields compared with non-titrated libraries as well as displaying a general trend of increased microbial diversity. In addition, titrating 16S rRNA libraries was associated with reductions in OTUs which had high representation in non-titrated 16S rRNA libraries and were observed to dominate a negative sequencing control. Together, the results of the present study demonstrate the potential of titrating bDNA in 16S rRNA libraries in microbiome studies as an additional measure to improve sequencing yield and enrichment of intestinal communities, whilst mitigating against the influence of foreign microbial DNA contamination in intestinal samples.