Inconsistent patterns of microbial diversity and composition between highly similar sequencing protocols: a case study with reef-building corals

16S rRNA gene profiling (amplicon sequencing) is a popular technique for understanding host-associated and environmental microbial communities. Most protocols for sequencing amplicon libraries follow a standardized pipeline that can differ slightly depending on laboratory facility and user. Given that the same variable region of the 16S gene is targeted, it is generally accepted that sequencing output from differing protocols are comparable and this assumption underlies our ability to identify universal patterns in microbial dynamics through meta-analyses. However, discrepant results from a combined 16S rRNA dataset prepared by two labs whose protocols differed only in DNA polymerase and sequencing platform led us to scrutinize the outputs and challenge the idea of confidently combining them for standard microbiome analysis. Using technical replicates of reef-building coral samples from two species, Montipora aequituberculata and Porites lobata , we evaluated the consistency of alpha and beta diversity metrics between data resulting from these highly similar protocols. While we found minimal variation in alpha diversity between platform, significant differences were revealed with most beta diversity metrics, dependent on host species. These inconsistencies persisted following removal of low abundance taxa and when comparing across higher taxonomic levels, suggesting that bacterial community differences associated with sequencing protocol are likely to be context dependent and difficult to correct without extensive validation work. The results of this study encourage caution in the statistical comparison and interpretation of studies that combine rRNA sequence data from distinct protocols and point to a need for further work identifying mechanistic causes of these observed differences. Importance Amplicon sequencing remains a popular technique for characterizing organism and environmental microbiomes. The publication of sequence data from microbiome studies on open-access repositories provides an opportunity to identify universal patterns in microbial dynamics. To this end, it has been widely accepted that sequencing output from differing protocols are comparable and can be combined for analysis, so long as the same gene region is targeted. While most protocols for amplicon sequencing follow standardized pipelines, they can differ slightly between laboratory facility and user. In this study, we compared technical replicates of coral samples to evaluate the efficacy of combining organism-associated microbial datasets derived from two differing protocols. We found inconsistencies in the differences between bacterial communities, which persisted following data manipulations intended to increase comparability. These results suggest caution must be taken in the statistical comparison and interpretation of studies that combine data derived from distinct protocols.