Abstract
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 gene 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 gene sequence data from distinct protocols and point to a need for further work identifying mechanistic causes of these observed differences.
Highlights
Microbial ecology has benefited tremendously from recent technological advances in areas such as high throughput sequencing (Schuster, 2008)
To test whether coral microbiome sequence data generated from the two protocols were comparable, we analyzed paired sequence libraries, combined for comparative downstream analyses, using four standard variables for assessing microbiome variations at both the amplicon sequence variants (ASVs) and OTU levels: alpha diversity, beta diversity, beta dispersion, and differential abundance measures
In the OTU dataset, two P. lobata HiSeq protocol samples contained less than 998 reads and were removed along with their MiSeq protocol counterparts leaving 22 samples for comparison consisting of 953,396 reads and 2,174 OTUs
Summary
Microbial ecology has benefited tremendously from recent technological advances in areas such as high throughput sequencing (Schuster, 2008). Available sequencing data resulting from these initiatives provide opportunities for the production of meta-analyses and for researchers with smaller scale projects to make comparisons and/or combine their dataset with a much broader set of samples, allowing increased impact of their finer sequencing efforts. The EMP standardized protocols for 16S rRNA gene sequencing are optimized for repeatedly processing large numbers of samples and benefit from automation and high throughput sequencing on an Illumina HiSeq platform. Smaller numbers of samples are more often sequenced on the Illumina MiSeq platform due to cost effectiveness and increased read length. Meta-analyses of 16S rRNA gene data across microbial study systems already utilize crossprotocol and platform data that are stored in public repositories (see Duvallet et al, 2017; Pammi et al, 2017; Mo et al, 2020)
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