The Bias Associated with Amplicon Sequencing Does Not Affect the Quantitative Assessment of Bacterial Community Dynamics

Abstract

The performance of two sets of primers targeting variable regions of the 16S rRNA gene V1–V3 and V4 was compared in their ability to describe changes of bacterial diversity and temporal turnover in full-scale activated sludge. Duplicate sets of high-throughput amplicon sequencing data of the two 16S rRNA regions shared a collection of core taxa that were observed across a series of twelve monthly samples, although the relative abundance of each taxon was substantially different between regions. A case in point was the changes in the relative abundance of filamentous bacteria Thiothrix, which caused a large effect on diversity indices, but only in the V1–V3 data set. Yet the relative abundance of Thiothrix in the amplicon sequencing data from both regions correlated with the estimation of its abundance determined using fluorescence in situ hybridization. In nonmetric multidimensional analysis samples were distributed along the first ordination axis according to the sequenced region rather than according to sample identities. The dynamics of microbial communities indicated that V1–V3 and the V4 regions of the 16S rRNA gene yielded comparable patterns of: 1) the changes occurring within the communities along fixed time intervals, 2) the slow turnover of activated sludge communities and 3) the rate of species replacement calculated from the taxa–time relationships. The temperature was the only operational variable that showed significant correlation with the composition of bacterial communities over time for the sets of data obtained with both pairs of primers. In conclusion, we show that despite the bias introduced by amplicon sequencing, the variable regions V1–V3 and V4 can be confidently used for the quantitative assessment of bacterial community dynamics, and provide a proper qualitative account of general taxa in the community, especially when the data are obtained over a convenient time window rather than at a single time point.

Performance Comparison of Illumina and Ion Torrent Next-Generation Sequencing Platforms for 16S rRNA-Based Bacterial Community Profiling

“…We found minimal variability among replicates (see Fig. S1 in the supplemental material), consistent with independent reports addressing the experimental variability of ampliconbased 16S rRNA surveys (32)(33)(34)(35)(36).…”

Evaluation of 16S rRNA amplicon sequencing using two next-generation sequencing technologies for phylogenetic analysis of the rumen bacterial community in steers

“…2. This has also been demonstrated in other studies that focus on bacterial community diversity analyses (Ibarbalz et al, 2014).…”

High-rate activated sludge communities have a distinctly different structure compared to low-rate sludge communities, and are less sensitive towards environmental and operational variables

“…The temporal scaling exponents for the high-rate (0.262, R 2 ¼ 0.960) and low-rate system (0.249, R 2 ¼ 0.968) were similar (p > 0.05) ( Supplementary Fig. S3), and fell within the lower range of values between 0.21 and 0.50 reported for activated sludge systems (Kim et al, 2013;Wells et al, 2011;Shade et al, 2013;Hai et al, 2014;Ibarbalz et al, 2014). The similarity of temporal scaling exponents of the high-and low-rate community is unexpected, given that these systems differed in species richness and selective pressure caused by differences in SRT.…”

“…Note that time may not be a true environmental factor, and community changes over time may reflect deterministic as well as neutral changes (Lynch and Neufeld, 2015) Assuming that this study included the environmental variables most relevant for the ecology of activated sludge communities (Valentin-Vargas et al, 2012;Wells et al, 2011;Hai et al, 2014;Ibarbalz et al, 2014), the percentage of unexplained variation was 52.5% in the high-rate and 44.1% in the low-rate system. This suggests that high-rate activated sludge communities are more shaped by neutral factors than low-rate communities.…”