High-Throughput Absolute Quantification Sequencing Advances Characterisation of Microbial Community Ecology.

Abstract

High-throughput sequencing has been extensively used in microbial ecology research, but this technology typically generates semi-quantitative relative abundance data. The discrepancies of community dynamics delivered by relative and absolute abundances have long been recognised by microbial ecologists. However, few studies have considered the discrepancies and their potential effects on related ecological interpretations. Here, the absolute copy numbers (absolute abundances) of taxa were quantified using a 'spike-in' based 16S absolute quantification sequencing to investigate the dynamics, co-occurrence patterns, and ecological assembly processes of microbial community in the river-connected Lake Dongting, Hunan province, China. Routine sequencing data analysis based on relative abundance was also obtained for comparison. The results indicated that outcomes based on relative and absolute abundances were comparable at the community level but differed significantly at the population level when dynamics and interactions of specific taxa were of interest. Specifically, at the population level, the dynamics of individual taxa were usually masked by their relative abundances, particularly for abundant taxa. The correlation-based co-occurrence network constructed from relative abundance largely underestimated the importance of rare taxa, consisted of massive false negative connections, and was less stable than the one constructed using absolute abundance. At the community level, the diversity and composition of microbial communities in Lake Dongting exhibited significant temporal rather than spatial variations, and temperature was determined to be the most important factor shaping microbial community composition and assembly processes, regardless of which abundance data were used. Nonetheless, relative abundance data yielded stronger environmental and deterministic effects on microbial assembly than absolute abundance. In summary, our study highlights the importance of incorporating absolute quantification to unveil the underlying microbial ecological patterns masked by relative abundance.