16S amplicon sequencing of AD microbiomes

The microbial community responsible for the biogas-producing anaerobic digestion (AD) process used to recover energy from waste is poorly characterised and likely contains many organisms not required for gas generation. Metagenomics approaches offer insights into AD communities that could potentially be exploited to improve the conversion of biomass to biogas, but distinguishing between the biological and technical variability of samples from different sources makes generalisation of results challenging. To determine how different DNA extraction methodologies and sequencing approaches influence the perceived microbiome, we analysed technical replicates of 16S rRNA amplicons and whole shotgun metagenomes from different AD systems. Here we show that diversity measurements of the microbiome in AD samples were strongly influenced by extraction regimes and analysis methodology. 16S rRNA amplification resulted in over- and under-estimation of several taxonomic phyla compared to a PCR-free metagenomics approach. Samples extracted using mechanical methods displayed significantly lower diversity and fewer observed phylotypes than samples extracted by chemical and thermal lysis, which produced higher molecular weight DNA and showed the greatest diversity in community profiles. Our results highlight that the most commonly used extraction and analysis protocols provide a skewed view of the diversity and species richness of the AD microbiome which is likely to extent to other samples. We suggest that methodological differences could be a significant source of variability among AD, and other, metagenomics studies.