Obtaining representative community profiles of anaerobic digesters through optimisation of 16S rRNA amplicon sequencing protocols

A reliable and reproducible method for identification and quantification of the microorganisms
involved in biogas production is important for the study and understanding of the microbial
communities responsible for the function of anaerobic digester systems. DNA based
identification using 16S rRNA gene amplicon sequencing is rapid, cheap, high throughput, and
has high taxonomic resolution. However, biases are introduced in multiple steps of this
approach, including non-representative DNA extraction and uneven taxonomic coverage of
selected PCR primers, potentially giving a skewed view of the community composition . As
such sample specific optimisation and standardisation of DNA extraction, as well PCR primer
selection, are essential to minimising the potential for such biases.
The aim of this study was to develop a protocol for optimized community profiling of anaerobic
digesters. The FastDNA SPIN kit was selected and the mechanical lysis parameters optimised
for extraction of genomic DNA from mesophilic and thermophilic anaerobic digester samples.
Different primer sets were compared for targeting the archaea and bacteria, both together and
individually . Shotgun sequencing of the metagenome was used to produce a reference
community profile for comparison of the 16S rRNA gene amplicon profiles prepared from the
same DNA samples. All samples were run on our in-house Illumina platform.
The DNA extraction showed to be a compromise between DNA yield and DNA integrity, with
optimal parameters involving harsher lysis conditions than recommended for the commercial
kit, and appeared to be similar for both the mesophilic and thermophilic reactor biomass
samples. The community profiling was found to be greatly influenced by the selected PCR
primers, and showed that in silico designed primers need experimental testing and validation.
The amplicon sequencing showed that DNA extraction and PCR had a high level of
reproducibility. Using indexed primers and liquid handling robotics to prepare multiple
samples in a reproducible manner makes amplicon sequencing a tool that provides a cheap and
fast link between microbial community structure and function. In conclusion, we have
optimised the different steps providing a promising approach for high throughput community
profiling of anaerobic digesters.