Optimisation of 16S rDNA amplicon sequencing protocols for microbial community profiling of anaerobic digesters

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 a TruSeq PCR free metagenome library was used to produce a reference community profile for comparison of the 16S rRNA gene amplicon profiles prepared from the same DNA extract. All libraries were sequenced on our in-house Illumina platform.
The DNA extraction showed to be a compromise between DNA yield and DNA integrity, with optimal parameters involving four times 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 of extraction level replicates showed that DNA extraction, PCR, and sequencing had a high level of reproducibility. Thus allowing one to distinguish even closely related communities. Using dual-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.