Do high throughput sequencing techniques provide the same phylogenetic result in anaerobic digestion system?

Anaerobic Digestion (AD) of organic matter is performed by an intricate set of microbial species which includes bacteria and archaea. Their activity and abundance is influenced by many operational parameters (e.g. temperature, pH, feedstock composition, etc.) and this can have a relevant effect on process efficiency. Due to the importance covered by the microbiome, numerous studies have been performed to investigate the microbial composition in biogas reactors. Investigations were performed both using traditional microbiological techniques based on isolation of microbes, and also with cultivation-independent approaches. Due to the difficulty in the isolation of strictly anaerobic species, cultivation-independent approaches won more approval among scientists. For the simplicity of use, the 16S rRNA amplicon sequencing was used in many different studies, however in the AD system it is still unverified if the use of degenerate primers during PCR amplification can provide an accurate representation of the microbial composition. To identify potential biases in 16S amplicon sequencing, we compared the results with those obtained using shotgun sequencing performed on total RNA and on genomic DNA. All the strategies were performed on the same samples and using the same sequencing strategy (Illumina). In order to minimize variability, both genomic DNA and total RNA were extracted simultaneously from the same samples. Three independent laboratory-scale reactors were used as biological replicates and were analysed before and after a perturbation of the system determined by oleate addition. This study evidenced that abundance of numerous genera (e.g. Methanofollis and Syntrophothermus) is systematically underestimated (<50 fold) in the analysis performed with 16S amplicons, suggesting potential biases in the amplification efficiency of universal primers.