A 16S rRNA gene Illumina–based barcoded assay design for high throughput characterisation of microbial communities from anaerobic digesters

High throughput sequencing of 16S/18S rRNA gene is becoming an indispensable tool to explore microbial community ecology. To date, most of the studies using next generation amplicon sequencing of microorganisms involved in the anaerobic digestion process (AD) are based on the 454 pyrosequencing. However, the cost per read obtained with the Illumina technology is currently less than 1/100 of that for the 454 pyrosequencing, thus enabling throughout sequencing and larger number of samples to be analysed per study (e.g. given the current Miseq output, around 100 000 reads per sample can be expected for a pool of 96 libraries). Moreover, the Illumina technology is less biased by the GC content of the template and currently allows for a relatively long sequence read of 600 bp (2 x 300bp). This consideration is particularly important, since longer sequences permit for more accurate assignment to a taxonomic group. Here, we designed and optimized an Illumina–based 16S rRNA amplicon approach for a high throughput characterization of microbial communities from different AD.