High throughput sequencing of full-length SSU rRNA sequences from complex microbial communities without primer bias and how it affects our ability to study microbial ecology

The small subunit (SSU) ribosomal RNA (rRNA) genes have been used to study of microbial diversity and evolution for the last 30 years. Today, databases containing full-length SSU rRNA reference sequences remains fundamental for many core tools in microbial ecology, such as community profiling using 16S/18S amplicon sequencing and in-situ studies based on fluorescence in situ hybridization (FISH) microscopy. The quality of the data produced relies heavily on the reference databases used, and it is widely recognized that the current databases are underpopulated, ecosystem skewed, and subject to primer bias. This is a consequence of the low throughput, primer based methods used to generate the sequences. Therefore, we developed a method which combines reverse transcription of environmental full-length SSU rRNA sequences and Illumina based synthetic long-read sequencing to obtain high quality, full-length SSU rRNA sequences in a high throughput manner. In our initial trials, we obtained more than 40,000 high quality SSU rRNA sequences (> 1200 bp) representing all domains of life from five different ecosystems (human gut, soil, fresh water, anaerobic digestion and activated sludge), showing the general application and the throughput of the method. Here we describe how the method works and demonstrate how the access to comprehensive ecosystem specific SSU databases affect our ability to study microbial ecology.