Retrieval of a million high-quality, full-length SSU rRNA sequences without primer bias. fSSU-RNAseq

Small subunit (SSU) ribosomal RNA (rRNA) genes have been the standard phylogenetic markers for the study of microbial evolution and diversity for decades1,2. However, the essential reference databases of full-length rRNA gene sequences are underpopulated, ecosystem skewed, and subject to primer bias; which gives an incomplete view of the true diversity3–6. Here we present an approach that combines poly(A)-tailing and reverse transcription of SSU rRNA molecules with synthetic long-read sequencing, to generate high quality, full-length SSU rRNA sequences without primer bias in high-throughput. We applied the approach to complex samples from seven different ecosystems and obtained more than a million SSU rRNA sequences from all domains of life with an estimated raw error rate of 0.17%. We observed a high fraction of novel diversity including several deeply branching phylum level lineages related to the Asgard Archaea. This approach will allow expansion of the SSU rRNA reference databases by orders of magnitude and will enable a comprehensive census of the tree of life.