Microbial diversity based on a multilocus and long amplicon sequencing approach using MinION(TM) sequencer.

We have recently explored the performance of MinION™ device regarding microbial diversity of complex samples demonstrating that data obtained from sequencing of nearly-full 16S rRNA gene amplicons is feasible to study microbial communities through nanopore technology. We wanted to move a step forward in this type of strategy by designing a multi-locus and ultra-long amplicon sequencing method to study the microbial diversity. As a consequence, we present a study of the 16S, 23S, and the internal transcribed spacer (ITS, that frequently encodes tRNA genes) simultaneous sequencing, using MinION™ MkI device and R9 chemistry, with prior generation of ~4.5kb DNA fragments by amplifying the nearly-full operon encoding the ribosomal RNA genes in bacteria, the rrn operon (rrn hereinafter). We have studied the rrn of two mock microbial communities composed of genomic DNA from 20 and 8 different bacterial species, obtained respectively from BEI Resources and ZYMO Research Corp., using the MinIONTM sequencing platform.