@article {Ben{\'\i}tez-P{\'a}ez117143, author = {Alfonso Ben{\'\i}tez-P{\'a}ez and Yolanda Sanz}, title = {Multi-locus and ultra-long amplicon sequencing approach to study microbial diversity at species level using the MinION{\texttrademark} portable nanopore sequencer}, elocation-id = {117143}, year = {2017}, doi = {10.1101/117143}, publisher = {Cold Spring Harbor Laboratory}, abstract = {Background The miniaturised and portable DNA sequencer MinION{\texttrademark} has demonstrated great potential in different analyses such as genome-wide sequencing, pathogen outbreak detection and surveillance, human genome variability, and microbial diversity. In this study, we tested the ability of the MinION{\texttrademark} platform to perform ultra-long amplicon sequencing in order to design new approaches to study microbial diversity using a multi-locus approach.Results Using R9 chemistry, we generated more than 17,000 reads in a single sequencing run of 40h. After compiling a robust database by parsing and extracting the rrn bacterial region from more than 67,000 complete or draft bacterial genomes, we were able to demonstrate that the data obtained during sequencing of the ultra-long amplicon in the MinION{\texttrademark} device was sufficient to study two mock microbial communities in a multiplex manner and to almost completely reconstruct the microbial diversity contained in the HM782D and D6305 mock communities.Conclusions Although nanopore-based sequencing produces reads with lower per-base accuracy compared with other platforms, we presented a novel approach consisting of multi-locus and ultra-long multiplex amplicon sequencing using the MinION{\texttrademark} MkIb DNA sequencer and R9 chemistry that helps to overcome the main disadvantage of this portable sequencing platform. Despite the technical issues impairing generation of 2D reads with more per-base accuracy, when processed with adequate alignment filters, the 1D reads obtained this way were sufficient to characterize the microbial species present in each mock community analysed. Improvements in nanopore chemistry, such as minimising base-calling errors and new library protocols able to produce rapid 1D libraries, will provide more reliable information. Such data will be useful for more comprehensive and faster specific detection of microbial species and strains in complex ecosystems.ECEuropean CommissionENAEuropean Nucleotide ArchiveHDFHierarchical Data FormatITSinternal transcribed spacerNCBINational Center for Biotechnology InformationONTOxford Nanopore TechnologiesPCRPolymerase Chain ReactionrDNADNA encoding for the Ribosomal RNArRNARibosomal RNArrnthe DNA region containing the 16S and 23S bacterial rRNA genes and its respective ITS regionSINASILVA Incremental AlignerUSBUniversal Serial Bus}, URL = {https://www.biorxiv.org/content/early/2017/03/15/117143}, eprint = {https://www.biorxiv.org/content/early/2017/03/15/117143.full.pdf}, journal = {bioRxiv} }