Microbiome Characterization and Genome Sequencing Using the Oxford Nanopore MinION Platform

The advent of high-throughput sequencing led to breakthroughs in microbiome analysis and to an explosion of bacterial genomes being released in public repositories. However, many microbiotas cannot be cultured in the lab without introducing drastic changes in the underlying bacterial communities and the distribution of genomes present in online repositories is heavily skewed towards organisms that are amenable to culturing. Those that cannot (or should not in the case of dangerous pathogens) be cultured are often underrepresented and culture-free methods require sequencing technologies capable of starting from tiny amounts of input DNA. While Illumina libraries starting from 1 ng of DNA have been available for a few years, investigating microbiomes using short reads is difficult. Bacterial genomes sequenced with this platform are also often incomplete due to the presence of repetitive elements. Longer sequencing reads such as those provided by the PacBio platform are very valuable when investigating complex bacterial communities and genomes, but the amounts of material required by this platform (in the 5-10 ug range) limits what can be done with it. One of the appeals of the latest long read technology from Oxford Nanopore is that it can starts from minute amounts of DNA. We used 16S amplicon-based nanopore sequencing to investigate diseased microbiomes and developed a quick pipeline to analyze the resulting data. We also used a hybrid illumina/nanopore approach to sequence various staphylococcal and streptococcal species from limited amounts of genomic DNA. Both approaches are simple enough to be performed by undergraduates with limited supervision.