Experimental Metagenome on MinION

This is an artificial metagenome created by pooling various proportions of DNA from four microbe cultures to simulate a simple metagenome for analysis by Oxford nanopore MinION. Single-species cultures of Escherichia coli, Pseudomonas fluorescens, Microcystis aeruginosa, and Synechococcus elongatus were grown to log phase then harvested for DNA extraction. Following mechanical shearing, templates were prepared using either equimolar amounts of each species or combinations of DNAs where one species was rare (0.05%). Libraries were created using his-tag bead purification according to MinION specifications and libraries were sequenced using MinION R7.x flow cells. For validation, a final library was prepared using a mock microbial community with 20 species included in “staggered” proportions (obtained from ATCC, the mixture had 1,000 to 1,000,000 16S rRNA operon copies per organism per µL of material supplied).