Event Description
Norman Dovichi, PhD,
University of Notre Dame
“Capillary Zone Electrophoresis Coupled With Next-Generation DNA Sequencing for Deep Microbiome Analysis”
The extreme complexity of environmental microbiomes presents formidable challenges to their characterization by next-generation sequencing. Highly abundant species dominate the sequencing data, and huge numbers of sequences must be generated to identify rare species. Microbiome analysis will benefit from segregation of rare species away from highly abundant species. We have coupled capillary zone electrophoresis with a sterile fraction collector to separate an environmental microbiome. Fractions were deposited onto a Petri dish for characterization of culturable microbes, into wells of a microtiter plate for next-generation sequencing of 16sRNA, and onto an E. coli lawn to visualize phage. While some wells of the microtiter plate were dominated by a small number of species, most fractions generated diverse species representation. Analysis of the fractionated microbiome generated 660 operational taxonomic units (OTUs) that mapped to known species, compared with 258 OTUs from the unfractionated sample. One well of the fractionated sample generated 419 OTUs, which is 66% larger than from the unfractionated sample; fractionation moved highly abundant species into different wells of the microtiter plate, allowing identification of rarer species. These results are the first demonstration of deep 16sRNA sequencing of a fractionated microbiome, and the first demonstration of a significant increase in the number of OTU identifications compared to analysis of the intact microbiome.
|
