Water raw sequence reads. freshwater metagenome

The limitation of 16S rRNA gene sequencing for microbial community analyses in fresh water is the inability to differentiate live (vital) and dead (inactive) cells, which can lead to false positive results in the absence of live microbes. Propidium monoazide (PMA) has been used in the past for selective removal of DNA from dead cells during downstream sequencing process. In comparison, 16S rRNA sequencing can target the active microbial cells in water as RNA is produced by metabolically active cells. The objective of this study was to compare the efficiency and sensitivity of DNA-based, PMA-based and RNA-based 16S rRNA gene/molecule Illumina sequencing in detecting live bacterial cells in water samples obtained from different sources (First Nation community drinking water; city of Winnipeg tap water; water from Red River, Manitoba, Canada; and experimentally spiked water samples).