DNA Microbial Screening of Air Filters Using Next-generation Sequencing

Air tilters collected as part of the Bio Watch program contain a large variety of microorganisms and large eukaryotes (humans. rodents. insects) present in proximity to the air tilters. DNA isolation and sequencing methods need to be optimized for specific DNA concentration and sequencing depth to ensure that all microbes can he properly sequenced in the presence of complex backgrounds. The quality and quantity of the DNA sample are crucial factors that determine the success of the sequencing experiment. A high-quality DNA isolation protocol must be developed to extract pure and intact microbial DNA from the sample, free of contaminants and inhibitors. The library preparation requirements need to he optimized to ensure that the DNA fragments are of the right size and quantity to initiate sequencing, avoiding over- and under-clustering sequencing issues. The sequencing depth required will depend on the complexity of the microbial community and the number of reads necessary to obtain a robust representation of the community. An optimization will be performed to identify optimal extraction and sample preparation conditions and develop standard operating procedures. We will employ robust analysis of sequenced data to ensure that the results arc accurate, reliable, and reproducible.