Optimization of Covaris Settings for Shearing Bacterial Genomic DNA by Focused Ultrasonication and Analysis Using Agilent 2200 TapeStation

Shearing of bacterial gDNA within a specific size range prior to sequencing library construction is a critical step in Next Generation Sequencing workflows. The quality control of the sheared bacterial gDNA is required in large multiplexed formats for large volume workflows, such as those used in the 100K Pathogen Genome Sequencing Project. Using the Covaris E220 instrument, the power and treatment time were varied to determine the effect on the optimal fragment size (150–350 bp) in the resulting sheared gDNA of four bacterial pathogens: Salmonella enterica subsp. enterica serovar Saint Paul strain Sp3 and serovar Typhimurium strain LT2, Klebsiella sp. and Vibrio spp. DNA fragment quantification and sizing were measured using an Agilent 2200 TapeStation system, and Agilent High Sensitivity D1000 ScreenTape assay. The 2200 TapeStation system was suitable to determine size distribution after fragmentation of gDNA in a 96-well plate format, a format suitable for high-throughput workflow and compatible with shearing technologies that use a 96-well plate multiplexed format. This approach enabled the measurement of gDNA and sheared DNA using a single technology. Lenore Kelly Agilent Technologies, Inc. Santa Clara, CA, USA