Eric Lee’s scientific contributions

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.

The initial step in Next Generation Sequencing is to construct a library from genomic DNA. To gain the optimum result, extracted DNA must be of high molecular weight with limited degradation. High-throughput sequencing projects, such as the 100K Pathogen Genome Project, require methods to rapidly assess the quantity and quality of genomic DNA extracts. In this study, assessment of the applicability of the Agilent 2200 TapeStation was done using genomic DNA from nine foodborne pathogens using several accepted high-throughput methods. The Agilent 2200 TapeStation System with Genomic DNA ScreenTape and Genomic DNA Reagents was easy to use with minimal manual intervention. An important advantage of the 2200 TapeStation over other high-throughput methods was that high molecular weight genomic DNA quality and quantity can be quantified apart from lower molecular weight size ranges, providing a distinct advantage in the library construction pipeline and over other methods available for this important step in the Next Generation Sequencing process.