Choice of library preparation and its effects on sequence quality, assembly, and precise in silico prediction of virulence genes in shiga toxin producing E.coli

Whole genome sequencing (WGS) provides essential public health information and is used worldwide for pathogen surveillance, epidemiology, and source tracking. The sequencing of foodborne pathogens is commonly performed with Illumina sequencing chemistry to obtain data with high accuracy. The choice of library preparation method for highly complex organisms is very critical and can affect the final data output.

The majority of Illumina sequencing platforms use rapid library preparation such as Nextera XT (transposon-based technology) (Illumina San Diego, CA), but this preparation has the potential to miss randomly distributed segments of genomes that might be important for downstream analyses. The Illumina Nextera DNA Prep library preparation kit, the successor of Nextera XT, shows better overall coverage of the complete genome.

This study compared the quality of sequence data generated using Nextera XT and Nextera DNA Prep kits for DNA library preparation on an Illumina MiSeq, using a set of 30 O121:H19 shiga-toxin positive Escherichia coli strains isolated from flour during a 2016 outbreak. The performance of the two kits were evaluated using several metrics including sequencing quality, assembly quality, uniformity of genome coverage, and virulence gene identification.

Overall, the results showed that in all of the analysed metrics, the Nextera DNA Prep kit performed outstanding in comparison to Nextera XT. The Nextera DNA Prep kit allowed for comprehensive detection of all virulence genes, which is of extremely high importance for making an educated assessment of the virulence potential of Escherichia coli.

This comprehensive side-by-side comparison will be of significance for those interested in improving their sequencing workflow for STECs and the determination of health risks using WGS data.