David Greig from Public Health England presented a session in London Calling 2019 with the title “Comparison of single nucleotide variants identified by Illumina and Oxford Nanopore technologies in the context of a potential outbreak of Shiga toxin producing.” They explained how they work on pathogen surveillance and focus on STEC: Shiga toxin-producing Escherichia coli. STEC can produce fatal complications. The team receives samples and performs a series of diagnostic PCRs. Then, they extract DNA for sequencing with a QIAGEN QiaSymphony. They sequence Nextera libraries with Illumina. The raw reads are analyzed to perform species identification and then MLST using the MOST program. Gene finder is used to detect genes encoding serotypes and toxins. A mapping-based approach is used to map to a reference and call variants. SnapperDB stores variants and compares samples within that database. Greig managed to introduce Oxford Nanopore sequencing into the pipeline. They used the Promega Wizard Genomic Purification kit with extra incubation time and less vigorous pipetting. Then, they prepare rapid sequencing libraries with the Rapid Barcoding kit. The bioinformatics pipeline was modified to include Porechop for quality control and adapter removal. Kraken was used for species identification. The team optimized variant calling to include parameters for ONT data. The ONT data was calling more variants than the Illumina sequencing data. They learned that 94/95 variants from ONT data were methylated. The team tested a real scenario in which two STEC samples arrived one evening and were sequenced on Nanopore and Illumina devices. The Nanopore results were obtained within 17 hours. The team concluded that the workflow can be successfully implemented with Oxford Nanopore Technologies devices and sequencing. They are applying the approach for outbreak surveillance analyses. I appreciate their work in optimizing the process and sharing their process. It helped me better understand the challenges with variant analysis for STEC and other pathogens that are public health risks.
