Abstract
We compared the consistency, accuracy and reproducibility of next-generation short read sequencing between ten laboratories involved in food safety (research institutes, state laboratories, universities and companies) from Germany and Austria. Participants were asked to sequence six DNA samples of three bacterial species (Campylobacter jejuni, Listeria monocytogenes and Salmonella enterica) in duplicate, according to their routine in-house sequencing protocol. Four different types of Illumina sequencing platforms (MiSeq, NextSeq, iSeq, NovaSeq) and one Ion Torrent sequencing instrument (S5) were involved in the study. Sequence quality parameters were determined for all data sets and centrally compared between laboratories. SNP and cgMLST calling were performed to assess the reproducibility of sequence data collected for individual samples. Overall, we found Illumina short read data to be more accurate (higher base calling accuracy, fewer miss-assemblies) and consistent (little variability between independent sequencing runs within a laboratory) than Ion Torrent sequence data, with little variation between the different Illumina instruments. Two laboratories with Illumina instruments submitted sequence data with lower quality, probably due to the use of a library preparation kit, which shows difficulty in sequencing low GC genome regions. Differences in data quality were more evident after assembling short reads into genome assemblies, with Ion Torrent assemblies featuring a great number of allele differences to Illumina assemblies. Clonality of samples was confirmed through SNP calling, which proved to be a more suitable method for an integrated data analysis of Illumina and Ion Torrent data sets in this study.
Highlights
Whole genome sequencing is a high resolution, high-throughput method for the molecular typing of bacteria
We present the results of an interlaboratory study for short-read bacterial genome sequencing with ten participating laboratories from German-speaking countries initiated by the §64 German Food and Feed Code (LFGB) working group “next-generation sequencing (NGS) Bacterial Characterization” chaired by the Federal Office of Consumer Protection and Food Safety (BVL)
Since base calling and quality prediction algorithms for Illumina and Ion Torrent are different, Ion Torrent reads are usually assessed with a Q score of ≥Q20, hampering a direct comparison
Summary
Whole genome sequencing is a high resolution, high-throughput method for the molecular typing of bacteria. Which sequencing platform different laboratories choose to acquire is dependent on financial resources, and on individual needs and routine applications, with throughput, error rates/error types, read lengths and run time as the main concerning parameters. This leads to an increased diversification of the sequencing community (Moran-Gilad et al, 2015), creating a natural competition between producers, which benefits users through an ongoing improvement of technology and equipment. The interlaboratory study was carried out by the German Federal Institute of Risk Assessment (BfR) in 2019, with the aim to answer the question whether different WGS technology platforms provide comparable sequence data, taking into account the routine sequencing procedures established in these laboratories
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have