Reproducibility of next-generation-sequencing-based analysis of a CRISPR/Cas9 genome edited oil seed rape

Abstract

Next-generation-sequencing (NGS) becomes increasingly important for laboratories tasked with the detection of genetically modified organisms (GMOs) in food, feed and seeds. Its implementation into standardized workflows demands reliable intra- and inter-laboratory reproducibility. Here, we analyze the reproducibility of short- and long-read targeted NGS and long-read whole genome sequencing (WGS) data between three independent laboratories. Replicate samples were submitted for sequencing and comparatively analyzed. The targeted-NGS-samples consisted of oil seed rape (OSR) sampled from a commodity shipment spiked with a genome edited (GE) OSR and the WGS-samples consisted of leaf material from the GMOs’ parental line. All laboratories delivered highly reproducible high-quality targeted NGS data with little variation. The detection of GMO-related sequences works well regardless of the facility, while the mapping to the complex genome is superior using long read data. Long read WGS is currently not suitable for routine use in enforcement laboratories, due to a large inter-laboratory variation.

Keywords

• NGS reproducibility
• NGS data quality
• NGT (New Genomic Techniques)
• GE (Gene Editing)
• GMO (Genetically Modified Organism)
• Detection