Direct metatranscriptome RNA-seq and multiplex RT-PCR amplicon sequencing on Nanopore MinION - promising strategies for multiplex identification of viable pathogens in food

Authors: YANG, MANYUN - University Of Massachusetts; COUSINEAU, ALYSSA - New England Biolabs; LIU, XIAOBO - University Of Massachusetts; SUN, DANIEL - Brandeis University; LI, SHAOHUA - Us Food & Drug Administration (FDA); GU, TINGTING - University Of Massachusetts; LUO, SUN - New England Biolabs; Luo, Yaguang - Sunny; XU, MING-QUN - New England Biolabs; ZHANG, BOCE - University Of Massachusetts

Submitted to: Frontiers in Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/10/2020
Publication Date: 4/9/2020
Citation: Yang, M., Cousineau, A., Liu, X., Sun, D., Li, S., Gu, T., Luo, S., Luo, Y., Xu, M., Zhang, B. 2020. Direct metatranscriptome RNA-seq and multiplex RT-PCR amplicon sequencing on Nanopore MinION - promising strategies for multiplex identification of viable pathogens in food. Frontiers in Microbiology. https://doi.org/10.3389/fmicb.2020.00514.
DOI: https://doi.org/10.3389/fmicb.2020.00514

Interpretive Summary: Pathogen contamination to food products poses significant threats to public health and the financial wellbeing of the industry. Technologies that enable early detection of food-borne human pathogens are urgently needed by the food industry. In this study, we report a novel strategy to achieve real-time multiplex identification of viable pathogen in food. By optimizing universal Nanopore sample extraction and library preparation protocol, we improved the accuracy of rapid detection of multiple harmful bacteria in the complex food system. This research benefits pathogen detection method developers and the food industry for timely detection and removal of contaminated foods from the supply chain with improved public health outcome.

Technical Abstract: Viable pathogenic bacteria are major biohazards that pose a significant threat to the public health. Despite the recent developments in detection platforms, multiplex identification of viable pathogens in food remains a major challenge. A novel strategy is developed through direct metatranscriptome RNA-seq and multiplex RT-PCR amplicon sequencing on Nanopore MinION to achieve real-time multiplex identification of viable pathogen in food. Specifically, this study reports an optimized universal Nanopore sample extraction and library preparation protocol applicable to both Gram-positive and Gram-negative pathogenic bacteria, demonstrated using a cocktail culture of E. coli O157:H7, Salmonella enteritidis, and Listeria monocytogenes. Further evaluation and validation confirmed the accuracy of direct metatranscriptome RNA-seq and multiplex RT-PCR amplicon sequencing using Sanger sequencing and selective media. The study also included a comparison of different bioinformatic pipelines for metatranscriptomic and amplicon genomic analysis. MEGAN without rRNA mapping showed the highest accuracy of multiplex identification using the metatranscriptomic data. EPI2ME also demonstrated high accuracy using multiplex RT-PCR amplicon sequencing. In addition, a systemic comparison was drawn between Nanopore sequencing of the direct metatranscriptome RNA-seq and RT-PCR amplicons. Both methods are comparable in accuracy and time. Nanopore sequencing of RT-PCR amplicon has higher sensitivity, but Nanopore metatranscriptome sequencing excels in read length and dealing with complex microbiome and non-bacterial transcriptome backgrounds. To the best of our knowledge, this is the first report of metatranscriptome sequencing of cocktail microbial RNAs on the emerging Nanopore platform. Direct RNA-seq and RT-PCR amplicons sequencing of metatranscriptome enable the direct identification of nucleotide analogs in RNAs.