Direct Metatranscriptome RNA-seq and Multiplex RT-PCR Amplicon Sequencing on Nanopore MinION – Promising Strategies for Multiplex Identification of Viable Pathogens in Food

Abstract

<jats:title>Abstract</jats:title><jats:p>Viable pathogenic bacteria are major biohazards that pose a significant threat to food safety. 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 <jats:italic>E. coli</jats:italic> O157:H7, <jats:italic>Salmonella enteritidis</jats:italic>, and <jats:italic>Listeria monocytogenes</jats:italic>, which were selected based on their impact on economic loss or prevalence in recent outbreaks. 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, which is highly informative for determining microbial identities while detecting ecologically relevant processes. The information pertained in this study could be important for future revelatory research, including predicting antibiotic resistance, elucidating host-pathogen interaction, prognosing disease progression, and investigating microbial ecology, etc.</jats:p>

Nanopore sequencing and its application to the study of microbial communities

“…In fact, RNA sequencing provides additional information, such as the functionality of AMR genes, allowing to identify a situation where the resistance gene is present but not transcribed, and therefore does not generate a resistant phenotype [106] . Metatranscriptomics is also very useful for the identification of viable pathogens since DNA-based approaches are unable to differentiate between viable and unviable bacterial cells [117] . This approach is particularly important in the detection of food pathogens, where food processing and storage often kill bacteria cells without removing their genomic DNA.…”

“…Direct RNA sequencing has recently been compared to multiplex real-time PCR amplicon sequencing for this purpose. Results suggest it to be especially applicable to complex microbiomes because it does not require assay customization for specific biohazards when a complete database is used during the bioinformatic analysis step [117] . Other applications of metatranscriptomics using nanopore sequencing also include pathogen detection from clinical samples, which is particularly useful for diseases caused by RNA viruses ( Table 4 ).…”

Insights into rRNA processing and modification mapping in Archaea using Nanopore-based RNA sequencing

“…Essentially, the requirements, but also the possibilities in eukaryotes and prokaryotes, are the same (31), with a poly(A) tail being an essential prerequisite, which is required to capture the RNAs. Using enzymatic polyadenylation of prokaryotic RNAs that in general lack poly(A) tails, the applicability of Nanopore RNAseq has already been demonstrated by metatranscriptomic sequencing of bacterial food pathogens (32) and by accurate estimation of gene expression levels in Klebsiella pneumoniae (33). Despite these initial studies, a comprehensive analysis of the applicability of Nanopore RNA-seq for the analysis of prokaryotic transcriptomes is lacking.…”