Comparing the significance of the utilization of next generation and third generation sequencing technologies in microbial metagenomics.

Background The implementation of fast, sensitive and specific diagnostic tools such as those for point-of-care testing (POCT) can assure public health security and food safety against foodborne infectious disease outbreaks. Norovirus is the most common pathogen causing foodborne outbreaks and the most frequent cause of acute gastroenteritis in humans. This frequency in outbreaks highlights the importance of the development and application of new portable diagnostic concepts and sensitive, multiplex, accurate, real-time (SMART) technologies considering the recommendations of Public Health Authorities accentuating personal and food hygiene measures to limit the spread of the virus. The development of POCT using handheld devices has significantly increased in recent years to detect norovirus due to the undeniable advantages of these methods such as rapidness, ultra-sensitivity and simplicity for norovirus diagnosis, in comparison to the ‘gold standard’ quantitative polymerase chain reaction (qPCR). Scope and approach Recent progress in the development of POCT devices and changing detection priorities in the food industry highlights the importance of efficient POCT methods selection, therefore, this review sought to outline the comparison of emerging POCT methods to be used for the analysis of food, water and clinical samples including molecular methods, biosensing technologies, immunodiagnostics, microarrays, and other prospective methods whereby implementation could aid the investigation of outbreaks of norovirus. Key findings In this study, advantages and disadvantages of POCT methods are illustrated and the prospective identification of POCT methods are compared and discussed. In summary, the concluding remarks illustrate the future trends and directions for POCT methods which can be applied to detect norovirus and other viruses.

Current state-of-the-art diagnostics for Norovirus detection: Model approaches for point-of-care analysis

The sequencing, assembly, and analysis of bacterial genomes is central to tracking and characterizing foodborne pathogens. The bulk of bacterial genome sequencing at the US Food and Drug Administration is performed using short-read Illumina MiSeq technology, resulting in highly accurate but fragmented genomic sequences. The MinION sequencer from Oxford Nanopore is an evolving technology that produces long-read sequencing data with low equipment cost. The goal of this study was to compare Campylobacter genome assemblies generated from MiSeq and MinION data independently, as well as hybrid genome assemblies combining both data types. Two reference strains and two field isolates of C. jejuni were sequenced using MiSeq and MinION, and the sequence data were assembled using the software programs SPAdes and Canu, respectively. Hybrid genome assembly was performed using the program Unicycler. Comparison of the C. jejuni 81-176 and RM1221 genome assemblies to the PacBio reference genomes revealed that the SPAdes assemblies had the most accurate nucleotide identity, while the hybrid assemblies were the most contiguous. Assemblies generated only from MinION data using Canu were the least accurate, containing many indels and substitutions that affected downstream analyses. The hybrid sequencing approach was the most useful for detecting plasmids, large genome rearrangements, and repetitive elements such as rRNA and tRNA genes. The full genomes of both C. jejuni field isolates were completed and circularized using hybrid sequencing, and a plasmid was detected in one isolate. Continued development of nanopore sequencing technologies will likely enhance the accuracy of hybrid genome assemblies and enable public health laboratories to routinely generate complete circularized bacterial genome sequences.

/pdf/comparison-of-miseq-minion-and-hybrid-genome-sequencing-for-tb3861y5t6.pdf

Comparison of MiSeq, MinION, and hybrid genome sequencing for analysis of Campylobacter jejuni.

Rapid, cost-effective identification of genetic variants in small candidate genomic regions remains a challenge, particularly for less well equipped or lower throughput laboratories. The application of Oxford Nanopore Technologies' MinION sequencer has the potential to fulfil this requirement. We demonstrate a proof of concept for a multiplexing assay that pools PCR amplicons for MinION sequencing to enable sequencing of multiple templates from multiple individuals, which could be applied to gene-targeted diagnostics. A combined strategy of barcoding and sample pooling was developed for simultaneous multiplex MinION sequencing of 100 PCR amplicons. The amplicons are family-specific, spanning a total of 30 loci in DNA isolated from 82 human neurodevelopmental cases and family members. The target regions were chosen for further interrogation because a potentially disease-causative variant had been identified in affected individuals following Illumina exome sequencing. The pooled MinION sequences were deconvoluted by aligning to custom references using the minimap2 aligner software. Our multiplexing approach produced an interpretable and expected sequence from 29 of the 30 targeted genetic loci. The sequence variant which was not correctly resolved in the MinION sequence was adjacent to a five nucleotide homopolymer. It is already known that homopolymers present a resolution problem with the MinION approach. Interestingly despite equimolar quantities of PCR amplicon pooled for sequencing, significant variation in the depth of coverage (127×-19,626×; mean = 8321×, std err = 452.99) was observed. We observed independent relationships between depth of coverage and target length, and depth of coverage and GC content. These relationships demonstrate biases of the MinION sequencer for longer templates and those with lower GC content. We demonstrate an efficient approach for variant discovery or confirmation from short DNA templates using the MinION sequencing device. With less than 130 × depth of coverage required for accurate genotyping, the methodology described here allows for rapid highly multiplexed targeted sequencing of large numbers of samples in a minimally equipped laboratory with a potential cost as much 200 × less than that from Sanger sequencing. 

/pdf/proof-of-concept-for-multiplex-amplicon-sequencing-for-17jupe3c.pdf

Proof of concept for multiplex amplicon sequencing for mutation identification using the MinION nanopore sequencer

Metagenomic next-generation sequencing (mNGS) holds promise as a diagnostic tool for unbiased pathogen identification and precision medicine. However, its medical utility depends largely on assay simplicity and reproducibility. In the current study, we aimed to develop a streamlined Illumina and Oxford Nanopore-based DNA/RNA library preparation protocol and rapid data analysis pipeline. The Illumina sequencing-based mNGS method was first developed and evaluated using a set of samples with known aetiology. Its sensitivity for RNA viruses (influenza A, H1N1) was < 6.4 × 102 EID50/mL, and a good correlation between viral loads and mapped reads was observed. Then, the rapid turnaround time of Nanopore sequencing was tested by sequencing influenza A virus and adenoviruses. Furthermore, 11 respiratory swabs or sputum samples pre-tested for a panel of pathogens were analysed, and the pathogens identified by Illumina sequencing showed 81.8% concordance with qPCR results. Additional sequencing of cerebrospinal fluid (CSF) samples from HIV-1-positive patients with meningitis/encephalitis detected HIV-1 RNA and Toxoplasma gondii sequences. In conclusion, we have developed a simplified protocol that realizes efficient metagenomic sequencing of a variety of clinical samples and pathogen identification in a clinically meaningful time frame.

/pdf/a-streamlined-clinical-metagenomic-sequencing-protocol-for-4ioop5gl3i.pdf

A streamlined clinical metagenomic sequencing protocol for rapid pathogen identification.

The capability of high-throughput sequencing (HTS) for detection of known and unknown viruses timely makes it a powerful tool for public health emergency response. Third-generation sequencing (TGS) offers advantages in speed and length of detection over second-generation sequencing (SGS). Here, we presented the end-to-end workflows for both Oxford Nanopore MinION and Pacbio Sequel on a viral disease emergency event, along with Ion Torrent PGM as a reference. A specific pipeline for comparative analysis on viral genomes recovered by each platform was assembled, given the high errors of base-calling for TGS platforms. All the three platforms successfully identified and recovered at least 85% Norovirus GII genomes. Oxford Nanopore MinION spent the least sample-to-answer turnaround time with relatively low but enough accuracy for taxonomy classification. Pacbio Sequel recovered the most accurate viral genome, while spending the longest time. Overall, Nanopore metagenomics can rapidly characterize viruses, and Pacbio Sequel can accurately recover viruses. This study provides a framework for designing the appropriate experiments that are likely to lead to accurate and rapid virus emergency response.

Comparison of third-generation sequencing approaches to identify viral pathogens under public health emergency conditions.

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Comparison of third-generation sequencing approaches to identify viral pathogens under public health emergency conditions.