Experts review NGS methods for crustacean norovirus

Scientists have evaluated the use of next-generation sequencing (NGS) methods against norovirus in crustaceans.

They found that such technology is poised to replace current methods, despite some limitations.

Norovirus contamination of bivalve molluscs (BMS) is a significant public health risk. Contaminated foods such as oysters may harbor concentrations and diverse sequences of viral genomes when contaminated by human sewage.

“Based on the results presented here, it is clear that too many European crustacean samples have been exposed to sewage contamination, as evidenced by the multiple sequences detected in the crustacean samples. , highlighting the need to improve coastal water quality,” the researchers said.

Using laboratory-prepared samples of known norovirus composition, scientists assess the sensitivity, reproducibility, reproducibility, and selectivity of metabarcoding, capture-based metagenomics, and long amplicon sequencing Did.

The three methods were capable of sequencing noroviruses in most of the test samples, but differed in their performance in obtaining the expected diversity. Experts also compared the strengths and weaknesses of each method, cost per sample versus equipment cost, time to results, and whether they are suitable for surveillance or outbreak sequencing.

A consortium of Institut Français de Recherche pour l’Exploitation de la Mer (Ifremer), Center for Environment, Fisheries and Aquaculture Science (Cefas), Danish Technical University (DTU) and Erasmus University Medical Center focuses on norovirus-contaminated oysters is guessing. sample. The findings were published by the European Food Safety Authority (EFSA).

Possibilities and Challenges of the Method
Metabarcoding, which separately amplifies the polymerase and capsid gene segments, followed by Illumina sequencing, was the most sensitive method. However, due to the amplification step and the high sensitivity of NGS, the method is prone to contamination and false positive results.

The work covered norovirus in crustaceans and patient samples from the routine monitoring and surveillance of oysters in the European Economic Area from November 2016 to December 2018 and from the survey of contamination.

Two artificially generated sample sets, 212 oyster samples from the survey, pairs of human and crustacean samples from 16 foodborne outbreaks in France and Denmark from 2012 to 2019, and a global There have been human norovirus sequences submitted to the surveillance network Noronet.

One metabarcoding approach uses Illumina technology and the other is based on long amplicon sequencing using Oxford Nanopore technology. Metabarcoding is highly sensitive and can better capture the diversity of noroviruses present in crustaceans.

Experts say metabarcoding is a more reliable, sensitive, reproducible, and easy-to-use method for sequence analysis.

Other methods, such as metagenomics, show promise but need further improvements in terms of sensitivity. Metagenomic sequencing is sensitive enough to investigate human developmental samples, but not samples from crustaceans. The VirCapSeQ metagenomics method requires optimization and should be used for highly contaminated or recently collected samples.

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