{"title":"Comparison of Three Viral Nucleic Acid\u0000 Preamplification Pipelines for Sewage Viral Metagenomics","authors":"Xavier Fernandez-Cassi, Tamar Kohn","doi":"10.1007/s12560-024-09594-3","DOIUrl":"10.1007/s12560-024-09594-3","url":null,"abstract":"<div><p>Viral metagenomics is a useful tool for detecting multiple human viruses in\u0000 urban sewage. However, more refined protocols are required for its effective use in\u0000 disease surveillance. In this study, we investigated the performance of three different\u0000 preamplification pipelines (specific to RNA viruses, DNA viruses or both) for viral\u0000 genome sequencing using spiked-in Phosphate Buffered Saline and sewage samples\u0000 containing known concentrations of viruses. We found that compared to the pipeline\u0000 targeting all genome types, the RNA pipeline performed better in detecting RNA viruses\u0000 in both spiked and unspiked sewage samples, allowing the detection of various mammalian\u0000 viruses including members from the <i>Reoviridae</i>,\u0000 <i>Picornaviridae</i>, <i>Astroviridae</i> and <i>Caliciviridae</i>.\u0000 However, the DNA-specific pipeline did not improve the detection of mammalian DNA\u0000 viruses. We also measured viral recovery by quantitative reverse transcription\u0000 polymerase chain reaction and assessed the impact of genetic background (non-viral\u0000 genetic material) on viral coverage. Our results indicate that viral recoveries were\u0000 generally lower in sewage (average of 11.0%) and higher in Phosphate Buffered Saline\u0000 (average of 23.4%) for most viruses. Additionally, spiked-in viruses showed lower genome\u0000 coverage in sewage, demonstrating the negative effect of genetic background on\u0000 sequencing. Finally, correlation analysis revealed a relationship between virus\u0000 concentration and genome normalized reads per million, indicating that viral metagenomic\u0000 sequencing can be semiquantitative.</p><h3>Graphical Abstract</h3><div><figure><div><div><picture><source><img></source></picture></div></div></figure></div></div>","PeriodicalId":563,"journal":{"name":"Food and Environmental Virology","volume":"16 3","pages":"1 - 22"},"PeriodicalIF":4.1,"publicationDate":"2024-04-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s12560-024-09594-3.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140636722","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}