Optimization of portable MinION sequencing technology for viral metagenome analysis

Author(s)

Victória Simionatto Zucherato

• Document type: Master's Dissertation
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2023-06-06

Examining board members

Svetoslav Nanev Slavov; Maurício Teixeira Lima; João Paulo Bianchi Ximenez

• Advisor: Svetoslav Nanev Slavov

Abstract

Next Generation Sequencing (NGS) technologies have shown accelerated growth in the recent years. One of the main applications NGS is metagenomics, which identifies emerging pathogens without established diagnosis. A promising and relatively low-cost technique is the portable MinION technology. One of the main problems of the MinION platform is the lack of optimized protocols (sample pre-preparation, viral concentration, extraction and amplification). Therefore, the objective of this work is to optimize viral metagenomics procedures applied to nanopore sequencing. We used two types of controls: for RNA viruses a blood unit of plasma obtained from HIV-positive blood donor with cycle threshold of Ct=25.8 and for DNA viruses: cultivated human cytomegalovirus (HCMV). These controls were used as a positive control to optimize the processes of viral concentration, extraction and preparation of metagenomic libraries. Initially, after enzymatic host nucleic acids degradation, viral concentration was performed by ultracentrifugation, which was subsequently confirmed with transmission electron microscopy. In addition, viruses were also concentrated by chemical method using linear polyacrylamide (LPA). Concentrated samples were extracted by two different strategies that were compared: QIAamp UltraSens Virus (QIAGEN) and High Pure Viral Nucleic Acid Large Volume (Roche Life Science). Subsequently, we compared different DNA fragmentation methods: mechanical fragmentation (Covaris), chemical fragmentation (endonucleases), amplification by the SMART-9N primer system and isothermal amplification without ligation. All samples were sequenced using flow cell 9.4.1 on the MinION device. The transmission electron microscopy showed the presence of HIV particles (expected size 80-100 nm) and abundance of phages, revealing its adequacy for viral concentration. The bioinformatic analysis revealed that the samples concentrated by LPA, extracted by the QIAamp UltraSens Virus kit and amplified by the SMART-9N method, showed higher viral abundance and a high number of HIV and HCMV reads. Therefore, we propose that nanopore sequencing metagenomics might be performed including the aforementioned steps and we conclude that the optimization of all sample pre-processing procedures is crucial for better sequencing results using MinION technology, allowing its application in the area of viral metagenomics and in the discovery of emerging or reemerging viral pathogens. (AU)

• FAPESP's process: 21/09446-8 - Application of portable minion sequencing technology for viral metagenomics analysis
• Grantee: Victória Simionatto Zucherato
• Support Opportunities: Scholarships in Brazil - Master