Compiti ed esercitazioni VERIFICATO
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- Introduction to SARS-CoV-2 and the Pandemic:
- A new betacoronavirus (SARS-CoV-2) was identified in January 2020, leading to the WHO declaring a pandemic on March 11, 2020.
- As of May 14, 2021, global figures included over 160 million confirmed cases and 3.3 million deaths across 223 territories.
- Common symptoms (SINTOMI) include fever, sore throat, dizziness, and lung infection.
- The first genomic sequence of SARS-CoV-2 was reported in December 2019, detailing its genome structure with ORFs and structural proteins.
- Sample Collection and RNA Extraction:
- Sample Collection: Includes clinical samples from respiratory tracts (upper/lower), non-respiratory clinical samples, infected cell lines, and environmental samples.
- RNA Extraction: Standard methodologies involve guanidine salt and phenol.
- Next Generation Sequencing (NGS) Strategies for SARS-CoV-2 Genomes:
- Shotgun Metatranscriptomics:
- Advantages: Sequences entire sample DNA, no prior sequence knowledge required.
- Disadvantages: Requires high viral load, high sequencing depth, is a slow and labor-intensive technique, and demands specialized laboratories.
- Characteristics: Detects co-infections, uses 20-50M reads, achieves ≥99% genome coverage, high SNV accuracy, and is high cost.
- Amplicon-based Sequencing:
- Advantages: Highly specific, requires small RNA quantities, low cost, and is effective with low viral load samples.
- Disadvantages: Requires significant prior sequence knowledge, and has potential primer inefficiency during amplification.
- Characteristics: Does not detect co-infections, uses 5-20M reads, achieves ≥95-99% genome coverage, high SNV accuracy, and is low cost.
- Hybrid Capture Enrichment Sequencing:
- Advantages: Highly specific, characterized by lower sequencing depth (potentially suitable for targeted approaches), and is robust to genomic variability.
- Disadvantages: Primarily used with high viral load samples.
- Characteristics: Uses 5-20M reads, achieves ≥95-99% genome coverage, moderate SNV accuracy, and is moderate cost.
- Direct RNA Sequencing:
- Advantages: Does not require reverse transcription or amplification, directly sequencing RNA.
- Disadvantages: Provides reads with a higher error rate.
- Characteristics: Detects SARS-CoV-2 and host transcriptome/epitranscriptome, uses 0.5M reads, achieves ≥99% genome coverage, low SNV accuracy, high cost, and typically uses viral cell cultures.
- Shotgun Metatranscriptomics:
- Genomic Assembly and Data Management:
- Genomic Assembly (ASSEMBLAGGIO GENOMICO): Compares coverage and assembly size across different library preparations (Amplicon, Hybrid capture, Metatranscriptomics) using technologies like Illumina and Oxford Nanopore.
- Data Deposition and Access (DEPOSIZIONE E ACCESSO AI DATI): Emphasizes the importance of sharing and accessing genomic data effectively.
- Key Platforms for Data and Analysis:
- GISAID EPICOV: Hosts over 100,000 SARS-CoV-2 genomes, providing genomic epidemiology and variant tracking, though with limited metadata.
- RESEARCH DATA ALLIANCE (RDA): Promotes data compliance with FAIR (Findability, Accessibility, Interoperability, Reuse) standards.
- BIO.TOOLS: Offers essential information on bioinformatics tools, databases, and workflows, including those related to COVID-19.
- NEXTSTRAIN & HYPHY: Tools for real-time monitoring of SARS-CoV-2 evolution.
- PROTOCOLS.IO: A platform for sharing reproducible methods in biology, chemistry, and health, including COVID-19 data.
- COVID-19 DATA PORTAL (Sweden): Facilitates research data sharing across various data types (genomics, proteomics, imaging).
- ELIXIR: Provides support and services for COVID-19 research.
- GITHUB (CDCgov/SARS-CoV-2_Sequencing): A repository for sequencing protocols and bioinformatics resources.
- COG-UK: Conducts large-scale, rapid whole-genome sequencing of SARS-CoV-2.
- NCBI VIRUS: A community portal for viral sequence data, offering quick access and dashboards for SARS-CoV-2.
- Conclusioni: The presentation concludes by summarizing the workflow from sample collection and sequencing to analysis, data sharing, and continuous monitoring, highlighting the comprehensive approach to understanding and combating the pandemic.