Episode 44: How to sequence SARS-CoV-2 using the ARTIC protocol with Joshua Quick

In this episode of the microbinfie podcast, Joshua Quick from the University of Birmingham provides an in-depth exploration of SARS-CoV-2 sequencing using the ARTIC protocol, highlighting the critical techniques and challenges in viral genome analysis during the COVID-19 pandemic.

Joshua Quick from the University of Birmingham presented on the topic "How to Sequence SARS-CoV-2 Using the ARTIC Protocol" during a workshop. This event was a collaborative effort between the ARTIC Network and CLIMB-BIG-DATA focused on COVID-19 data analysis. The session was chaired by Nick Loman.

Useful Links

• Twitter Announcement
• Primal Scheme Tool
• nCOV-2019 Sequencing Protocol V3 (LoCost) on Protocols.io
• ARTIC Network GitHub (RAMPART)

Key Concepts

• SARS-CoV-2: The virus responsible for COVID-19.
• ARTIC Protocol: A widely used protocol for sequencing SARS-CoV-2.
• Sequencing: A method used to determine the genetic sequence of an organism.
• COVID-19 Data Analysis: Involves analyzing viral genetic sequences to track mutation and transmission.

The workshop aimed at equipping researchers with protocol knowledge to enhance their understanding and ability to sequence SARS-CoV-2 efficiently.

Extra notes

• The podcast discusses the use of amplicon sequencing, particularly the ARTIC method, which is economical and scalable for recovering genomes even with high host levels.

• The multiplex PCR technique is highlighted as it allows the generation of multiple amplicons in a single reaction, which is essential for high-throughput sequencing needs.

• The Primal Scheme is a web-based tool developed for designing primers for multiplex PCR. Users can upload a FASTA file of references to design suitable primers for diverse viral genomes.

• The tool uses Parasail for aligning references and selecting conserved primers and allows for adjustments to design primers against specific reference genomes.

• The technique allows the production of 96 amplicons in two reactions covering approximately 30,000 base pair genomes, with high plexity managed through primer efficiency and overlaps.

• Amplification dropouts, which result in no data for specific genome regions, are a noted challenge and can occur due to poor primers or mutations in the primer binding site.

• The protocol initially developed for SARS-CoV-2 showed early challenges in primer dropouts, prompting refinements for improved consistency.

• The Primal Scheme tool's success rate improves significantly with diverse genomes due to a multi-alignment approach, although there are limits with highly diverse sets.

• Native barcoding protocols are preferred for high specificity in demultiplexing, necessary due to amplicon dropouts and avoiding cross-sample contamination.

• High GC content in bacterial genomes presents additional challenges in primer design. A high GC mode is available in Primal Scheme to adjust parameters suitable for such genomic contexts.

• Different commercialization acts include support and products provided by organizations like ONT, QIAGEN, and Illumina based on these PCR primers.

• The use of the direct RNA kit for sequencing is limited due to high input requirements and the necessity for polyadenylation in non-polyadenylated viruses, reducing practical utility in clinical settings.

• The discussion includes managing equimolar pools and balancing pools for improved genome completeness, with techniques available on protocols.io for calculating primer weighting based on coverage.

• The importance of stringent demultiplexing is emphasized to prevent cross-sample contamination leading to incorrect variant calling.

Key Points

1. ARTIC Sequencing Method

• Utilizes multiplex PCR for generating viral genome amplicons
• Enables efficient genome recovery even with high cycle threshold (CT) values
• Allows sequencing of 96 amplicons covering 30,000 base pair genomes

2. Primal Scheme Tool

• Web-based tool for designing multiplex PCR primers
• Uses multi-alignment approach to cover diverse viral genome references
• Allows flexible primer design with conservation and specificity considerations

3. Genomic Surveillance Challenges

• Manages amplicon dropouts and primer binding site mutations
• Requires careful primer pool balancing for genome completeness
• Supports decentralized sequencing networks like COG-UK

Take-Home Messages

• Multiplex PCR enables rapid, cost-effective viral genome sequencing
• Primer design is a critical step in successful viral genome recovery
• Collaborative, diverse methodological approaches enhance genomic surveillance