Episode 23: CoronaHiT: large scale multiplexing of SARS-CoV-2 genomes on Nanopore

The microbinfie podcast explores CoronaHiT, an innovative method for multiplexing SARS-CoV-2 genome sequencing on Oxford Nanopore technology, dramatically reducing sequencing costs and increasing sample throughput.

In this engaging discussion, we chat with the authors behind CoronaHiT, an innovative method that allows for sequencing up to 94 SARS-CoV-2 samples on a single MinION flowcell. This approach significantly reduces sequencing costs by three-fold and employs a simpler, faster protocol.

Guests

• Justin O'Grady
• David Baker

Topics Covered

Here’s a summary of the key points discussing the CoronaHIT method for multiplexing SARS-CoV-2 sequencing on Oxford Nanopore technology:

1. CoronaHIT Overview:

   • The method is designed to increase throughput and decrease costs by allowing the simultaneous processing of multiple samples.
   • The original method processed 24 samples per run at about £40 each, while CoronaHIT reduces this cost to between £13 and £18 per sample.

2. Technical Innovations:

   • CoronaHIT uses a low input Nextera method to insert tagmentation adapters during PCR, eliminating the need for a ligation reaction, which traditionally requires a higher amount of DNA.
   • This method allows for the rapid generation of multiple overlapping fragments for sequencing.

3. Comparison with the Arctic Protocol:

   • The Artic protocol, which is widely used for sequencing SARS-CoV-2, takes about 24 hours from library preparation to sequencing.
   • CoronaHIT aims to maintain the speed of the Arctic protocol while enhancing multiplexing capabilities.

4. Multiplexing Capacity:

   • The team successfully multiplexed 48 samples in a single flow cell and even attempted 94 samples, although the latter raised concerns about barcode crosstalk and data de-multiplexing accuracy.
   • The barcode design evolved from 16 to 24 base pairs to improve data recovery and reduce errors during sequencing.

5. Adaptations and Bioinformatics:

   • Switching from the Artic protocol to CoronaHIT is relatively straightforward, requiring only a few new reagents.
   • The method is compatible with existing nanopore de-multiplexing software.

6. Challenges with Sequencing Data:

   • The podcast addresses challenges with the Nextera method, specifically regarding potential loss of sequence data at the ends of amplicons and the importance of masking out synthetic sequences.
   • They discuss how the coverage of the SARS-CoV-2 genome can be uneven due to varying efficiencies of primer pairs, necessitating careful design and optimization of multiplexed reactions.

7. Performance Comparison:

   • The group evaluates the comparative performance of nanopore sequencing with standard Illumina sequencing, noting the advantages of nanopore in field applications where Illumina access may be limited.

This episode highlights the innovative approaches in SARS-CoV-2 sequencing, addressing both practical applications and underlying technical challenges. For more detailed information, check out their preprint on bioRxiv: DOI: 10.1101/2020.06.24.162156