Sequencing (e.g. Sanger, Illumina or Nanopore technologies) provides a deeper insight into the genome of SARS-CoV-2 and can be used for many different purposes during an outbreak, such as the path of transmission through a community, monitoring the evolution of strains both geographically and temporally and identifying whether a patient may have co-infections. The number of samples you wish to sequence, plus the availability of each platform in your country is likely to affect the sequencing platform you choose. The following table should help to break down the differences between the main platforms currently available:
|
Sequencing platform |
Sanger |
Illumina |
Nanopore |
|
Sequencing type |
First generation |
Third generation |
Fourth generation |
|
Principle of sequencing |
chain-termination method |
Sequencing by synthesis |
Nanopore |
|
Read length |
Short read (~900 nt) |
Short read (50-300 nt) |
Long read (variable <1,000 kb) |
|
Scale |
Large: ~96 samples |
Large: each MiSeq machine can run ~96 samples |
Small – large: a few samples up to approximately 550 samples |
|
Paired reads? |
Not applicable (overlapping strategy) |
Yes |
Yes |
|
Run time |
30 minutes – 3 hours |
Between 4 hours – 6 days, depending on the machine and throughput capacity |
Dependent on the application as sequencing is in real time: can be run up to 48 hours |
|
Error rate |
0.1% |
0.1% |
5-20% |
|
Advantages |
High quality |
High throughput, low cost per sample |
Rapid and almost ‘point of care’ sequencing |
|
Disadvantages |
Low throughput, high cost per sample |
Short read assembly, depending on desired outcome. Potentially high cost and large infrastructure required |
Higher error rate, higher cost per sample (dependent on equipment) |
|
DNA requirements |
50-500 ng |
50-1000 ng |
400-1000 ng |
Nanopore webinar on SARS-CoV-2 sequencing
The Artic Network have published a SARS-CoV-2 Nanopore sequencing protocol on their website.
Sample collection
Sequencing relies on high quality nucleic acid, so it is important to ensure good extraction.
Oxford Nanopore Technologies extraction web page | Illumina extraction web page