Overview
1a. What is metagenomics?
Full length vs partial
16s/18s/ITS, etc
Workflows
https://onlinelibrary.wiley.com/doi/full/10.1111/1755-0998.13847
1b. Illumina vs Nanopore sequencing technologies
This overview is based on this excellent review article:
https://www.nature.com/articles/s41587-021-01108-x
Nanopore is part of the ‘third generation sequencing’ suite of sequencing technologies, producing longer (~500bp - 100,000bp), fewer reads than the more commonly used ‘second generation’ of sequencing, most typically represented by Illumina fixed-length, short read technology (~50bp - 400bp).
Whereas 2nd generation technology uses massively parallel sequencing - i.e. simultaneous sequencing millions of small DNA fragments annealed to a flow cell - 3rd generation sequencing includes several competing technologies, which differ substantially in terms of underlying technology. The two main companies are Pacific Biosystems ('PacBio') and Oxford Nanopore Technology ('ONT' or ‘Nanopore’).
An overview of 2nd and 3rd gen sequencing technologies can be seen here: https://www.sciencedirect.com/science/article/pii/S0198885921000628
It’s important to note that a significant difference between 2nd and 3rd generation technology is accuracy. The error rate (i.e. the number of bases with low sequencing quality scores) of 3nd gen has been considerably higher than 2nd gen, with typically ~0.1% error rate for Illumina sequences and >5% error rate for Nanopore. The Nanopore error rate has improved dramatically in recent years though, but still is considerably lower than Illumina. This higher error rate can cause issues, such as in metagenomics when identifying species that differ by a small number of base pairs.
Functionally, the longer 3rd gen reads can counter the higher error rates through increased number of potential base matches. For 16S rRNA sequencing, short read Illumina sequences typically cover two 16S hypervariable regions, whereas Nanopore sequences the full 1.5 kilobase 16S sequences, which includes all nine hypervariable regions.
1c. Amplicon vs shotgun sequencing
This 2023 paper compared Illumina and Nanopore shotgun sequencing for identifying bacteria strains with little genomic variation between them. Both Illumina and Nanopore were able to correctly identify the bacteria strains, despite the higher error rate of the Nanopore sequences.
1d. Reference paper
Data used in this workshop is from a paper that compared Illumina and Nanopore 16S datasets.
https://www.mdpi.com/2073-4425/11/9/1105
2.8. Sequence Data Availability
The Illumina and nanopore sequence datasets of the nose swab samples, generated and analyzed in the current study, are available in the European Nucleotide Archive (ENA) under accession number PRJEB28612
https://www.ebi.ac.uk/ena/browser/view/PRJEB28612
