Overview
What is metagenomics?
Amplicon vs shotgun (whole genome) sequencing
Full length amplicon vs hypervariable regions
16s/18s/ITS, etc
Workflows
https://onlinelibrary.wiley.com/doi/full/10.1111/1755-0998.13847
ASV vs OTU
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.
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.
Amplicon analysis workflow
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 analysed 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
Why Nextflow?
What is Nextflow was covered in session 1 of these workshops (Installing Nextflow ).
“scalable and reproducible scientific workflows using software containers.” https://www.nextflow.io/
Bioinformatics workflows are complicated, and are becoming more complicated. Nextflow enables managed, reproducible curated bioinformatics workflows to be used by non-bioinformatician researchers.
Why do we use Nextflow?
Complexity: Analysis workflows are becoming more complex, with more steps (but producing more accurate, publishable results).
100’s of published workflows
Curated, multi-tool analyses
Optimised and improved over multiple versions
Detailed output with results, tables, figures, data files
Curated: Managed workflows are typically tested and assembled by experts in the field. Often are improved over multiple versions.
Nfcore: central repository for Nextflow pipelines: https://nf-co.re/pipelines
Nanopore has developed Nextflow workflows via epi2me: https://labs.epi2me.io/wfindex/
Reproducibility: Most published studies can’t be reproduced. The ‘reproducibility crisis’. Nexflow has version control, for both the workflow, and the multitude of software tools within.
The Nextflow workflows we’ll be running today are:
nfcore/ampliseq for the Illumina data: https://nf-co.re/ampliseq/2.9.0
wf-metagenomics for the Nanopore data: https://github.com/epi2me-labs/wf-metagenomics