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An example - a gut content analysis examining the community structure of bacteria (microbiome) via 16S amplicon sequencing would typically be referred to as a metagenome study. Whereas if the assessment of the gut content was instead exploring what the animal’s diet was (what plants they have eaten, for example), using another amplicon marker (e.g. Cytochrome b) would be an eDNA study.
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Amplicon vs shotgun (whole genome) sequencing
Metagenomic shotgun sequencing[edit]
Having reads of 400-500 base pairs length is sufficient to determine the species or strain of the organism where the DNA comes from, provided its genome is already known, by using for example a k-mer based taxonomic classifier software. With millions of reads from next generation sequencing of an environmental sample, it is possible to get a complete overview of any complex microbiome with thousands of species, like the gut flora. Advantages over 16S rRNA amplicon sequencing are: not being limited to bacteria; strain-level classification where amplicon sequencing only gets the genus; and the possibility to extract whole genes and specify their function as part of the metagenome.[19] The sensitivity of metagenomic sequencing makes it an attractive choice for clinical use.[20] It however emphasizes the problem of contamination of the sample or the sequencing pipeline.[21]
Full length amplicon vs hypervariable regions
16s/18s/ITS, etc
Workflows
https://onlinelibrary.wiley.com/doi/full/10.1111/1755-0998.13847While whole genome sequencing provides a comprehensive view of all the genetic variations within a sample, amplicon sequencing focuses on sequencing specific genomic regions (like the 16s rrna gene). This targeted approach makes amplicon sequencing more cost-effective than whole genome sequencing.
Shotgun metagenomic sequencing, unlike 16S rRNA sequencing, can read all genomic DNA in a specimen rather than just one portion of a particular gene. Shotgun sequencing can simultaneously identify and profile bacteria, fungi, viruses, and a variety of other microorganisms, which is useful for microbiome research.
Pros and cons of amplicon vs whole genome sequencing:
Amplicon | Whole genome | |
|---|---|---|
Dataset size | Very small | Medium to very large |
Computational resources | Small | Medium to very large |
Price | Low | Medium to high |
Taxonomic resolution | Mostly genus | Species or strain |
Functional analysis | Limited | Greater detail |
Database curation | Detailed | Minimal |
Taxonomic coverage | Specific (e.g. 16s = bacteria) | All taxa |
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Analysis of the microbiome: Advantages of whole genome shotgun versus 16S amplicon sequencing
Full length amplicon vs hypervariable regions
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https://nf-co.re/ampliseq/2.9.0/docs/usage#taxonomic-classification
ASV vs OTU
Illumina vs Nanopore sequencing technologies
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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.
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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
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