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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/10.1111/1755-0998.13847
https://nf-co.re/ampliseq/2.9.0/docs/usage#taxonomic-classification
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While 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 |
Analysis of the microbiome: Advantages of whole genome shotgun versus 16S amplicon sequencing
Our study demonstrates that whole genome shotgun sequencing has multiple advantages compared with the 16S amplicon method including enhanced detection of bacterial species, increased detection of diversity and increased prediction of genes. In addition, increased length, either due to longer reads or the assembly of contigs, improved the accuracy of species detection.
Full length amplicon vs hypervariable regions
The 16S rRNA gene is about 1,500 bases in length. Illumina reads are much shorter than this, therefore amplicon analysis typically involves sequencing 2-3 ‘hypervariable’ 16S regions.
https://www.nature.com/articles/s41598-023-30764-z
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Different groups of bacteria are better represented by specific regions.
With the development of 3rd generation long read sequencing, such as Nanopore and PacBio, the full 16S length can be sequenced. This reduces bias and improves taxonomic resolution.
https://www.nature.com/articles/s41598-020-80826-9
NOTE: For eukaryotic (e.g. fungi) metagenomic amplicon sequencing, ITS (Internal transcribed spacer) regions are used.
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https://data.eresearchqut.net/paulw/public/mahsa_manuscript2/index.html
ASV vs OTU
Taxonomic assignments in nfcore/ampliseq are based on Amplicon sequence variants (ASV), inferred using the DADA2 software package by matching the sample sequences to the SILVA ribosomal RNA sequence database.
DADA2 infers sample sequences exactly and resolves differences of as little as 1 nucleotide.
SILVA provides comprehensive, quality checked and regularly updated datasets of aligned small (16S/18S, SSU) and large subunit (23S/28S, LSU) ribosomal RNA (rRNA) sequences for all three domains of life (Bacteria, Archaea and Eukarya).
Traditionally Operational Taxonomic Units (OTUs) have been used in 16S ampicon studies. More recently ASVs have been used, due their improved accuracy in identifying taxa, particularly genus and species. Basically, OTUs utilise a similarity clustering method to identify taxa, whereas ASV are generated by quantifying exact sequence matches to an amplicon database (e.g. Silva or Greengenes) and then statistically adjusting this using confidence thresholds.
The OTU method typically can identify 97% similarity (with any accuracy) whereas the ASV method can identify even single base-pair differences. This enables a finer resolution of taxa down to the genus and species level. Note that there is increasing ‘fuzziness’ toward the lower taxonomic levels, as the diversity within some taxa is greater than the diversity between this and other taxa (in other words, even with ASV, not all taxa can be resolved to lower taxonomic levels and this is highly dependent on the taxonomic group involved).
https://www.zymoresearch.com/blogs/blog/microbiome-informatics-otu-vs-asv
Illumina vs Nanopore sequencing technologies
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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 3rd 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.
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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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