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Full length amplicon vs hypervariable regions

https://data.eresearchqut.net/paulw/public/mahsa_manuscript2/index.html

https://nanoporetech.com/resource-centre/epi2me-16s-workflow-real-time-identification-bacteria-and-archaea

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https://nf-co.re/ampliseq/2.9.0/docs/usage#taxonomic-classification

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 highly depedant on the taxonomic group involved).

https://www.zymoresearch.com/blogs/blog/microbiome-informatics-otu-vs-asv

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Illumina vs Nanopore sequencing technologies

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