MAME ch.4 review

Cards cover key concepts from Chapter 4 notes: amplicon sequencing, 16S/18S/mtCOI/ITS, OTU/ASV/ESV, rarefaction, Baas Becking, Kill the Winner, SAR11, culturing methods, and taxonomy basics.

What is an amplicon?

DNA produced by PCR (in vitro) from a target region for sequencing or analysis.

What is an OTU (Operational Taxonomic Unit)?

A cluster of similar 16S rRNA gene sequences used as a proxy for taxonomic groups, commonly defined at ≥97% similarity.

What is an ASV/ESV?

Exact Sequence Variant (ASV/ESV) – a unique, exact sequence resolved after error correction, representing distinct sequences without clustering.

What does a rarefaction curve show?

The number of observed OTUs (or diversity units) as a function of the number of sequences sampled; used to assess sampling effort and richness.

Differentiate cultivation-dependent from cultivation-independent methods.

Cultivation-dependent methods grow organisms on plates and identify them from colonies; cultivation-independent methods use DNA-based techniques (e.g., PCR of environmental DNA) without culturing.

Which gene is most commonly used to assess bacterial community structure?

The 16S rRNA gene, which has conserved regions for primers and variable regions for taxonomic discrimination.

Which gene is used for identifying eukaryotic microbes (and what are common alternatives)?

18S rRNA gene for broad eukaryotes; mtCOI for animal barcoding; ITS region is often used for fungi; eukaryotes also have 18S in mitochondria/chloroplasts.

What is mtCOI?

Mitochondrial cytochrome c oxidase subunit I; a barcode gene used for animal identification.

What is the ITS region and where is it located?

Internal Transcribed Spacer region between rRNA genes; used for fungal barcoding in many eukaryotes (ITS location can vary by organism).

State the Baas Becking hypothesis.

Everything is everywhere, but the environment selects.

Explain the Kill the Winner (KtW) hypothesis.

Viruses that infect bacteria help determine which bacterial taxa dominate by preferentially killing the most abundant hosts.

What is the SAR11 clade and why is it important?

Pelagibacter ubique; a ubiquitous, abundant marine clade of bacteria, extremely small in size with a small genome; highly successful oligotrophs.

What is the Seed Bank hypothesis in microbial ecology?

Rare biosphere taxa act as a reservoir that can become dominant when environmental conditions change (e.g., oil spills).

Why is 16S rRNA gene used as a barcode for prokaryotes, and what features does it have?

Because it is present in all bacteria/archaea, with conserved regions for primers and hypervariable regions for distinguishing taxa.

Give examples of universal primers used in 16S rRNA gene amplification.

27F and 1492R; commonly used universal primers that flank the conserved regions to amplify near-full-length 16S rRNA gene.

What is the typical length of a 16S amplicon used for taxonomy, and what range can near-full-length sequences reach?

Typical amplicons are about 200–300 bp; near-full-length sequences can be ~1,500 bp.

What taxonomic levels are shown in standard microbial classifications?

Domain, Phylum, Class, Order, Family, Genus, Species, and sometimes Strain.

What does a ‘phylotype’ refer to in microbial taxonomy?

A taxonomic unit defined by phylogenetic relationships based on sequence data, often used interchangeably with OTU in some contexts.

Explain the difference between a clade and a taxon.

A clade is a monophyletic group descended from a common ancestor; a taxon is a named group at any taxonomic level (domain, phylum, etc.).

What is the significance of conserved vs. variable regions in the 16S rRNA gene?

Conserved regions enable universal primer binding across taxa; hypervariable regions provide taxonomic discrimination among bacteria.

What is the conceptual difference between cloning-based amplicon methods and direct sequencing?

Clone libraries involve cloning PCR fragments into a host (e.g., E. coli) before sequencing; direct sequencing sequences PCR products directly without cloning.

Why are mitochondria and chloroplasts relevant to 16S rRNA gene discussions?

Mitochondria and chloroplasts contain 16S-like rRNA genes due to ancient endosymbiosis, linking organelle genomes to bacterial ancestry.

What is a ‘universal primer’ and give examples for 16S amplification?

Primers designed to bind conserved regions across many taxa to amplify variable regions; examples include 27F and 1492R, and 515F/806R.

What is the reason many microbes are difficult to culture, and how does this impact diversity studies?

Many are unculturable with standard lab media; cultivation-independent methods reveal greater diversity and detect abundant taxa missed by cultivation.

What does ‘relative abundance’ mean in microbial community analyses?

The proportion of the total community contributed by a particular taxon, usually expressed as a percentage of reads or sequences.

How is OTU vs ASV/ESV defined in sequencing analyses?

OTU clusters sequences by a similarity threshold (commonly 97%); ASV/ESV resolves exact sequences without clustering, representing unique variants.

Which depth-related microbial groups gain an advantage and why?

Chemolithoautotrophs that can oxidize ammonia for energy in deep, dark environments where sunlight is absent.

Why is the term ‘species’ problematic in prokaryotes?

Many prokaryotes do not mate; 16S rRNA is not universally sufficient to define species; horizontal gene transfer complicates species concepts.