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16S ribosomal RNA is the ribosomal RNA of the small ribosomal subunit in bacteria and archaea. Together with ribosomal proteins, this RNA forms the small subunit's architecture, which positions messenger RNA for translation. The name refers to a sedimentation coefficient of about16 svedbergs: it is neither a count of subunits nor a length in nucleotides. The RNA molecule and the DNA gene encoding it are different molecules, even when a DNA sequencing assay is informally described as “16S sequencing”.^[ref]

A structural example is the bacterial30S small ribosomal subunit: the2000 X-ray crystallography study of B. T. Wimberly and colleagues resolved its RNA–protein architecture at3Å resolution. At a different scale, 16S ribosomal RNA sequences can serve as inherited characters for phylogenetic analysis. The1977 study by George E. Fox and colleagues compared ribosomal RNA oligonucleotide cataloguing results from methane-producing organisms with those from other organisms, helping expose a deep evolutionary division.^[ref][ref]

A useful caution is that one genome need not contain just one16S gene, and its copies need not have identical nucleotide sequences. Silvia G. Acinas and colleagues explicitly measured this variation across sequenced genomes. As a hypothetical example, two distinct16S DNA sequences recovered from a sample could represent two organisms, or two copies in one organism. Counting distinct sequences therefore requires more interpretation than counting organisms.^[ref]

Silvia G. Acinas is a researcher in marine microbial ecology, genomics and evolution. Her institutional research profile describes work connecting the genetic diversity of marine microorganisms with their ecological capabilities, using approaches including metagenomics and cultivation.^[ref]

Acinas was first author of a 2004 study of variation among 16S ribosomal RNA sequences encoded within individual genomes. A genome can contain multiple copies of a 16S ribosomal RNA gene; those copies can be identical or differ in sequence. The study compared copy numbers, sequence divergence and redundancy. These properties complicate the interpretation of diversity estimates based on the marker.^[ref]

An invented example separates two effects. Suppose three microbial cells each have two marker-gene copies and two other cells each have five. With one complete genome per cell and no other biases, there are $3\times 2+2\times 5=16$ marker copies among five cells. Their contributions to the copy pool are $6/16$ and $10/16$, whereas their contributions to cell number are $3/5$ and $2/5$. Marker abundance and cell abundance have different denominators. If copies within a single genome also differ in sequence, multiple sequence variants can originate from one organism.

This work illustrates a recurring measurement issue in microbial ecology: a molecular marker is a biological object whose own variation affects the inference. Acinas and colleagues investigated that variation directly, rather than simply equating each recovered sequence with a separate kind of microorganism.^[ref]