16S Ribosomal RNA Amplicon Sequencing

This set of vocabulary flashcards covers the fundamental concepts, structural components, advantages, disadvantages, and technical workflows of 16S ribosomal RNA amplicon sequencing.

Molecular clocks

A term used by Woese and colleagues to describe bacterial rRNA genes due to their universality, activity, cellular functions, and extremely conserved structure and nucleotide sequence.

23S ribosomal RNA

A type of prokaryotic ribosomal RNA with a sequence length of about $3300$ nucleotides.

16S ribosomal RNA

A type of prokaryotic ribosomal RNA with a sequence length of about $1550$ nucleotides, serving as a standard in bacterial taxonomic classification.

5S ribosomal RNA

A type of prokaryotic ribosomal RNA with a sequence length of about $120$ nucleotides.

Variable regions

The nine regions across the bacterial domain in the 16S gene that correlate to lower-level taxonomy such as genus and species.

Conserved regions

The eight regions in the 16S gene that correlate to higher-level taxonomy.

Sequence divergence

A range of $0.5\%$ to $1\%$ used to delineate the species taxonomic rank in 16S rRNA gene sequence similarity comparisons.

Horizontal gene transfer

A process that is cited as not being a significant problem for 16S ribosomal RNA sequencing.

Shotgun metagenomics

A comprehensive functional representation via whole genome sequencing that microbiome research is moving towards as costs drop.

DNA isolation

The initial step in the complete 16S sequencing workflow followed by library preparation, sequencing, and data analysis.

27F and 1492R

A pair of primers usually used to amplify the full-length 16S ribosomal RNA gene.

V1 to V3 region

The region identified as most useful for distinguishing skin bacterial genus Staphylococcus and generally used for skin microbiome studies.

PacBio SMRT sequencing

A technology used for sequencing the full-length 16S ribosomal RNA gene typically after amplification by 27F and 1492R primers.

V3 and V4 region

The regions commonly amplified using a limited cycle PCR when sequencing is performed with Illumina MiSeq.

Nextera XT indices

A system of adapters and dual index barcodes used to pool up to $96$ libraries together for sequencing.

Operational Taxonomic Units (OTU)

Clusters of high-quality sequences commonly based on $97\%$ identity of the reads used for species annotation and phylogeny.