What is 16s rRNA?
16s rRNA sequencing is a type of sequencing that specifically targets the 16s rRNA gene. It is frequently used for metagenomics - the sequencing of genetic material directly from an environment (such as sea water or the gut microbiome via stool samples) rather than the sequencing of genes from a specific organism. rRNA stands for “ribosomal RNA”.
Ribosomes are the bits inside the cell that convert mRNA into proteins. It is made up of rRNA and (chicken-and-egg-ily) proteins. Names have been given to the different bits of rRNA in a ribosome. 16s rRNA is the name for one of the parts, specifically in prokaryotic species.
16s rRNA sequencing targets the gene that codes for 16s rRNA, because it tends to evolve slowly - thus it can act as a fingerprint for different species, allowing us to get an overview of the prokaryotic species in a sample.
How is 16s rRNA sequencing done?
We use the high-fidelity Phusion polymerase for amplification of 16s and ITS2 marker genes. If extractions contain some carry-over inhibition or a high concentration of DNA, typically we test 1:1 and 1:10 dilutions and run the PCR products on gels for verification. PCR is done with dual-barcoded primers (Kozich et al. 2014) targeting either the prokaryotic (16S V4, V1-V3) or fungal (ITS2) regions. Our barcoding strategy enables multiplexing up to 384 samples per run. PCR products are verified visually by running a representative subset of samples on a gel. Samples with failed PCRs (or spurious bands) are re-amplified by optimizing PCR conditions. The PCR reactions are cleaned up and normalized using the high-throughput SequalPrep 96-well Plate Kit. Samples are then pooled to make one library that is quantified accurately with the KAPA qPCR Library Quant kit.
– 16S rRNA Sequencing Service page; Library Preparation Section, Microbiome Insights
A polymerase is a molecule that can create new strands of DNA/RNA (so its no wonder that it is used for amplification). Notably, polymerase is the “P” in PCR (Polymerase Chain Reaction). Primers are used to encourage amplification of only the 16s rRNA gene (we can ignore the bit about the ITS2 gene as they are talking about fungi there).
The 16S rRNA gene is approximately 1600 base pairs long and includes nine hypervariable regions of varying conservation (V1-V9). More conservative regions are useful for determining the higher-ranking taxa, whereas more quickly evolving ones can help identify genus or species.
Bukin et al. (2019)