16S和ITS rRNA测序

16s rRNA is a subunit of a ribosome found in all bacteria and archaea. It is 1500 nucleotides long and contains nine variable regions interspersed between conserved regions.

16s rRNA sequencing is a culture-free method to identify and compare bacterial diversity from complex microbiomes or environments that are difficult to study. It is commonly used to identify bacteria present within a given sample down to the genus and/or species level. Specifically, it is an amplicon-based sequencing method that targets the 16s rRNA bacteria-specific genetic marker using a single amplicon focused on a single gene.

Because the 16s rRNA sequence is ubiquitous in bacteria and archaea, it can be used to identify a wide diversity of microbes within a single sample and single workflow. Through 16s rRNA sequencing, one can identify taxa present in a sample. This leads to a greater understanding of our microbial communities and their interactions with us.

Both the ribosome and its subunits are characterized by their sedimentation coefficients, expressed in Svedberg units (symbol: S). In this case, 16s means it takes 16 Svedberg units of time for the ribosome to sediment in a solution.

All bacteria and archaea have a 16s rRNA sequence.

In amplicon sequencing of 16S rRNA, the primers used bind within regions that are not 100% conserved across bacteria. This leads to some regions of certain bacteria not being included in sequencing. Additionally, chloroplasts have some homology to 16s rRNA genes and may amplify. Because microbiome samples can come from a wide variety of sources with variable compositions, it’s recommended to use control samples when investigating new sample types.

16S DNA refers to the gene in the bacterial genome that codes for the 16S rRNA. 16S rRNA is the rRNA that is transcribed from the 16S DNA gene. The Illumina 16S Metagenomic Sequencing Library Preparation protocol uses DNA as input, and the PCR primers target the variable regions V3 and V4 of the 16S DNA gene for the amplicon PCR.

The 16S Demonstrated Protocol provides an option for creating Illumina compatible libraries from the target of your choice. Fungi and other organisms do not have 16s rRNA genes, however, they have other conserved regions such as 18S and ITS regions. Any amplicon can be used to do similar diversity analysis studies.

The internal transcribed spacer 1 (ITS1) region of the rRNA cistron is a commonly used DNA marker for identification of fungal species in metagenomic sample. ⁵