16S和ITS rRNA测序（内部转录间隔区核糖体RNA测序）是常用的扩增子测序方法，可用于鉴定和比较给定样本中的细菌或真菌。基于NGS的ITS和16S rRNA基因测序可比较来自复合微生物组或环境中难以研究甚至不可能研究的样本的系统发育和分类，它们都是该应用领域的成熟方法。
原核生物的16S rRNA基因长约1500 bp，包含9个散布在保守区域之间的可变区。16S rRNA的可变区经常用于不同微生物群落的属或种系统进化分类1。rRNA顺反子的ITS1区域是鉴定宏基因组样本中真菌物种的常用DNA标记2。
微生物学家通过16S rRNA NGS可以将菌群宏基因组学研究的灵敏度提升至属水平。使用NGS进行ITS分析可以快速鉴定真菌，有助于我们深入了解真菌微生物组。此外，NGS还能在单次测序运行中分析多个样本。
使用BaseSpace Apps进行分类学分类16S Metagenomics
利用Illumina审核的GreenGenes分类数据库对16S rRNA靶向扩增子read进行分类学分类。DRAGEN Metagenomics Pipeline
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