16S sequencing

Microbial diversity is based on the Illumina NovaSeq sequencing platform, using paired end sequencing to construct small fragment libraries for sequencing. By concatenating and filtering Reads, clustering or denoising, and performing species annotation and abundance analysis, the species composition of the sample can be revealed; Further analysis of alpha diversity, beta diversity, significant species differences, correlation, functional prediction, etc. can uncover differences between samples. Microbial diversity research is the study of microbial systematics and species composition in environmental samples using amplified sequence variants as molecular markers. Mainly performed on the conserved regions of nucleic acid sequences encoding ribosomal RNA. Bacteria are mainly based on the 16S region, fungi are mainly based on the ITS region (internal transcriptome region), and eukaryotes are mainly based on the 18S region for sequencing analysis. 16S rDNA is the DNA sequence encoding the prokaryotic ribosomal small subunit rRNA (16S rRNA), 18S rDNA is the DNA sequence encoding the eukaryotic ribosomal small subunit rRNA (18S rRNA), and ITS is the DNA internal transcriptome region sequence encoding the eukaryotic ribosomal small subunit rRNA. These sequences contain both conservative and variable regions. Conservative sequence regions reflect the phylogenetic relationships between biological species, while high variable sequence regions can reflect the differences between species. Due to its relatively conservative evolutionary rate, 18S rDNA is more suitable for taxonomic classification at the species level and above in phylogenetic studies. ITS commonly used for microbial classification research are divided into two types: ITS1 and ITS2. ITS1 is located between 18S and 5.8S of the eukaryotic ribosomal rDNA sequence, while ITS2 is located between 5.8S and 28S of the eukaryotic ribosomal rDNA sequence. Due to its cleavage during ribosomal RNA processing, the ITS region does not play a functional role and has relatively low selection pressure during evolution. Its evolution rate is about 10 times that of 18S rDNA, making it a moderately conserved region. It can be used to study taxonomic levels of species and below. In addition, simultaneous amplification of 18S rDNA and ITS can be achieved by selecting primers. By analyzing the 18S rDNA sequence, the attribution of the sample can be determined at a higher level, and then the fungi can be classified at the species or subspecies level based on the ITS sequence.