Principles and Workflow of 16S/18S/ITS Amplicon Sequencing

This article shows what 16S/18S/ITS amplicon sequencing is and how it works. Let’s get ready to learn.

16S/18S/ITS amplification sequencing uses the next/third generation sequencing platform and performs high throughput sequencing of PCR products from specific regions such as 16S rDNA/18S rDNA/ITS/ functional genes. It overcomes the disadvantage of some microorganisms that is difficult or impossible to culture, and obtains the information of microbial community structure, evolutionary relationships and microbial correlation with environment in environmental samples.

• 16s rDNA: 16S rDNA is a DNA sequence encoding small subunit rRNA of prokaryotes with a length of about 1542bp. With a moderate molecular size and low mutation rate, 16S rDNA is the most commonly used marker in the study of bacterial systematics. The 16S rDNA sequence consists of 9 variable regions and 10 conservative regions, the conserved region sequences reflect the genetic relationships between species, while the variable region sequences reflect the difference between species. 16S rDNA sequencing is mainly used to analyze the diversity of bacteria or archaea.

Fig.1 16S rDNA and amplification primers

• 18S rDNA: 18S rDNA is a DNA sequence encoding small subunit rRNA of eukaryotic ribosomes. Like 16S rDNA, 18S rDNA sequence also consists of conservative regions and variable regions (V1-V9, absence of V6). Among variable regions, V4 has the most complete database information and the best classification effect, it is the mostly used and the best choice for 18S rRNA gene analysis notes. 18S rDNA sequencing reflects the species differences among eukaryotic organisms in given samples.

Fig.2 18S rDNA and amplification primers

• ITS: ITS (Internal Transcribed Spacer) is part of the non-transcriptional region of the fungal rRNA gene. The ITS sequences used for fungal identification usually include ITS1 and ITS2. Because in fungi, 5.8S, 18S, and 28S rRNA genes are highly conserved, whereas ITS can tolerate more mutations in the evolutionary process due to less natural selection pressure, and exhibits extremely wide sequence polymorphism in most eukaryotes. At the same time, the conservative type of ITS is relatively consistent within species, and the differences between species (or ever stains) are obvious. ITS sequence fragments are small (350 bp and 400 bp in length, respectively) and easy to analyze. They have been widely used in phylogenetic analysis of different fungi.

Fig.3 ITS and amplification primers

What is 16S/18S/ITS amplicon sequencing?

16S/18S/ITS amplicon sequencing uses Illumina or PacBio sequencing to read the PCR products which are amplified with suitable universal primers of one or several regions of 16S/18S/ITS. By detecting the sequence variation and abundance of the target area, the information of species classification and abundance, population structure, phylogenetic evolution and community comparison of environmental samples could be obtained.

How to conduct a 16S/18S/ITS amplicon sequencing?

In short, the main steps of 16S/18S/ITS amplicon sequencing include library construction, sequencing and bioinformatics analysis.

• Library Construction: We recommend the fusion primer library construction method, that is, the primers fused with the target sequence primers and the adapter, index and other sequences are synthesized in advance, then the genomic DNA targets are directly amplified by PCR. Amplicon libraries are purified and an equimolar pool of the amplicon libraries is prepared. The dilution required for template preparation is determined and followed by sequencing.
• Sequencing: The current sequencing platforms mainly include Illumina Miseq/HiSeq and third-generation sequencing platform.
• Illumina NGS (MiSeq/HiSeq2500/HiSeq4000): Due to the limitation of reading length, the NGS platform can only select a single variable region, double variable regions or triple variable regions as the target regions for the sequencing. When sequencing, only the completely sequenced Reads (Tags) can be used for further analysis, so different amplification regions should strictly follow the corresponding sequencing strategy. For example, if you chose V4 for analysis, the PE250 sequencing is needed, but for V1-V3 regions, the sequencing strategy should be PE300. Only in this way can the completeness of sequences be ensured. The original data is filtered out to remove low-quality reads and leave high-quality clean data for later analysis.
• PacBio SMRT Sequencing: Unlike NGS, the third generation sequencing platform can carry out full-length sequencing for 16S/18S/ITS, and it’s sequence alignment rate and identification accuracy rate are higher than that of the NGS.
• Bioinformatics Analysis: Reads are spliced into Tags according to the Overlap relationship between reads, and tags are aggregated into OTUs with a given similarity, and then OTUs are annotated by comparing OTUs with databases.

Operational taxonomic units (OTUs) are often used to classify groups of closely related individuals. In general, if the similarity of different 16S rDNA/18S rDNA/ITS sequences is higher than 97%, those sequences can be defined as an OTU. Each OTU corresponds to a different 16S rDNA/18S rDNA/ITS sequence, that is, each OTU corresponds to one species. By OTU analysis, the microbial diversity and the abundance of different microorganisms in the sample can be known.

Then based on OTU and species annotation results, sample species complexity analysis and species difference analysis are conducted. Species based analysis, LDA effect size analysis and more analysis are provided too.

Fig.4 The workflow of 16S/18S/ITS amplicon sequencing

At CD Genomics, our expert team with extensive experience can help you fully understand microbial communities and take advantage of them. In addition to 16S/18S/ITS Amplicon Sequencing, we also provide other microbial genomics services, including:

Metagenomic Shotgun Sequencing
Viral Metagenomic Sequencing
Metatranscriptomic Sequencing
Microbial Whole Genome Sequencing
Viral Genome Sequencing

References:

1. Michelsen, C. F., Pedas, P., Glaring, M. A., Schjoerring, J. K., & Stougaard, P. (2014) ‘Bacterial diversity in greenlandic soils as affected by potato cropping and inorganic versus organic fertilization’, Polar Biology, 37(1), 61-71.
2. Edwards, J., Johnson, C., Santosmedellí, C., Lurie, E., Podishetty, N. K., & Bhatnagar, S., et al. (2015) ‘Structure, variation, and assembly of the root-associated microbiomes of rice’, Proceedings of the National Academy of Sciences of the United States of America, 112(8), E911.
3. Evans, C. C., Lepard, K. J., Kwak, J. W., Stancukas, M. C., Laskowski, S., & Dougherty, J., et al. (2014) ‘Exercise prevents weight gain and alters the gut microbiota in a mouse model of high fat diet-induced obesity’, Plos One, 9(3), e92193.
4. Shehab, N., Li, D., Amy, G. L., Logan, B. E., & Saikaly, P. E. (2013) ‘Characterization of bacterial and archaeal communities in air-cathode microbial fuel cells, open circuit and sealed-off reactors’, Appl Microbiol Biotechnol, 97(22), 9885-9895.
5. Man, K. C., Au, C. H., Chu, K. H., Kwan, H. S., & Chong, K. W. (2010) ‘Composition and genetic diversity of picoeukaryotes in subtropical coastal waters as revealed by 454 pyrosequencing’, Isme Journal, 4(8), 1053.
6. Lie, A. A. Y., Liu, Z., Hu, S. K., Jones, A. C., Kim, D. Y., & Countway, P. D., et al. (2014) ‘Investigating microbial eukaryotic diversity from a global census: insights from a comparison of pyrotag and full-length sequences of 18s rrna genes’, Appl Environ Microbiol, 80(14), 4363-4373.
7. Lu, L., Yin, S., Liu, X., Zhang, W., Gu, T., & Shen, Q., et al. (2013) ‘Fungal networks in yield-invigorating and -debilitating soils induced by prolonged potato monoculture’, Soil Biology & Biochemistry, 65, 186-194.
8. Orgiazzi, A., Lumini, E., Nilsson, R. H., Girlanda, M., Vizzini, A., & Bonfante, P., et al. (2012) ‘Unravelling soil fungal communities from different mediterranean land-use backgrounds’, Plos One, 7(4), e34847.
9. Lakshmanan, V., Ray, P., & Craven, K. D. (2017) ‘Rhizosphere sampling protocols for microbiome (16s/18s/its rrna) library preparation and enrichment for the isolation of drought tolerance-promoting microbes’, Methods Mol Biol, 1631, 349-362.