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Principals and Workflow of 16S/18S/ITS

Amplicon Sequencing
This article shows what is 16S/18S/ITS amplicon sequencing 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.

What is 16S rDNA /18S rDNA/ITS?

⚫ 16s rDNA: 16S rDNA is a DNA sequence encoding small subunit rRNA of prokaryotes in 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
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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 analyzed.

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.

1) 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 the dilution
required for template preparation is determined and followed by sequencing.

2) 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 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 analysing, 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.
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3) Bioinformatics Analysis: Reads is 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.

OTU, operational taxonomic units (OTUs), is often used in the microbiological culture-free analysis. In
general, the similarity of different 16S rDNA/18S rDNA/ITS sequences is higher than 97% and 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

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,
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2. Edwards, J., Johnson, C., Santosmedellín, 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.
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3. Evans, C. C., Lepard, K. J., Kwak, J. W., Stancukas, M. C., Laskowski, S., & Dougherty, J., et al. (2014)
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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.