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Application note

Full-length HiFi
sequencing of eukaryotic
ITS and rRNA amplicons
Introduction
Taxonomic precision is critical for community ecology
studies on microbial eukaryotes. For taxa such as fungithat are difficult to culture, the taxonomic precision ofculture-free approaches affords greater understandingof their role in the mycobiome. Molecular-based
approaches for classification include sequencing target
regions chosen based on their rates of evolution andease of amplification and detection. The regions mostcommonly used as barcode sequences for eukaryotes
are the internal transcribed spacer region (ITS1 andITS2) and the 18S and 28S rRNA genes (Figure 1).
PacBio® HiFi long-read amplicon sequencing has
demonstrated advantages for high-throughput, highresolution eukaryotic barcode sequencing.

In contrast to short reads, in which coverage is limited
to the ITS1 or ITS2 regions only, HiFi reads span the fulllength of fungal ITS regions. This encompasses 800
bp, with the opportunity for amplicons up to ~5 kb if
18S and/or 28S genes are included to span the operon.
Along with more conserved rRNA genes, these regions
allow for reduced sequence similarity and higherresolution taxonomic information at the species level(Tedersoo et al., 2018, Tedersoo et al., 2021, Figure 2).
In addition to yielding greater taxonomic value, HiFi fulllength amplicon sequencing is cost comparable toshort-read, partial amplicon sequencing. In thisApplication note, we present general guidance for theamplification of full-length eukaryotic ITS and rRNA
regions from complex community DNA samples.

Figure 1. (from Tedersoo et al., 2018) Map of primers used to amplify eukaryotic rRNA genes and the internal transcribed spacer (ITS)
therein. Lines below the map indicate amplicons. Colors of primers and lines indicate specificity: black, universal; red, Fungi; green,Glomeromycota; blue, Oomycetes.
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102-326-580 REV02 JAN2024

Page 2Figure 2. (from Tedersoo et al., 2018.) Relative operational taxonomic unit (OTU) richness of different primer pairs of (a) full internaltranscribed spacer (ITS), (b) ITS1 and (c) ITS2 data subsets. White bars, PacBio MagBead loading on RSII instrument; light grey bars, PacBio
Diffusion loading on RSII instrument; orange bars, Diffusion loading on PacBio Sequel® instrument; dark grey bars, Illumina MiSeq. Thisprimer design can be easily adapted for the Sequel IIe instrument using the same steps as outlined in the 16S amplificiation protocol.

Workflow overview
Following amplification of the ITS and/or rRNA primers,amplification workflows (Figure 3) proceed according
to the PacBio 16S bacterial amplification protocol.
Note:

The target region primer sequences need tobe replaced in this protocol to suit that of the
project’s needs and some PCR conditions
may need to be adjusted.

The amplification workflow should begin withextracted, normalized DNA. PCR amplification of thedesired target regions should be performed withbarcoded primers for multiplexing, and should include
extraction, negative, and positive controls.
Amplification results should be confirmed with agarose
gel or another approach. Next, barcoded amplicons arepooled together, and multiplexed amplicon libraries
should be prepared for HiFi sequencing according to
the protocol for the SMRTbell® prep kit 3.0. Finally, HiFi
microbiome amplicon data can be analyzed withseveral bioinformatics tools (see Data analysis
section).

This protocol currently supports up to 192 uniquesample barcodes with dual indexing. Amplicons arepooled prior to SMRTbell library construction following
the PacBio procedure and the library is run on a SMRT®
Cell.
Extracted, normalized DNA

PCR amplification of desired target

Confirm amplification results

Pool barcoded amplicons

Prepare multiplexed ampliconlibraries
HiFi sequencing

Multiplexing approachThis assay uses a one-step PCR workflow withbarcodes added to the target region primer sequences.

Data analysis
Figure 3. HiFi sequencing ITS and rRNA amplicon workflow.

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Amplicon selection and PCR
conditions

The resulting library can be prepared for sequencing on
PacBio long-read systems following instructions in theSample Setup module of SMRT® Link.

There are many primer options available for eukaryotic
rRNA and ITS amplicons. To date, many studies havecompared the ability of multiple primer sets to best
determine eukaryotic diversity with PacBio long-readsequencing. Here we give example primer sets with
PCR conditions available in the references (Table 1).
Note:

These primer sequences and PCR conditions
have neither been verified nor are supported
by PacBio.

It is recommended that you do further review of theliterature provided here (see References) to determine
which primers will best suit your needs. For example,primer performance can vary depending on ampliconsize, sample environment, and ability to amplify certaintaxa.

SMRTbell library construction andsequencing
It is recommended to QC the PCR products for poolingprior to SMRTbell library construction. The guidance forconstructing multiplexed amplicon libraries can befound here.

Data analysis tools
HiFi microbiome amplicon data can be analyzed witheither DADA2, QIIME2, Microbiome Helper, NextITS,One Codex, EzBiome, or the PacBio pipeline availableon GitHub.

Conclusion
Studying fungi and other eukaryotes in complex
communities is challenging because they can bedifficult to identify and are therefore underrepresented
in environmental microbiome studies. Full-length
sequencing of ITS and/or rRNA amplicons provide aconvenient approach to barcoding eukaryotes andyields greater taxonomic resolution compared to shortreads. Here we demonstrate a modified HiFi 16S
sequencing protocol for use with ITS and rRNA
amplicon regions. This adapted workflow allows for thetaxonomic precision needed to improve ourunderstanding of microbial community ecology.

Target region andapproximate size

PrimerSequence (5’–3’)

Primary target

Reference

ITS*~800 bp

F: ITS1catta‖
R: ITS4ngsUni

5’-ACCWGCGGARGGATCATTA-3’
5’-CCTSCSCTTANTDATATGC-3’

Fungi, oomycetes,and stramenophiles

Tedersoo and Anslan (2019)
Tedersoo and Lindahl (2016)

18S-ITS†~1 kb

F: ITS9MUNngs
R: ITS4ngsUni

5’-TACACACCGCCCGTCG-3’5’-CCTSCSCTTANTDATATGC-3’

Most eukaryotes

Tedersoo and Lindahl (2016)
Tedersoo and Lindahl (2016)

ITS-28S‡~1.8 kb

F: ITS1catta‖
R: LR5_TW14ngs5’-ACCWGCGGARGGATCATTA-3’
5’-TCCTGAGGGAAACTTCG-3’

Most eukaryotes

Tedersoo and Anslan (2019)
Tedersoo and Lindahl (2016)

18S-ITS-28S§~5 kb

F: V4F
R: D11_3143R

5’-CCAGCASCYGCGGTAATTCC-3’
5’- RCCACAAGCYARTTATCC-3’

Most eukaryotes

Stoeck et al. (2010)
Latz et al. (2022)

Table 1. Example primer sets by target region and taxa. *Flanks at the extreme 3’ terminus of 18S at position 1790, spans the entire ITSregions 1 and 2, and flanks at the extreme 5’ terminus of 28S at position 40. †Flanks in 18S at position 1635 before V9 (last variable region),spans the entire ITS regions 1 and 2, and flanks at the extreme 5’ terminus of 28S at position 40. ‡Flanks at the extreme 3’ terminus of 18S atposition 1790, spans the entire ITS regions 1 and 2, and flanks in 28S at position 968 after D4. §Flanks in 18S before V4 at position 565, spansthe entire ITS regions 1 and 2, and flanks in 28S at position 3143 after D11. ‖ITS1catta primer selects against amplifying plant DNA.
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Resources
Procedure & checklist – Amplification of bacterial fulllength 16S gene with barcoded primers
Procedure & checklist – Preparing multiplexed
amplicon libraries using SMRTbell® prep kit 3.0

References
These references represent a subset of publications
that use PacBio full-length amplicon sequencing, as
categorized by gene/region and taxa.

16S and fungal ITS
Wang, Q., Li, J., Yang, J., Zou, Y., & Zhao, X. Q. (2022).
Diversity of endophytic bacterial and fungal microbiota
associated with the medicinal lichen Usnea longissima
at high altitudes. Frontiers in Microbiology, 13, 958917.
https://doi.org/10.3389/fmicb.2022.958917
Zhou, J. C., Wang, Y. F., Zhu, D., & Zhu, Y. G. (2023).
Deciphering the distribution of microbial communities
and potential pathogens in the household dust. Scienceof The Total Environment, 872, 162250.
https://doi.org/10.1016/j.scitotenv.2023.162250

16S and fungal ITS-28S
Li, J., He, Z., Yan, L., He, Y., & Yang, J. (2023). Analysisof the microbial community structure and flavor
components succession during salt-reducing picklingprocess of zhacai (preserved mustard tuber). Food
Science & Nutrition. https://doi.org/10.1002/fsn3.3297

18S and fungal ITS
Latz, M. A., et al. (2022). Short-and long-readmetabarcoding of the eukaryotic rRNA operon:evaluation of primers and comparison to shotgun
metagenomics sequencing. Molecular Ecology
Resources, 22(6), 2304-2318.
https://doi.org/10.1111/1755-0998.13623

Runnel, K., et al. (2022). DNA barcoding of fungal
specimens using PacBio long-read high-throughput
sequencing. Molecular Ecology Resources, 22(8), 28712879. https://doi.org/10.1111/1755-0998.13663

16S and 18SZhang, X., Bi, L., Gentekaki, E., Zhao, J., Shen, P., &Zhang, Q. (2023). Culture-Independent Single-Cell
PacBio Sequencing Reveals Epibiotic Variovorax andNucleus Associated Mycoplasma in the Microbiome ofthe Marine Benthic Protist Geleia sp. YT (Ciliophora,Karyorelictea). Microorganisms, 11(6), 1500.
https://doi.org/10.3390/microorganisms11061500

18SZhu, H., Li, S., Hu, Z., & Liu, G. (2018). Molecularcharacterization of eukaryotic algal communities in thetropical phyllosphere based on real-time sequencing of
the 18S rDNA gene. BMC Plant Biology, 18(1), 1-14.
https://doi.org/10.1186/s12870-018-1588-7

16S, 18S, and ITS
Tedersoo, L., Albertsen, M., Anslan, S., & Callahan, B.
(2021). Perspectives and benefits of high-throughput
long-read sequencing in microbial ecology. Applied andenvironmental microbiology, 87(17), e00626-21.
https://doi.org/10.1128/AEM.00626-21

18S, 28S, and ITS
Tedersoo, L., Tooming-Klunderud, A., & Anslan, S.
(2018). PacBio metabarcoding of Fungi and othereukaryotes: errors, biases and perspectives. New
Phytologist, 217(3), 1370-1385.
https://doi.org/10.1111/nph.14776

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© 2024 PacBio. All rights reserved. Research use only. Not for use in diagnostic procedures.
102-326-580 REV02 JAN2024