May 14, 2026
16S rRNA Amplicon Sequencing Using Ion GeneStudio S5
- Grace Semabia Kpeli1,
- Prince Agyirey-Kwakye2,
- Counseller Nutifafa Livingstone1,
- Lillian Teye Cudjoe1,
- Emmanuel Edem Dotse1,
- Ebenezer Nyarko1,
- Ebenezer Zar1,
- Obed Nyarko-Otu1,
- Hubert Kwame Agbogli1,
- Godwin Glilekpeh1,
- Solomon Korankye1,
- Priscilla Essandoh1,
- Daniel Elorm Kabotso3
- 1Department of Biomedical Science, University of Health and Allied Sciences, Sokode Lokoe, Ho, Ghana;
- 2AttoDiagnostics Limited, Norwich, UK;
- 3Department of Basic Science, University of Health and Allied Sciences, Sokode Lokoe, Ho, Ghana
- GSK's workspace
Protocol Citation: Grace Semabia Kpeli, Prince Agyirey-Kwakye, Counseller Nutifafa Livingstone, Lillian Teye Cudjoe, Emmanuel Edem Dotse, Ebenezer Nyarko, Ebenezer Zar, Obed Nyarko-Otu, Hubert Kwame Agbogli, Godwin Glilekpeh, Solomon Korankye, Priscilla Essandoh, Daniel Elorm Kabotso 2026. 16S rRNA Amplicon Sequencing Using Ion GeneStudio S5. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl84468g2w/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 14, 2026
Last Modified: May 14, 2026
Protocol Integer ID: 317104
Keywords: 16S rRNA, amplicon sequencing, Ion GeneStudio S5, Ion AmpliSeq, microbiome, environmental DNA, ion ampliseq microbiome health research kit, ion genestudio s5 this protocol, using ion genestudio s5, ion genestudio, rrna amplicon, environmental water dna extract, amplicon preparation, hv9
Abstract
This protocol describes 16S rRNA amplicon sequencing of environmental water DNA extracts covering hypervariable regions HV2–HV9, using the Ion AmpliSeq Microbiome Health Research Kit on the Ion GeneStudio™ S5 platform. It includes amplicon preparation, chip loading, sequencing, and initial demultiplexing and quality control using Ion Reporter™ Software.
Materials
Equipment
- Ion GeneStudio™ S5 System (Thermo Fisher Scientific, U.S.)
- Ion 540™ Chip (Cat# 46496, Thermo Fisher Scientific)
- Standard thermal cycler (for amplicon amplification)
- Qubit™ Fluorometer (for library quantification)
Reagents and Consumables
- Ion AmpliSeq™ Microbiome Health Research Kit (Thermo Fisher Scientific)
- Ion 540™ Chip (Cat# 46496, Thermo Fisher Scientific)
- Extracted environmental DNA (from Protocol 2), minimum quality: A260/280 ≈ 1.8–2.0
Procedure
Review input DNA quality: Confirm DNA concentration and purity from Qubit and NanoDrop results meet the Ion AmpliSeq kit's input requirements.
Amplify 16S rRNA amplicons covering hypervariable regions HV2 to HV9 using the Ion AmpliSeq™ Microbiome Health Research Kit, following the manufacturer's protocol.
Quantify amplicon libraries using the Qubit™ Fluorometer. Record concentrations for all samples.
Normalise individual sample libraries to equimolar concentrations as specified by the manufacturer's protocol, then pool for sequencing.
Load the pooled, normalised library onto the Ion 540™ Chip following the Ion GeneStudio™ S5 manufacturer's protocol.
Perform targeted sequencing on the Ion GeneStudio™ S5 platform according to the manufacturer's run parameters.
Use Ion Reporter™ Software version 5.18 to demultiplex raw sequencing reads by sample barcode.
Apply quality control filters within Ion Reporter™ Software v5.18. Review output metrics (read counts, quality scores) before proceeding to bioinformatics analysis.
Quality Control
Only samples meeting minimum DNA quality thresholds should proceed to library preparation.
Library concentrations should be confirmed by Qubit prior to chip loading.
Review Ion Reporter™ QC metrics post-sequencing before proceeding to downstream analysis.
Protocol references
Ion AmpliSeq™ Microbiome Health Research Kit — Thermo Fisher Scientific.
Ion GeneStudio™ S5 System User Guide — Thermo Fisher Scientific.
Ion Reporter™ Software v5.18 — Thermo Fisher Scientific.