Jul 01, 2026
Version 1
OceanOmics Centre: eDNA Amplicon Library Preparation using Illumina TruSeq DNA PCR-Free V.1
- Laura Missen1,
- Marcelle Ayad2,
- Anna Depiazzi1,
- Ebony Thorpe1,
- OceanOmics UWA1
- 1Minderoo OceanOmics Centre at UWA;
- 2Minderoo Foundation
External link: https://www.uwa.edu.au/oceans-institute/partnerships/minderoo-oceanomics-centre-at-uwa
Protocol Citation: Laura Missen, Marcelle Ayad, Anna Depiazzi, Ebony Thorpe, OceanOmics UWA 2026. OceanOmics Centre: eDNA Amplicon Library Preparation using Illumina TruSeq DNA PCR-Free. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj16bbvk5/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: January 07, 2026
Last Modified: July 01, 2026
Protocol Integer ID: 237687
Keywords: eDNA, eDNA Metabarcoding, Amplicon Library Preparation, Illumina, Next-Generation Sequencing , TruSeq, Tru-Seq DNA PCR-Free , Library Preparation, Ligation, UD Indexes, Marine eDNA
Abstract
This protocol describes a modified amplicon library preparation workflow using the Illumina TruSeq DNA PCR-Free Library Preparation Kit. This method has been optimised for environmental DNA (eDNA) metabarcoding of marine vertebrates and is compatible with five assays: 16S Fish/D, MiFish-U, MiFish-E2, MarVer1, and COI-Leray. The workflow converts indexed PCR amplicons into sequencing-ready libraries through a ligation approach.
This protocol is employed by the OceanOmics Centre to generate amplicon sequencing data from metabarcoding. It represents a compiled and modified protocol based on Illumina's published methods, adapted for our specific samples. Library Preparation takes approximately 4–5 hours to complete, with an additional 3 hours for fragment analysis and library QC.
Guidelines
Standard lab set-up:
- Ensure competency and training requirements are met.
- Wear gloves and a lab coat.
- Use tips with filters to avoid contamination.
- Wipe workspace with 10% bleach, leave to decontaminate for 10 minutes before wiping with ddH20.
- Sterilise all equipment (vortex, minispin, pen markers, outside of tip boxes, pipettes) before use.
- UV pipettes, racks, tubes (lids open), and plates for 15 minutes before use.
- Wipe the inside of the centrifuge and rotor with 70% ethanol.
- Ensure all reagents are aliquoted in appropriate amounts, and stored according to manufacturers' recommendations.
- All waste must be disposed of following institutional biosafety regulations.
Materials
Equipment
- ThermalCycler
- Magnetic stand for 1.5/2.0 mL tubes
- Magnetic stand for 96-well plate
- Vortex with plate adapter
- Minispin
- Centrifuge with plate adapter
- UV sterilisation cabinet
- Ice block
- Qubit 4.0 Fluorometer
Equipment
Femto Pulse System
NAME
Automated Gel electrophoresis
TYPE
Agilent
BRAND
M5330AA
SKU
Consumables
- Eppendorf 1.5 mL DNA LoBind Tubes, PCR Clean
- 50 mL Falcon tubes for making-up 80% ethanol and for waste
- Semi-skirted PCR plate (Eppendorf twin.tec semi-skirted PCR plate 96)
- Plate seals (Axygen CyclerSeal PCR sealing film)
- Nuclease-free water
- TE buffer for Femto dilutions
Reagents
TruSeq DNA PCR-Free Library Preparation KitCatalog #FC-121-3001 IDT for Illumina- TruSeq UD Indexes (96 indexes, 96 samples (v2))Illumina, Inc. 100% Molecular grade ethanol Qubit dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q32851
Femto Pulse Ultra Sensitivity NGS KitAgilent TechnologiesCatalog #FP-1101-0275
Safety warnings
This protocol involves the use of potentially hazardous biological materials, chemicals (including ethanol and enzymatic reagents), and electrical laboratory equipment. Always wear appropriate personal protective equipment (PPE), including a lab coat, gloves, and eye protection. Be familiar with the relevant Safety Data Sheets (SDS) and laboratory risk assessments before commencing work. Follow institutional safety guidelines and dispose of waste in accordance with local regulations. Exercise caution when handling sharp instruments, thermal cyclers, and high-speed centrifuges to prevent injury or equipment damage.
Relevant SDS documents: 
Resuspension Buffer.pdf73.7KB Sample Purification Beads.pdf73KB Stop Ligation Buffer.pdf170.2KB
Resuspension Buffer.pdf73.7KB Sample Purification Beads.pdf73KB Stop Ligation Buffer.pdf170.2KB Before start
Prepare working environment as per the guidelines.
Identify the gDNA pools that are to be library prepped and then assign each pool an adapter using the Illumina Pooling Guidelines for the IDT for Illumina TruSeq UD Index kit (96 indexes, 96 samples): https://support-docs.illumina.com/SHARE/IndexAdaptersPooling/Content/TruSeqIDTIlluminaUDIndexes.htm
Prepare Reagents
Thaw Resuspension Buffer (RSB) at room temperature for 30 minutes.
Remove Sample Purification Beads (SPB) from the fridge and allow to equilibrate at room temperature for 30 minutes.
Remove End Repair Mix (ERP2) from the freezer, thaw at room temperature and then place on ice.
Thaw A-Tailing Mix (ATL) at room temperature and then place on ice. Vortex and spin-down before use.
NOTE: this can be done at step 37.
Thaw Stop Ligation Buffer (STL) at room temperature and then place on ice. Vortex and spin-down before use.
NOTE: this can be done at step 50.
Thaw the adapters required at room temperature and then place on ice. Flick mix and spin-down before use.
NOTE: this can be done at step 43.
Prepare the required volume of 80% ethanol (960 µL 100% molecular grade ethanol, 240 µL nuclease-free water per library +10% excess).
Prepare Plate
Prepare DNA for library preparation in a total volume of 50 µL.
- For 16S-Fish/D, MiFish-U/E2 and MarVer1, a 250ng input is required for library preparation.
- For COI-Leray, a 100ng input is required for library preparation.
For instructions on how to blend DNA for library preparation see the following protocol:
Transfer 50 µL of each pool into a PCR plate. Label this plate CEP (Clean-Up End Repair Plate).
NOTE: it is recommended to record pool positions on a plate map e.g. in Excel
Convert Overhangs
Add 10 µL of RSB to each pool.
Add 40 µL of ERP2 to each pool.
Seal the plate with a PCR sealing film.
Using a vortex, shake at 1800 rpm for 1 minute.
Centrifuge at 300 x g for 1 minute.
Incubate the plate in a ThermalCycler using the following conditions:
Total volume: 100 µL
Place ERP2 back into the freezer, it will not be required again.
Remove Small Fragments
Remove plate from ThermalCycler and centrifuge at 300 x g for 1 minute.
Transfer each pool into a clean and labelled 1.5 mL DNA LoBind tube. The CEP plate can be discarded.
Vortex SPB until fully resuspended.
NOTE: ensure the beads are at room temperature.
Add 180 µL of SPB to each sample (1.8x ratio). Pipette mix 10x.
Incubate at Room temperature for 5 minutes.
Place on a magnetic stand for 1.5 mL/2.0 mL tubes and leave for 5 minutes until the supernatant becomes clear.
Carefully remove and discard the supernatant into a designated waste container. Do not disturb the pellet.
Add 200 µL of 80% ethanol to each sample.
Incubate at room temperature for 30 seconds.
Carefully remove and discard the supernatant into a designated waste container. Do not disturb the pellet.
Repeat steps 24-26 for a total of two washes.
Briefly spin-down tubes to collect the remaining ethanol.
Place tubes back on the magnetic rack and remove any remaining ethanol with a 10 µL pipette.
Air-dry on the magnetic rack, with the lids open, for 5 minutes.
NOTE: do not leave for longer as beads can over-dry.
Remove tubes from the magnetic rack.
Add 17.5 µL of RSB to each pool. Pipette the liquid over the beads from the side of the tube, this can be done multiple times until fully resuspended.
Incubate at Room temperature for 5 minutes.
Place on the magnetic rack and leave for 5 minutes.
Transfer 15 µL of each pool into a designated well on a PCR plate. Label this plate ALP (Adapter Ligation Plate).
NOTE: it is recommended to record pool positions on a plate map e.g. in Excel
This is a safe stopping point.
Seal the plate and store at -20°C for up to 7 days, or proceed immediately to the next step immediately.
Adenylate 3' Ends
If removing plate from the freezer, bring to room temperature.
Centrifuge plate at 300 x g for 1 minute.
Add 2.5 µL of RSB to each pool.
Add 12.5 µL of ATL to each pool.
Seal the plate with a PCR sealing film.
Using a vortex, shake at 1800 rpm for 1 minute.
Centrifuge at 300 x g for 1 minute.
Incubate the plate in a ThermalCylcer using the following conditions:
Total volume: 30 µL
Place the ATL buffer back in the freezer, it will not be required again.
Once complete, remove the plate and centrifuge at 300 x g for 1 minute.
Ligate Adapters
In the order listed below, add the following reagents to each pool:
NOTE: LIG2 is temperature sensitive, remove from the freezer immediately before use, place in an ice block while pipetting and then place immediately back into the freezer.
Seal the plate with a PCR sealing film.
Using a vortex, shake at 1800 rpm for 1 minute.
Centrifuge at 300 x g for 1 minute.
Incubate the plate in a ThermalCycler using the following conditions:
Immediately after 10 minutes, remove the plate from the ThermalCylcer, centrifuge at 300 x g for 1 minute, remove seal, and then add 5 µL of STL to each pool.
NOTE: it is very important to add the STL buffer as soon as the incubation finishes, do not let the ThermalCycler hold!
Re-seal the plate and shake at 1800 rpm for 1 minute using a vortex.
Centrifuge at 300 x g for 1 minute.
Proceed immediately to the clean-up step. The STL buffer can be placed back into the freezer, it will not be required again.
Library Clean-Up
Label one 1.5 mL DNA LoBind tube for each library.
Vortex SPB until fully resuspended.
NOTE: ensure beads are at room temperature.
Add 42.5 µL of SPB to each library. Pipette mix 10x.
Incubate at Room temperature for 5 minutes.
Place on a magnetic stand for 96-well plates and leave for 5 minutes until supernatant becomes clear.
Using a 200 µL pipette, aspirate and discard the supernatant into a designated waste container.
Add 200 µL of 80% ethanol to each library.
Incubate on the magnetic stand for 30 seconds.
Remove and discard the supernatant.
Repeat steps 61-63 for a total of two washes.
Using a 10 µL pipette, remove any residual ethanol in the well.
Air-dry on the magnetic rack for 2 minutes.
Remove plate from the magnetic rack and and 52.5 µL of RSB to each library. Pipette mix 10x.
Incubate at Room temperature for 5 minutes.
Place on the magnetic stand and leave for 5 minutes until supernatant becomes clear.
Transfer 50 µL of supernatant into a new well in the plate, or if there is no room, transfer to a new plate.
Vortex SPB to fully resuspend.
Add 50 µL of SPB to each library.
Incubate at Room temperature for 5 minutes.
Place on the magnetic stand and leave for 5 minutes until supernatant becomes clear.
Using a 200 µL pipette, aspirate and discard the supernatant into a designated waste container.
Add 200 µL of 80% ethanol to each library.
Incubate on the magnetic stand for 30 seconds.
Remove and discard the supernatant.
Repeat steps 76-78 for a total of two washes.
Using a 10 µL pipette, remove an residual ethanol in the well.
Air-dry on the magnetic rack for 2 minutes.
Remove plate from the magnetic rack and and 22.5 µL of RSB to each library. Pipette mix 10x.
Incubate at Room temperature for 5 minutes.
Place on the magnetic stand and leave for 5 minutes until supernatant becomes clear.
Transfer 20 µL into the designated 1.5 mL DNA LoBind tube. This is now your final ligated library.
This is a safe stopping point.
Store library at 4ºC. Libraries should be sequenced within 7 days. Avoid freeze/thawing as this can degrade DNA.
Library Quantification
Use the Qubit High Sensitivity dsDNA kit to quantify 2 µL of each library. Libraries should be around 2-10 ng/µL.
NOTE: if sequencing the library more than 7 days after library prep, repeat quantification.
Library Fragment Analysis
Use the Femto Pulse Ultra Sensitivity NGS Kit to check the fragment length of the final cleaned and size-selected libraries.
NOTES:
- Other fragment analysis instruments can be used for this step, however, Femto Pulse is the most accurate for libraries with Y-shaped adapters. Other instruments would need to be optimised to account for this.
- A 0.2 ng/µL dilution should be used for input into the Femto Pulse. A total volume of 2 µL of your library should be used for this dilution.
Visually check the Femto results: at this stage, a single peak around the expected size range for the assay should be observed.
Example of a successful library preparation for 16S-Fish/D assay:
Expected result
Expected size-ranges of ligated libraries for OceanOmics assays as are follows:
For each library, calculate the % expected peak by entering the concentration values for the expected size range and the total concentration in the workbook below. Library preparation is successful if the % expected peak is above 50%
%_Peak_Calculator.xlsx64.2KB These concentrations can be found in the Femto report, an example is shown below. The total concentration is highlighted in orange, and the concentration of the expected peak is highlighted in pink.
If successful, proceed to sequencing. If unsuccessful, investigate and repeat library preparation if required.
Protocol references
Illumina TruSeq DNA PCR-Free Library Preparation protocol: https://support.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/samplepreps_truseq/truseq-dna-pcr-free-workflow/truseq-dna-pcr-free-workflow-reference-1000000039279-00.pdf
MiFish-U:
Miya, M., Sato, Y., Fukunaga, T., Sado, T., Poulsen, J., Sato, K., Minamoto, T., Yamamoto, S., Yamanaka, H., & Araki, H. (2015). MiFish, a set of universal PCR primers for metabarcoding environmental DNA from fishes: detection of more than 230 subtropical marine species. Royal Society open science, 2(7), 150088.
MiFish-E2:
Miya, M. a. S., T. (2019). Multiple species detection using MiFish primers. Pages 55–92 in
Environmental DNA sampling and experimental manual version 2.1. Ed. by eDNA Methods
Standardization Committee, The eDNA Society, Otsu, Japan.
MarVer1:
Valsecchi, E., Bylemans, J., Goodman, S. J., Lombardi, R., Carr, I., Castellano, L., Galimberti, A., & Galli, P. (2020). Novel universal primers for metabarcoding environmental DNA surveys of marine mammals and other marine vertebrates. Environmental DNA, 2(4), 460-476.
16S-Fish/D:
Berry, T. E., Osterrieder, S. K., Murray, D. C., Coghlan, M. L., Richardson, A. J., Grealy, A. K., Stat, M., Bejder, L., & Bunce, M. (2017). DNA metabarcoding for diet analysis and biodiversity: A case study using the endangered Australian sea lion (Neophoca cinerea). Ecology and evolution, 7(14), 5435-5453.
Deagle, B. E., Gales, N. J., Evans, K., Jarman, S. N., Robinson, S., Trebilco, R., & Hindell, M. A. (2007). Studying seabird diet through genetic analysis of faeces: a case study on macaroni penguins (Eudyptes chrysolophus). PloS one, 2(9), e831.
COI-Leray:
Leray, M., J. Y. Yang, C. P. Meyer, et al. 2013. A New Versatile Primer Set Targeting a Short Fragment of the Mitochondrial COI Region for Metabarcoding Metazoan Diversity: Application for Characterizing Coral Reef Fish Gut Contents. Frontiers in Zoology 10: 34.