Jun 30, 2026
OceanOmics Centre: eDNA Amplicon Library Preparation with Lucigen NxSeq AMPFree Low DNA Kit
- Laura Missen1,
- Marcelle Ayad2,
- Anna Depiazzi1,
- Ebony Thorpe1,
- Georgia Nester1,
- 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, Georgia Nester, OceanOmics UWA 2026. OceanOmics Centre: eDNA Amplicon Library Preparation with Lucigen NxSeq AMPFree Low DNA Kit. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgq136ovk5/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 15, 2026
Last Modified: June 30, 2026
Protocol Integer ID: 238690
Keywords: eDNA, environmental DNA, Ligation, Amplicon library preparation, Next-generation sequencing , UD indexes, NxSeq AMPFree Low-DNA, library preparation, amplicon, environmental DNA
Abstract
This protocol describes a modified amplicon library preparation workflow for Illumina sequencing, adapted from the Lucigen NxSeq AMPFree Low DNA Library Preparation Kit. The 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 for high-throughput metabarcoding and incorporates modifications to the manufacturer's instructions to improve performance with short amplicons and multiplexed environmental DNA samples. Library preparation takes approximately 6–7 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 and tubes (lids open) 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
- Vortex
- Minispin
- UV sterilisation cabinet
- Incubator
- Ice bucket and ice
- 96-well ice block
- Qubit 4.0 Fluorometer
Equipment
Femto Pulse System
NAME
Automated Gel electrophoresis
TYPE
Agilent
BRAND
M5330AA
SKU
Consumables
- Eppendorf 1.5 mL LoBind Tubes, PCR Clean
- 50 mL Falcon tubes for making-up 80% ethanol and for waste
- 0.2 mL PCR tubes of strips
Reagents
Lucigen NxSeq AMPFree Low DNA Library Prep KitBiosearch TechnologiesCatalog #14000-2
NxSeq Adapters Biosearch Technologies 100% Molecular grade ethanol Qubit dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q32851 CleanNGS Beads LABOGENECatalog #CNGS-0500 Nuclease-free WaterThermo FisherCatalog #AM9906
Femto Pulse Ultra Sensitivity NGS KitAgilent TechnologiesCatalog #FP-1101-0275
Resuspension BufferIllumina, Inc.Catalog #FC-131-1096
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: 
Lucigen NxSeq AMPFree Low DNA Library Prep Kit SDS.pdf116KB
Lucigen NxSeq AMPFree Low DNA Library Prep Kit SDS.pdf116KB Before start
Prepare working environment as per the guidelines.
Identify the amplicon pools that are to be library prepped and then assign each pool an adapter using the adapter pooling guidelines:
This library preparation uses 8bp indexes.
Prepare Reagents and Samples
Remove Clean NGS Beads from the fridge and allow to equilibrate at room temperature for 30 minutes.
Thaw Elution Buffer (EB) at room temperature for 30 minutes.
Prepare the required volume of 80% ethanol (1200 µL 100% molecular grade ethanol, 300 µL nuclease-free water per library, this includes a 10% excess).
Thaw 2X Buffer (2XB) from the Lucigen kit at room temperature and then place on ice.
Thaw the required adapters at room temperature and then place on ice.
Prepare DNA for library preparation in a total volume of 17 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:
Library Preparation
Set-up end repair and A-tailing reaction for each library in a 0.2 mL PCR tube. Add reagents in the
following order:
NOTE: Enzyme Mix (EM) is temperature sensitive. Remove EM from Lucigen kit only when required, then place back in -20°C immediately after use. Pipette to mix before use.
Pipette mix 10x then briefly spin-down.
Place tubes in a ThermalCycler and incubate according to the following parameters:
While the program is running, remove a 96-well ice block for PCR tubes from the freezer and leave at room temperature to partially defrost.
The 2XB reagent can be placed back into the freezer, it will not be required again.
Once program is complete, remove tubes from the ThermalCycler, spin-down, then place in the 96-well ice block.
Add the following reagents to each tube in the order listed below:
NOTE: Ligase (LIG) is temperature sensitive. Remove LIG from Lucigen kit only when required, then place back in -20°C immediately after use. Pipette to mix before use.
Pipette mix 10x then briefly spin-down.
Place tubes in a ThermalCycler and incubate according to the following parameters:
Once complete, remove tubes from the ThermalCylcer and transfer the total 57 µL to a 1.5 mL DNA LoBind tube. Proceed immediately to clean-up.
Clean-Up
Ensure CleanNGS beads are at room temperature and vortex until fully resuspended.
Add 57 µL of CleanNGS beads to each library (1:1 ratio), pipette 10x to mix.
Incubate at Room temperature for 5 minutes.
Place tubes into a magnetic rack and incubate for 5 minutes.
With the tube in the magnetic rack, aspirate and discard the supernatant into a designated waste container. Do not disturb the pellet.
With the tube in the magnetic rack, add 750 µL of 80% ethanol and incubate for 30 seconds.
Aspirate and discard the supernatant into a designated waste container. Do not disturb the pellet.
Repeat steps 22-23 for a total of two washes.
Briefly spin-down tubes than place back into the magnetic rack.
Remove any residual ethanol with a 10 µL pipette.
With the lids open, leave the tubes in the magnetic rack to air-dry for 5 minutes.
NOTE: do not leave for longer as beads can over dry which could effect yield.
Remove tubes from the magnetic rack and add 102 µL of EB. Pipette 10x to mix.
Incubate at 37 °C for 5 minutes.
Place tubes into the magnetic rack and incubate for 5 minutes.
Transfer 100 µL to a labelled 1.5 mL DNA LoBind tube.
NOTE: tube should be labelled LL (ligated library) and include project, assay, type (sample/control), date and initials.
This is a safe stopping point.
Ligated libraries can be stored at 4°C if processing within the same week or at -20°C for long-term storage. Avoid freeze/thawing as this can degrade DNA.
Size-Selection
Transfer 50 µl of ligated library from the previous step to a new 1.5mL LoBind tube. Label accordingly.
NOTE: the entire 100 µL can be used but it is best practice to proceed with half the volume in case of any issues during size-selection.
Add the required volume of Clean NGS beads to each library according to the table below. Pipette mix 10x.
NOTE: these ratios are optimised for these specific assays.
Incubate at Room temperature for 5 minutes.
Place the tube in the magnetic rack and incubate for 5 minutes at Room temperature until the supernatant becomes clear.
With the tube in the magnetic rack, aspirate and discard the supernatant. Do not disturb the bead pellet.
With the tube in the magnetic rack, add 750 µL of 80% ethanol to each tube and incubate for 30 seconds.
With the tube in the magnetic rack, aspirate and discard the supernatant. Do not disturb the bead pellet.
Repeat steps 37-38 for a total of two washes.
Briefly spin-down then place the tubes back on the magnetic rack.
Remove any residual ethanol with a 10 µL pipette.
With the lids open, leave the tubes in the magnetic rack to air-dry for 5 minutes.
NOTE: do not leave for longer as beads can over dry which could effect yield.
Remove tubes from the rack and add 24 µL of EB. Pipette 10x to mix.
Incubate at 37 °C for 5 minutes.
Place tubes on the magnetic rack and incubate for 5 minutes.
Transfer 22 µL to a new 1.5 mL DNA LoBind tube. Label accordingly.
NOTE: tube should be labelled SSL (size-selected library) and include project, assay, type (sample/control), date and initials.
This is a safe stopping point.
Size-selected libraries can be stored at 4°C and should be sequenced within 7 days.
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 preparation, 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.
NOTE: 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.
Visually check the Femto results: 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 are 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
Lucigen NxSeq Kit and Adapters:
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.