Jul 14, 2025
COI library preparation for Illumina MiSeq eDNA metabarcoding - rocky intertidal seawater samples
- Mary McElroy1
- 1University of California, Santa Barbara
External link: https://www.ncbi.nlm.nih.gov/sra/PRJNA1289101
Protocol Citation: Mary McElroy 2025. COI library preparation for Illumina MiSeq eDNA metabarcoding - rocky intertidal seawater samples. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxzordv8j/v1
Manuscript citation:
In prep
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 18, 2022
Last Modified: July 14, 2025
Protocol Integer ID: 62780
Keywords: metabarcoding, environmental DNA, COI, rocky intertidal, library preparation, Illumina MiSeq, amplicon sequencing, seawater sampling, rocky intertidal seawater samples this protocol, rocky intertidal seawater sample, dna metabarcoding, coi library preparation for illumina miseq, amplicon metabarcoding with the illumina miseq system, seawater sample, library preparation for coi amplicon metabarcoding, library preparation for illumina miseq, coi amplicon metabarcoding, rocky intertidal habitat, amplicon metabarcoding, california rocky intertidal habitat, illumina miseq system, illumina miseq, coi library preparation, illumina
Funders Acknowledgements:
Zegar Family Foundation
Grant ID: SB200094
Abstract
This protocol describes library preparation for COI amplicon metabarcoding with the Illumina MiSeq system. Seawater samples were collected from California rocky intertidal habitats, filtered using Sterivex capsules, and extracted with Qiagen DNeasy Blood & Tissue kits (see related protocols). This protocol is modified from:
Wangensteen, O.S., Palacín, C., Guardiola, M. and Turon, X., 2018. DNA metabarcoding of littoral hard-bottom communities: high diversity and database gaps revealed by two molecular markers. PeerJ,6, p.e4705.
Materials
This protocol requires a laboratory set up for standard PCR, gel electrophoresis and imaging, DNA quantitation, and cold storage.
See CALeDNA Library Preparation protocols and Illumina 16S Sample Prep Guide for detailed lists of instruments and consumables.
Troubleshooting
Before start
Order Leray XT primer set (Wangensteen et al. 2018) with Illumina overhangs:
Forward overhang: 5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
Reverse overhang: 5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG
Forward primer (mlCOIintF-XT): 5′-GGWACWRGWTGRACWITITAYCCYCC
Reverse primer (jgHCO2198): 5′-TAIACYTCIGGRTGICCRAARAAYCA
Amplicon PCR
Dilute primers to 5 uM in PCR water. Dilute BSA to 4 mg/ml in PCR water. Set up triplicate PCRs for each sample.
Perform PCRs in 20 ul volumes with the following reaction chemistry:
10 ul 2X AmpliTaq Gold 360 Master Mix (Applied Biosystems)
0.75 ul 4 mg/ml bovine serum albumin (BSA)
1 ul 5 uM fwd primer
1 ul 5 uM rvs primer
1.25 ul undiluted DNA template
6 ul PCR grade water
Thermocycling conditions:
Initial denaturation, 95C for 10 min
35 cycles of denaturation, 94C for 1 min; annealing, 45C for 1 min; extension, 72C for 1 min
Final extension, 72C for 5 min, then hold at 4C
Check PCR products
Run gels for all samples to check for successful amplification.
Amplicon PCR clean-up
Clean PCR products using 0.8X ratio and AMPure XP beads (Beckman Coulter). Use fresh 70% ethanol and elute DNA in 30 ul ultra pure PCR water.
Bead prep: x samples * (0.8)(45 ul sample) * 1.1(10% error) = x ul beads
Product QC and quantification
Check PCR product sizes for a random mix of samples and controls.
Quantify all pooled sample yields.
Normalize
Using product yields, dilute samples in a new plate to 10 ng in up to 11.25 ul PCR water per sample. If yield is less than 10 ng, then use 11.25 ul product for indexing reactions.
Index PCR
Use IDT for Illumina DNA/RNA Unique Dual Indexes (Sets A-D, 20027213-6).
For each sample, perform PCR in 25 ul reactions with the following reaction chemistry:
12.5 ul KAPA HiFi Hot Start Ready mix (Roche Diagnostics 07958927001)
1.25 ul Illumina UD indexing primer mix
11.25 ul normalized product
Thermocycling conditions:
Initial denaturation, 95C for 5 min
8 cycles of denaturation at 98C for 20 sec; annealing at 56C for 30 sec; extension at 72C for 3 min.
Final extension, 72C for 5 min, then hold at 4C.
Index PCR clean-up
Check product sizes again (see Step 6).
Determine bead ratio using observed fragment sizes after index PCR. Use bead prep calculation from Step 5, adjusted for 25 ul product and appropriate bead ratio from product size check. We used 0.8X.
Clean PCR products with AMPure XP beads (Beckman Coulter). Use fresh 70% ethanol and elute DNA in 30 ul ultra pure PCR water.
Quantify all indexed sample yields.
Pool libraries
Pool cleaned, indexed samples by equal mass, targeting a final library concentration of 10-15 nM in at least 200 ul. Use fragment size to calculate nM from ng/ul.
Quantify pooled library and check fragment size (same methods as in previous steps).
Sequencing
Prep PhiX Control Kit v3 for 15% spike-in.
Sequence with MiSeq v3 600-cycle kit, 2 x 300 PE reads.
Protocol references
Wangensteen, O.S., Palacín, C., Guardiola, M. and Turon, X., 2018. DNA metabarcoding of littoral hard-bottom communities: high diversity and database gaps revealed by two molecular markers. PeerJ,6, p.e4705.