eDNA Metabarcoding Lab Workflow

SOP-L706 Revision 0.1
Author

Marc Los Huertos

EA Program, Pomona College

Published

2026-06-26

1 Purpose

This SOP describes the end-to-end laboratory workflow for environmental DNA (eDNA) metabarcoding, from receipt of field-collected water filter samples through DNA extraction, PCR amplification, library preparation, sequencing, and bioinformatics analysis. eDNA metabarcoding combines universal or group-specific primers with high-throughput sequencing to detect and characterize aquatic biodiversity from water samples without capturing organisms (Deiner et al., 2017; Taberlet et al., 2012).

This SOP serves as the integrating document that ties together the field collection procedures in SOP-F700 with the molecular and computational SOPs in the L700 series. It does not repeat detailed procedures described in other SOPs but provides the eDNA-specific modifications, QC requirements, and decision points that distinguish eDNA metabarcoding from standard amplicon sequencing of soil or tissue samples.

2 Scope

  • Applies to: Laboratory processing of eDNA water samples for multi-species detection via amplicon sequencing on Illumina platforms; target taxa include fish (12S), amphibians (16S/12S), macroinvertebrates (COI), and general biodiversity surveys (18S, COI)
  • Does not apply to: Species-specific eDNA detection by qPCR or ddPCR (single-species assays); soil microbial community analysis (see SOP-L704 and SOP-L705); field collection procedures (see SOP-F700)
  • Location: Molecular biology bench, SGM 133

3 Responsibilities

Role Responsibility
Lab PI Approve target markers, primers, and sequencing plan
Trained Personnel Process samples per this SOP; maintain contamination controls
New Students Complete eDNA-specific contamination training before participating

4 Definitions

  • Amplification blank (no-template control, NTC): A PCR reaction containing all reagents except sample DNA; run with every PCR plate; any amplification in the NTC indicates contamination and invalidates the entire plate.
  • Blocking primer: A modified oligonucleotide added to a PCR reaction to selectively suppress amplification of abundant non-target DNA (e.g., host DNA in gut or tissue eDNA samples); allows rare target sequences to be amplified competitively.
  • eDNA metabarcoding: A method that uses universal or group-specific primers to amplify and sequence a short, taxonomically informative DNA region (barcode) from environmental DNA, yielding a community- level inventory of taxa present in a sample.
  • Environmental DNA (eDNA): Genetic material shed into the environment by organisms through mucus, feces, skin cells, eggs, and other biological material; collected non-invasively from water, sediment, or air.
  • Extraction blank: A tube containing only extraction reagents and no biological material, processed through the full extraction procedure; detects reagent or handling contamination; must yield no target amplification.
  • Field blank: A filter or collection vessel processed through all field collection steps but with no environmental sample; detects airborne or handling contamination during field work.
  • Metabarcode locus: The specific genomic region amplified for community identification; common loci include 12S rRNA (fish), 16S rRNA (bacteria, invertebrates), 18S rRNA (eukaryotes), COI (invertebrates, fish), and ITS2 (fungi, plants).
  • Occupancy model: A statistical model that accounts for imperfect detection in eDNA surveys by estimating the probability that a species is truly present given detection/non-detection across replicate samples or PCR replicates.
  • PCR replicate: One of multiple independent PCR reactions run from the same DNA extract; increases detection sensitivity for rare targets and allows estimation of false-negative rates; standard eDNA practice is 3–8 replicates per sample.
  • Sequence read depth (coverage): The total number of sequencing reads assigned to a sample; directly affects the number of rare taxa detected; EA Program target is a minimum of 10,000 reads per sample after quality filtering.

5 Safety and Hazards

WarningHazards
  • Chemical: Lysis buffers (guanidine salts), ethanol, SPRI beads, and DNA stains. See individual SOP safety sections for details.
  • Biological: eDNA samples may originate from water bodies containing pathogens. Handle with standard precautions.
  • UV radiation: If using UV transilluminator for gel imaging.

5.1 Required PPE

5.2 Emergency Procedures

Refer to SOP-L003 Hazardous Materials Handling.

6 Workflow Overview

The eDNA metabarcoding workflow consists of six stages. Each stage references a detailed SOP.

Stage Description SOP Reference
1 Field collection and preservation F700
2 DNA extraction from filters L700
3 PCR amplification and verification L703
4 Library preparation and sequencing L704
5 Bioinformatics (QIIME 2 / DADA2) L705
6 Taxonomic interpretation and reporting This SOP, Section 6.6

7 Procedure: eDNA-Specific Modifications

7.1 Sample Receipt and Logging

  1. Upon receipt from the field, verify that all samples match the chain of custody form per SOP-F008.
  2. Inspect Sterivex capsules or filter bags for integrity (no leaks, caps intact, preservative present).
  3. Log each sample in the lab notebook and Freezer Inventory Google Sheet.
  4. Store preserved filters at -20 C (short-term) or -80 C (long-term) until extraction.

7.2 DNA Extraction from Filters

Follow SOP-L700 with the following eDNA-specific modifications:

  • Sterivex capsules: Open the capsule aseptically using a sterile razor blade or the Sterivex extraction adapter. Transfer the membrane into the PowerBead Pro tube using sterile forceps. If Longmire’s buffer was used as preservative, add the buffer to the bead tube as well.
  • Open membrane filters: Transfer the folded filter directly into the bead tube using sterile forceps.
  • Extraction blanks: Process one extraction blank per batch. For eDNA, this means running an empty bead tube through the full protocol.
  • Field blanks: The field blank filter (from SOP-F700) is extracted alongside samples and serves as the primary contamination control for the entire field-to-lab chain.

7.3 Inhibition Testing

eDNA extracts from natural waters frequently contain PCR inhibitors (humic acids, tannins, metal ions) (McKee et al., 2015). Test for inhibition before committing to full library preparation:

  1. Run an internal positive control (IPC) qPCR assay on a subset of extracts (or all extracts, if the batch is small). The IPC uses a synthetic DNA target spiked into the PCR reaction at a known concentration.
  2. If the IPC Ct value shifts by more than 2 cycles compared to the uninhibited control, the extract is inhibited.
  3. For inhibited samples, dilute the extract 1:5 or 1:10 with nuclease-free water and retest. Alternatively, clean the extract using a OneStep PCR Inhibitor Removal Kit (Zymo Research).

7.4 Primer Selection for eDNA Metabarcoding

Target Taxa Marker Primers Amplicon Reference
Fish 12S rRNA MiFish-U-F / MiFish-U-R ~170 bp Miya et al. (2015)
Amphibians 16S rRNA 16Smam / 16Smam-R (modified) ~250 bp Vences et al. (2005)
Macroinvertebrates COI mlCOIintF / jgHCO2198 ~313 bp Leray et al. (2013)
General eukaryotes 18S rRNA Euk1391f / EukBr ~130 bp Amaral-Zettler et al. (2009)
General eukaryotes COI fwhF2 / fwhR2n ~205 bp Vamos et al. (2022)
Note

Primer choice determines which taxa can be detected. Discuss target taxa and primer selection with the PI before beginning library preparation. Multi-marker approaches (e.g., 12S + COI) provide broader taxonomic coverage but require separate library preparations and sequencing runs (Deiner et al., 2017).

7.5 PCR and Library Preparation

Follow SOP-L703 for initial PCR screening and SOP-L704 for library preparation, with these eDNA-specific considerations:

  • Replicates: Run 3 independent PCR replicates per sample to improve detection probability for rare species (Ficetola et al., 2015). Pool the replicates after PCR1 before cleanup and indexing.
  • Cycle number: Use 35 cycles for PCR1 (higher than standard 16S amplicon work) because eDNA concentrations are typically very low. Be aware that additional cycles increase the risk of chimera formation and tag-jumping (Schnell et al., 2015).
  • Blocking primers (optional): If non-target taxa (e.g., human DNA) dominate the PCR product, consider using blocking primers or PNA (peptide nucleic acid) clamps to suppress amplification of non-target sequences.
  • Controls: In addition to the standard NTC and extraction blank, include the field blank and (if available) a positive control consisting of a mock community of known species at known concentrations.

7.6 Bioinformatics and Taxonomic Assignment

Follow SOP-L705 for denoising and taxonomic classification, with these modifications:

  • Reference database: Use a curated database matched to the marker and target taxa:
    • 12S fish: MitoFish or MIDORI2
    • COI: BOLD / MIDORI2
    • 16S/18S: SILVA or GenBank nt
  • Species-level assignment: For eDNA, species-level identification is the primary goal (unlike microbiome studies where genus-level is often sufficient). Use BLAST-based classification (classify-consensus- blast in QIIME 2) in addition to or instead of the Naive Bayes classifier for improved species-level resolution (Goldberg et al., 2016).
  • Decontamination: Use the decontam R package to identify and remove contaminant ASVs using the field blank and extraction blank data (Davis et al., 2018).
  • Detection criteria: An ASV is considered a positive detection for a species only if:
    1. It matches a reference sequence at greater than or equal to 97–99% identity (marker-dependent)
    2. It is absent from all negative controls (field blank, extraction blank, NTC), or its abundance in the sample exceeds 10x the abundance in the highest-read control
    3. It is detected in at least 2 of 3 PCR replicates (if replicates were sequenced independently) (Ficetola et al., 2015)

7.7 Reporting

eDNA metabarcoding results are reported as a species detection list per site with the following metadata:

  • ASV sequence and percent identity to reference
  • Number of reads per ASV per sample
  • Detection across replicates (e.g., detected in 2/3 replicates)
  • Negative control results
  • Any taxa flagged as potential contaminants
Important

eDNA metabarcoding provides presence/detection data. Quantitative interpretation of read counts as species abundance is not recommended without careful validation, because PCR amplification bias, primer affinity, and DNA shedding rates vary among taxa (Kelly et al., 2019).

8 Quality Control

  • Field blanks: Must be negative for all target taxa. Any target taxon detected in a field blank compromises the detection claim for that taxon across all samples in the same sampling event.
  • Extraction blanks: Must be negative. Positive extraction blanks indicate lab contamination; the entire extraction batch is suspect.
  • NTC: Must be negative. Positive NTCs indicate PCR contamination.
  • Positive control (mock community): Must recover all expected species. Failure to detect a known species in the mock community indicates primer bias or PCR failure.
  • Replication: A minimum of 3 PCR replicates per sample is recommended for occupancy-based detection analyses.
  • Document all control results in the project data file and report alongside species detections.

9 Waste Disposal

Refer to waste disposal tables in SOP-L700, SOP-L703, and SOP-L704.

10 References

Amaral-Zettler, L. A., McCliment, E. A., Ducklow, H. W., & Huse, S. M. (2009). A method for studying protistan diversity using massively parallel sequencing of V9 hypervariable regions of small-subunit ribosomal RNA genes. PLOS ONE, 4(7), e6372. https://doi.org/10.1371/journal.pone.0006372
Davis, N. M., Proctor, D. M., Holmes, S. P., Relman, D. A., & Callahan, B. J. (2018). Simple statistical identification and removal of contaminant sequences in marker-gene and metagenomics data. Microbiome, 6(1), 226. https://doi.org/10.1186/s40168-018-0605-2
Deiner, K., Bik, H. M., M"achler, E., Seymour, M., Lacoursiere-Roussel, A., Altermatt, F., Creer, S., Bista, I., Lodge, D. M., Vere, N. de, Pfrender, M. E., & Bernatchez, L. (2017). Environmental DNA metabarcoding: Transforming how we survey animal and plant communities. Molecular Ecology, 26(21), 5872–5895. https://doi.org/10.1111/mec.14350
Ficetola, G. F., Pansu, J., Bonin, A., Coissac, E., Giguet-Covex, C., De Barba, M., Gielly, L., Lopes, C. M., Boyer, F., Pompanon, F., Ray0̆0e9, G., & Taberlet, P. (2015). Replication levels, false presences and the estimation of the presence/absence from eDNA metabarcoding data. Molecular Ecology Resources, 15(3), 543–556. https://doi.org/10.1111/1755-0998.12338
Goldberg, C. S., Turner, C. R., Deiner, K., Klymus, K. E., Thomsen, P. F., Murphy, M. A., Spear, S. F., McKee, A., Oyler-McCance, S. J., Cornman, R. S., Laramie, M. B., Mahon, A. R., Lance, R. F., Pilliod, D. S., Strickler, K. M., Waits, L. P., Fremier, A. K., Takahara, T., Herder, J. E., & Taberlet, P. (2016). Critical considerations for the application of environmental DNA methods to detect aquatic species. Methods in Ecology and Evolution, 7(11), 1299–1307. https://doi.org/10.1111/2041-210X.12595
Kelly, R. P., Shelton, A. O., & Gallego, R. (2019). Understanding PCR processes to draw meaningful conclusions from environmental DNA studies. Scientific Reports, 9(1), 12133. https://doi.org/10.1038/s41598-019-48546-x
Leray, M., Yang, J. Y., Meyer, C. P., Mills, S. C., Agudelo, N., Ranwez, V., Boehm, J. T., & Machida, R. J. (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(1), 34. https://doi.org/10.1186/1742-9994-10-34
McKee, A. M., Spear, S. F., & Pierson, T. W. (2015). The effect of dilution and the use of a post-extraction nucleic acid purification column on the accuracy, precision, and inhibition of environmental DNA samples. Biological Conservation, 183, 70–76. https://doi.org/10.1016/j.biocon.2014.11.031
Miya, M., Sato, Y., Fukunaga, T., Sado, T., Poulsen, J. Y., Sato, K., Minamoto, T., Yamamoto, S., Yamanaka, H., Araki, H., Kondoh, M., & Iwasaki, W. (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. https://doi.org/10.1098/rsos.150088
Schnell, I. B., Bohmann, K., & Gilbert, M. T. P. (2015). Tag jumps illuminated – reducing sequence-to-sample misassignments in metabarcoding studies. Molecular Ecology Resources, 15(6), 1289–1303. https://doi.org/10.1111/1755-0998.12402
Taberlet, P., Coissac, E., Hajibabaei, M., & Rieseberg, L. H. (2012). Environmental DNA. Molecular Ecology, 21(8), 1789–1793. https://doi.org/10.1111/j.1365-294X.2012.05542.x
Vamos, E. E., Giesen, C., Tränkner, M., Germanis, O., & Fonseca, V. G. (2022). A review of bioinformatic tools and databases for eDNA metabarcoding. Environmental DNA, 4(5), 967–990. https://doi.org/10.1002/edn3.325
Vences, M., Thomas, M., Meijden, A. van der, Chiari, Y., & Vieites, D. R. (2005). Comparative performance of the 16S rRNA gene in DNA barcoding of amphibians. Frontiers in Zoology, 2(1), 5. https://doi.org/10.1186/1742-9994-2-5

12 Training Record

Before processing eDNA samples independently, personnel must:

  1. Read this SOP and all related SOPs (F700, L700, L703, L704, L705)
  2. Demonstrate aseptic filter handling and extraction under supervision
  3. Complete an eDNA-specific contamination awareness session covering pre-PCR/post-PCR separation, filter handling, and control interpretation
  4. Process a supervised batch including field blank, extraction blank, and positive control with clean results
  5. Be signed off by the PI or lab manager