COI DNA Barcoding for Consumer Fish Samples
Identifies teleost fish to species via the Folmer COI barcode region using the Ward et al. 2005 primer cocktail. Designed for citizen-science fish-mislabeling studies using consumer sushi/sashimi samples. Not a clinical or regulatory-grade assay.
Version History
Version 0.1.3 Latest
Effective: 2026-05-18Documentation patch — no protocol body changes. v0.1.3 marks the canonical transition point for the (measurement_type, technology_type) ISA Assay classification. F1 backfill (2026-05-18) retroactively walked v0.1.0–v0.1.2 back from assay-method OBI subclasses (OBI:0002767 'amplicon sequencing assay' / OBI:0000695 'chain termination sequencing assay') to the classic ISA-Tab split (EDAM:data_3494 'DNA sequence' / OBI:0000626 'DNA sequencing assay'). Original (a)-rationale preserved for audit: assay-method specificity gave per-procedure semantic richness. Walk-back rationale: classic m_type/t_type split groups measurement events into meaningful ISATabExporterV2 buckets — DISTINCT (m_type, t_type) per Study collapses Sanger/Nanopore/Illumina assays into a shared ISA-Tab file rather than singleton-per-procedure files. Also matches FAIR-consumer expectations for external ISA-Tab archive publication. Convention ratified 2026-05-18 between Kevin + LibreBiotech dev during F1 planning.
Version 0.1.2
Effective: 2026-04-19Adds ISA Assay classification at the procedure_version level per PR 6a: measurement_type=OBI:0002767 (amplicon sequencing assay), technology_type=OBI:0000695 (chain termination sequencing assay). No content changes — steps, equipment, materials, parameters, references, and narrative text carried verbatim from v0.1.1 via submit_coi_protocol.py import. Required for PR 6b's exporter to correctly group COI measurement events under a single ISA-Tab Assay Table.
Version 0.1.1
Effective: 2026-04-19Adds two per-run parameters (annealing_temp_c, chelex_incubation_time_min) for per-Assay capture on the measurement matrix. No changes to steps, equipment, materials, or references — content inherited verbatim from v0.1.0 via submit_coi_protocol.py.
Version 0.1.0 Viewing
Effective: 2026-04-18Initial pilot release. Monolithic procedure (sample → Chelex extraction → COI PCR → agarose gel → Sanger submission → BLAST species call). Planned v1.0 splits into atomic Sample Prep / Measurement & QC / Sequencing protocols once the dry run identifies the natural seams.
Procedure Details
- BSL-1 work only. No human-subject, clinical, or regulated samples.
- 95–100°C heat step. Use a dedicated heat block with closed lids; never open flame. Eye protection.
- Pipetting hygiene. Gloves and filter tips throughout. Change gloves between extraction and PCR setup.
- SYBR Safe is safer than ethidium bromide but still a DNA intercalator — gloves, no skin contact, seal gel waste per local biohazard rules.
- Blue-LED transilluminator with amber filter — NOT UV. Required if the protocol will be taught in schools.
- PCR waste. Bleach-treat pipette tips and tubes; discard in sealed bag.
- No clinical interpretation. Mislabeling calls are for open-dataset research, not consumer complaints or legal action.
Sample specification. ~20 mg teleost tissue per sample, rice-grain size. Raw or sashimi-grade preferred; lightly seared acceptable. Fully cooked, heavily processed, or surimi products require mini-barcode primers (see Completion Notes / Troubleshooting).
Primer prep. Order FishF1, FishF2, FishR1, FishR2 as separate 25-nmol oligos from IDT. Resuspend each to 100 µM in TE; dilute working stocks to 10 µM. Make two mixes: F-mix = equimolar F1+F2 at 10 µM total; R-mix = equimolar R1+R2 at 10 µM total.
Chelex prep. 5% w/v Chelex-100 in low-EDTA TE. Keep the suspension mixed — resin settles fast; pipette immediately after vortexing.
Bench prep. Bleach-wipe pre- and post-PCR areas. Dedicated pipette sets if available; otherwise filter tips plus strict glove changes. No-template controls are mandatory.
LibreBiotech setup. Before wet lab: create the Investigation ("Sushi Truth: Consumer Fish Mislabeling"), nested Study ("Pilot Season 1 — [city]"), and pre-register Sample records with claimed species, vendor metadata, and photos.
- Day 1 (active wet lab, ~3.5 h): extraction 45 min • PCR setup 15 min • thermocycling 2 h (hands-off) • gel run + image 45 min
- Day 1→3 (external): Sanger sequencing 24–48 h turnaround
- Day 3 (analysis, ~1 h): trim, BLAST, species call, log to LibreBiotech
- Time-sensitive: Chelex extract is best used within 4 h fresh or stored at –20°C. Run the gel the same day as cycling — do not leave PCR plates at room temp overnight.
-
Gel imager / UV transilluminator
Specs: Blue-LED transilluminator + amber filter; Pi HQ camera
DIY build in repo; NOT UV -
Microcentrifuge
Centrifugation
Specs: ≥12,000 × g
Benchtop -
Gel electrophoresis system
Electrophoresis
Specs: Horizontal, ≥100 V PSU
Mini gel format -
Micropipette
Liquid handling
Specs: P10, P200, P1000
Calibrated; filter tips only -
Heat block
Thermal regulation
Specs: 56°C and 95–100°C capable -
Thermal cycler
Thermal regulation
Specs: 25 µL tubes, programmable ramp, heated lid
Pi-controlled OpenPCR-compatible build; schematics in repo
-
Proteinase K
Enzyme
Qty: 5 µL
NEB P8107S, 20 mg/mL -
Primer (custom)
Primer
Qty: 0.25 µL
IDT custom; 5'-TCAACCAACCACAAAGACATTGGCAC-3' (Ward 2005) -
Primer (custom)
Primer
Qty: 0.25 µL
IDT; 5'-TCGACTAATCATAAAGATATCGGCAC-3' -
Primer (custom)
Primer
Qty: 0.25 µL
IDT; 5'-TAGACTTCTGGGTGGCCAAAGAATCA-3' -
Primer (custom)
Primer
Qty: 0.25 µL
IDT; 5'-ACTTCAGGGTGACCGAAGAATCAGAA-3' -
Agarose
Reagent
Qty: 0.4 g per gel
Molecular grade -
Buffer
Reagent
Qty: 500 mL per gel -
DNA ladder
Reagent
Qty: 5 µL per lane
NEB N3231S -
Loading dye
Reagent
Qty: 1 µL per well
NEB B7024S -
Lysis solution
Reagent
Qty: 200 µL
Bio-Rad 142-1253, 5% w/v in low-EDTA TE -
Nuclease-free water
Reagent
Qty: q.s. µL
To 25 µL per reaction -
Nucleic acid stain
Reagent
Qty: 1 X (final)
Invitrogen S33102 from 10,000X stock -
PCR master mix
Reagent
Qty: 12.5 µL
NEB OneTaq 2X M0482S; alt: NEB Q5 2X
Protocol Parameters Captured per-assay on each run; exported as ISA-Tab Parameter Value columns
| Name | Type | Required | Default | Unit | Description |
|---|---|---|---|---|---|
annealing_temp_c |
number | — |
52
|
degree Celsius (UO:0000027) | Annealing temperature for COI PCR cycling. Default 52°C works for the Ward 2005 primer cocktail. Raise to 54°C if the gel shows multiple bands (non-specific priming). |
chelex_incubation_time_min |
number | — |
30
|
minute (UO:0000031) | Incubation time at 56°C for Chelex/Proteinase K lysis. Default 30 min suffices for fresh tissue. Extend to 60–120 min for cooked or heavily processed samples. |
Procedure Steps (Version 0.1.0)
Collect 2–3 tissue pieces per sample, ideally before sauces are applied. Create a Sample record in your Study with claimed species, vendor, price, date/time, photo, and claim source.
Transfer ~20 mg tissue (rice-grain size) to a labeled 1.5 mL tube and cut into small fragments.
Add 200 µL of freshly vortexed 5% Chelex-100 suspension and 5 µL of 20 mg/mL Proteinase K to the tube.
Incubate at 56°C for 30 min. Vortex briefly once at the 15 min mark.
Transfer to 95°C and incubate for 10 min.
Vortex 10 s; centrifuge at 12,000 × g for 2 min.
Carefully transfer ~100 µL of the clear upper phase to a new labeled tube, avoiding the Chelex pellet. This is the DNA template.
Prepare a PCR master mix for n+1 reactions (always include a no-template control). Per reaction: 12.5 µL OneTaq 2X, 0.5 µL F-mix (10 µM), 0.5 µL R-mix (10 µM), 10.5 µL nuclease-free water.
Aliquot 24 µL master mix into each PCR tube. Add 1 µL template to sample tubes; add 1 µL water to the NTC tube.
Run the thermocycler: 94°C 2 min; 35 cycles of [94°C 30 s, 52°C 30 s, 72°C 45 s]; 72°C 10 min; 4°C hold.
Export the thermocycler temperature log from the Pi controller. Attach it to the PCR Process record in LibreBiotech.
Cast a 1.5% agarose gel in 1X TBE containing 1X SYBR Safe.
Load 5 µL PCR product + 1 µL 6X loading dye per well. Include a 100 bp DNA ladder lane.
Run at 100 V for ~30 min, until the dye front is two-thirds down the gel.
Image the gel on a blue-LED transilluminator with amber filter. Expected: single sharp band at ~655 bp. Upload the image to the PCR Process record.
For clean single-band samples, submit 10–20 µL unpurified PCR product to a Sanger service (e.g. Plasmidsaurus Premium PCR) with FishF1 as the sequencing primer. Label tubes with the LibreBiotech Sample ID exactly.
When sequences return, trim reads to Q20 and BLAST the consensus against BOLD (primary) and NCBI nr (secondary).
Accept a species-level call if top hit ≥98% identity, query coverage ≥90%, and gap to next species ≥1%. Otherwise report to genus only.
Log the BLAST call, accession, %ID, query coverage, and rationale on the Sample record. Set mislabeled=true when the call differs from the claimed species.
Expected outcome. Single sharp ~655 bp band per sample on gel; clean Sanger trace (≥Q30 across amplicon); BLAST top hit to BOLD ≥98% identity, query coverage ≥90%, gap to next species ≥1% → species-level call.
Storage. Chelex extracts: –20°C, up to 1 year. PCR products: –20°C, up to 6 months.
Data logging. Every sample should end with a Sample record in LibreBiotech containing: claimed species, BLAST call, %ID, accession, thermocycler log, gel image, Sanger read. Flag mislabeled=true when the call differs from the claim.
Troubleshooting.
| Symptom | Likely cause | Fix |
|---|---|---|
| No band (cooked sample) | DNA degraded | Mini-barcode primers (Shokralla 2015); extend Proteinase K to 2 h |
| Band in no-template control | Reagent / bench contamination | Bleach bench, fresh aliquots, new tips — do not proceed |
| Multiple bands | Non-specific priming | Raise annealing to 54°C; dilute template 1:10 |
| Smear | Template overload or degradation | Dilute Chelex extract 1:10 |
| Low Sanger quality | Primer-dimer / PCR carryover | Request cleanup service; or gel-extract the 655 bp band |
| BLAST call ambiguous (top two hits within 1%) | Closely related species | Report to genus; note on Sample record |
References
- Ward RD, Zemlak TS, Innes BH, Last PR, Hebert PDN (2005). DNA barcoding Australia's fish species. Phil Trans R Soc B 360:1847–57. DOI paper
- Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R (1994). COI primers for invertebrates. Mol Mar Biol Biotechnol 3:294–9. paper
- Shokralla S et al. (2015). Next-generation DNA barcoding: using nanopore sequencing to enhance species monitoring. Sci Rep 5:9687. DOI paper
- Walsh PS, Metzger DA, Higuchi R (1991). Chelex 100 as a medium for simple extraction of DNA. BioTechniques 10:506–13. paper
- BOLD Systems — Barcode of Life Data System Link paper
- NCBI BLAST Link paper