Sanger Sequencing Sample Submission (Outsourced)
Preparation and submission of purified PCR products for outsourced Sanger sequencing. Covers concentration measurement, sample labelling, primer selection, shipping format, and vendor-specific requirements (Plasmidsaurus Premium PCR, Azenta/Genewiz, AGRF). Atomic technique supporting procedure 56 (COI Fish Barcoding) and any workflow requiring species-ID or variant confirmation. Written for a novice audience. Most Australian DIY-bio workflows route through Plasmidsaurus (international shipping, no ABN required) or BioQuisitive-pooled AGRF access.
Version History
Version 0.1.1 Latest
Effective: 2026-04-20Cross-reference fixes: BLAST refs 72→76, SPRI ref 70→74. All `procedure N` references converted to Markdown hyperlinks pointing at https://librebiotech.org/?action=show&id=N — enables in-app click-through to referenced sibling procedures. Text content otherwise preserved.
Version 0.1.0 Viewing
Effective: 2026-04-20Initial release. Sanger sequencing method (Sanger 1977) is ~45 years old; sample submission is logistics + concentration matching. Fresh original prose with practical vendor-specific guidance for Australian workflows.
Procedure Details
- No hazardous reagents inherent in the procedure. DNA samples are biologically inert at submission concentrations.
- Shipping considerations. PCR products ship ambient temperature internationally (confirmed by Plasmidsaurus workflow). Do not freeze — ice-pack shipping can cause damage.
- Pipetting hygiene. Filter tips; avoid amplicon cross-contamination when preparing multiple submissions.
- Privacy for custom primers. Sequencing vendors see your primer sequences; do not submit sensitive IP without an NDA.
Vendor selection (Australian DIY-bio context):
| Vendor | Format | AU access | Turnaround | Typical cost |
|---|---|---|---|---|
| Plasmidsaurus Premium PCR | US, international ambient shipping | ✅ individual orders accepted | 2–3 days after receipt | $5–10 per read |
| Azenta/Genewiz | US, international shipping | ✅ individual orders | 3–5 days | $5–8 per read |
| AGRF (Australian Genome Research Facility) | AU institutional | ❌ institutional only | 2–3 days | $8–12 per read |
| BioQuisitive-pooled AGRF access | AU pooled | ✅ via BioQuisitive community | Varies by batch | $8–12 per read + BioQuisitive membership |
For the Sushi Truth pilot, Plasmidsaurus Premium PCR is the default — international ambient shipping works in practice, no Australian ABN or institution required, and rates are competitive.
Concentration requirement:
- Plasmidsaurus Premium PCR: 10–20 ng/µL in 10–20 µL sample. Primer provided separately in a separate tube (1 µL of 5 µM stock).
- Azenta/Genewiz: 10–30 ng/µL, 10 µL sample + primer at 5–10 µM. Pre-mixed samples (sample + primer in one tube) are also accepted.
- AGRF: 20 ng/µL minimum, 10 µL sample, primer pre-mixed at 3.2 µM final in the submission tube.
Primer selection.
- For a PCR amplicon, use ONE of the two PCR primers as the sequencing primer. Choice depends on which end of the amplicon you want to sequence first — reads are typically cleanest in the first ~800 bp, so sequence from the end closer to your critical region.
- For COI barcoding: FishF1 is the standard forward sequencing primer; gives a clean read across the Folmer region.
Labelling.
- Every tube: unique sample identifier. Link to LibreBiotech Sample ID exactly.
- Tube caps: plain, non-coloured where possible (vendors can't read through colour).
- External tube labels: short identifier + vendor-required format (usually alphanumeric only).
Shipping.
- 0.2 mL PCR strips or 0.5 mL microcentrifuge tubes — vendor-specific.
- Ambient temperature shipping; no ice packs.
- Padded envelope or small box.
- International shipping: include invoice marked "DNA samples for sequencing; no commercial value" — avoids customs delays.
Mental model: Sanger sequencing reads the DNA base sequence one nucleotide at a time using dideoxy terminator chemistry. The vendor runs the reaction on a capillary sequencer and returns you an electropherogram (peak trace) + FASTA file. Clean reads give sharp, well-separated peaks across 600–1000 bases.
- Prep (15–30 min per sample batch): measure concentrations, dilute if needed, label, package.
- Shipping (1–3 days for international ambient): hands-off.
- Sequencing (2–3 days after vendor receipt): hands-off.
- Data retrieval (5 min): download FASTA and electropherogram.
- Total: ~1 week from PCR product to species call for international shipping.
-
Microcentrifuge
Centrifugation
Specs: ≥13,000 × g
Brief spin-down before shipping -
Micropipette
Liquid handling
Specs: P10, P20, P200; filter tips
For aliquotting and primer dilution -
Spectrophotometer
Measurement
Specs: Nanodrop or similar for DNA
Concentration verification before submission
-
Primer (custom)
Primer
Qty: 1-2 µL of 5 µM stock
Use one of the two PCR primers; for COI, FishF1 standard. IDT or equivalent supplier
Protocol Parameters Captured per-assay on each run; exported as ISA-Tab Parameter Value columns
| Name | Type | Required | Default | Unit | Description |
|---|---|---|---|---|---|
sample_concentration_ng_per_ul |
number | — |
15
|
— | Target DNA concentration for submission. 10–30 ng/µL typical; vendor-specific. |
sample_volume_ul |
number | — |
10
|
microliter (UO:0000101) | Volume of DNA sample per tube. 10 µL minimum (Azenta); 20 µL preferred (Plasmidsaurus). |
primer_concentration_uM |
number | — |
5
|
— | Sequencing primer concentration. 5 µM standard; 3.2 µM for AGRF pre-mix format. |
primer_volume_ul |
number | — |
1
|
microliter (UO:0000101) | Volume of sequencing primer per tube. 1 µL Plasmidsaurus; 2 µL Azenta. |
Procedure Steps (Version 0.1.0)
Identify which samples need sequencing. Pull the purified PCR products (from procedure 70 SPRI cleanup or procedure 56 directly if clean single-band).
Measure concentration on a Nanodrop or Qubit. Record value as a Sample annotation.
Verify concentration is in the vendor's accepted range (typically 10–30 ng/µL). If too dilute: concentrate via shorter elution (re-elute SPRI into 10 µL instead of 20 µL) or Qubit-only verification. If too concentrated: dilute with water or TE to target range.
Label sample tubes with the vendor-required identifier format. Typically alphanumeric, no spaces, 8–12 characters. Match the LibreBiotech Sample ID exactly for downstream traceability.
Prepare sequencing primer. Dilute from 100 µM stock to 5 µM working concentration in nuclease-free water. Label a separate primer tube per vendor format (Plasmidsaurus wants it in its own tube; AGRF wants it pre-mixed).
Package per vendor specifications:
- Plasmidsaurus Premium PCR: 10–20 µL sample in one tube + 1 µL of 5 µM primer in a separate tube, both labelled with the same ID.
- Azenta/Genewiz: 10 µL sample + 2 µL of 5 µM primer, either separately or premixed in one tube.
- AGRF: 10 µL sample + primer pre-mixed at 3.2 µM final concentration in one tube.
Double-check every label matches between tube and your submission sheet.
Complete the vendor's submission form online. Paste the sample list, provide primer sequence (if the vendor requires it), choose the sequencing direction (forward/reverse primer).
Package samples in the vendor-supplied envelope or a padded mailer. Ambient temperature; no ice packs.
For international shipping: attach the vendor's customs form or include an invoice marked 'DNA samples for sequencing; no commercial value; CAS 63231-63-0 (DNA)'. Ship via a trackable international carrier.
Record the submission in LibreBiotech: Process record with each submitted sample as an annotated Sample input, vendor name, submission date, tracking number, estimated return date.
Monitor email for the read result. When received, download the FASTA and chromatogram. Upload to the corresponding Sample record as annotations.
Forward to procedure 72 for BLAST analysis and species call.
Expected outcome. An email from the vendor with:
- FASTA file of the sequence read (one record per tube submitted).
- Electropherogram (AB1 or chromatogram image) showing peak quality across the read.
Quality assessment.
- Read length: expect 600–1000 bases of high-quality sequence; the final 100–200 bases of any read degrade.
- Q-score (quality): >Q30 across the useful region indicates sharp, well-separated peaks. Visible in the chromatogram as clear single peaks of one colour per position.
- Poor-quality signs: multiple peaks at same position (primer-dimer, sample contamination), low-intensity peaks (insufficient template), or high baseline noise (contamination).
Downstream. Run the FASTA through BLAST (procedure 72) for species identification. Record the call, Q-score, read length, and any anomalies on the Sample record in LibreBiotech.
Troubleshooting.
| Symptom | Likely cause | Fix |
|---|---|---|
| No read returned | Sample too dilute; wrong primer sent | Verify concentration; resubmit with correct primer |
| Read starts 50+ bases in | Primer mismatch or primer-dimer carryover | Use different sequencing primer; SPRI-cleanup sample more aggressively |
| Double peaks throughout | Mixed sample (two PCR products); contamination | Gel-extract target band; resubmit |
| Short read (<200 bp) | Degraded template | Fresh PCR; cleaner template |
| Peaks of varying heights | Sample concentration off | Submit at ~15 ng/µL; verify with Nanodrop before shipping |
| Low-quality end of read | Normal (all reads degrade after 800 bp) | Acceptable; trim to Q20 before analysis |
References
- Sanger F, Nicklen S, Coulson AR (1977). DNA sequencing with chain-terminating inhibitors. PNAS 74(12):5463–7. (Original Sanger method). DOI paper
- Plasmidsaurus — Premium PCR Sanger sequencing service documentation. Link paper
- LibreBiotech procedure 56 — COI Fish Barcoding (upstream); procedure 72 — BLAST species call (downstream). Link protocol