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Laboratory Workflow: Sample Reception to Final Report

Overview

This document describes the end-to-end workflow for processing avian genetic testing samples at SENO's laboratory, from sample reception through to final report delivery.

Workflow Diagram

Sample Receipt → Registration → Inspection → DNA Extraction
                                            PCR/qPCR Setup
                                            Amplification
                                          Post-PCR Analysis
                                          Result Interpretation
                                               QC Review
                                              Reporting

Step 1: Sample Reception

1.1 Physical Receipt

TaskResponsibleAction
Package inspectionReceiving clerkCheck for leakage, damage, temperature (if cold pack required)
Documentation checkReceiving clerkVerify submission form: species, test type, client ID, number of samples
Photo documentationReceiving clerkPhotograph package condition (for customs claims if needed)
Chain of custodyReceiving clerkSign and date receipt; scan into LIMS

1.2 Sample Acceptance Criteria

Sample TypeAcceptReject (with notification)
FeathersFreshly plucked, follicle intact, 3–5 per birdShed feathers, no visible follicle, < 2 feathers
SwabsIn stabilization solution, sealed tubeLeaking, dry swab without solution, expired stabilizer
Blood (EDTA)0.1–0.5 mL, not hemolyzedClotted, hemolyzed, > 14 days at room temp
Blood (FTA card)Visible blood spot, dryNo visible spot, still moist (mold risk)

Step 2: Registration & Labeling

TaskDetail
Create case in LIMSEach client = 1 case; each bird = 1 sample entry
Assign sample IDFormat: SENO-YYYYMMDD-NNNN (date + sequential)
Barcode generation2D barcode printed and affixed to primary tube
Scan linkageBarcode linked to client ID, species, test type in LIMS
Batch assignmentGrouped by test type and extraction method

Step 3: Sample Inspection

3.1 Laboratory Inspection

TestMethodRecord
Feather follicle checkMagnifying lamp (10×)5-point scale: 0=no follicle → 5=excellent
Swab conditionVisualNormal / dried / leaking / contaminated
Blood qualityVisualNormal / hemolyzed / clotted / insufficient

3.2 Chain of Custody Continuation

Each transfer between technicians is logged: time, technician ID, sample count.

Step 4: DNA Extraction

See DNA Extraction SOP for detailed protocol.

4.1 Batch Processing

ParameterSpecification
Batch size48 samples + 2 controls (1 extraction positive, 1 extraction blank)
Extraction positiveKnown female parrot feather (ZW)
Extraction blankLysis buffer only (no sample)
Time per batch~45 min hands-on + 30 min incubation

4.2 Post-Extraction QC

TestCriteriaAction if Fail
NanoDrop concentration> 5 ng/µLRe-extract with additional feathers
A260/280 ratio1.7–2.0Re-purify if < 1.6; check if > 2.1
A260/230 ratio> 1.5Re-purify if < 1.2
Extraction blankNo DNA by NanoDropIf DNA detected: batch contamination investigation

Step 5: PCR/qPCR Setup

5.1 Pre-PCR Preparation

  • All work performed in Class 1,000 pre-PCR room
  • Dedicated PCR pipettes (filter tips only)
  • UV-decontaminated workstation (30 min before use)
  • Reagents thawed on ice, vortexed 5 s, spun 10 s

5.2 Plate Layout

RowContentPurpose
A1–H1Positive controls (duplicate)Assay validation
A2–H2Negative control (triplicate)Contamination check
A3–H3No-template control (triplicate)Reagent purity
A4–H12Samples (single if sexing, duplicate if qPCR)Test subjects
H11–H12Extraction blankExtraction quality

5.3 Thermal Cycling

See specific protocol: - Sexing: CHD PCR Protocol - Disease: PBFD qPCR Validation or panel-specific protocol

Step 6: Post-PCR Analysis

6.1 Sexing — Gel Electrophoresis

TaskDetail
Gel documentationUV transilluminator + digital image capture
Band sizingCompare to 100 bp ladder using gel analysis software
Result entryMale/Female/Inconclusive entered into LIMS
Image storageGel image filed with LIMS record

6.2 Disease — qPCR Analysis

TaskDetail
Baseline correctionAuto baseline (3–15 cycles) with manual override if needed
Threshold setting10× SD of baseline fluorescence (or manual: 0.1 RFU)
Ct value extractionExport to LIMS via automated data interface
Amplification curve reviewManual review of all curves for artifacts

6.3 DNA Fingerprinting — Capillary Electrophoresis

TaskDetail
Fragment analysisGeneMapper or equivalent software
Allele callingAutomated with manual bin review
Profile exportTab-delimited file for LIMS import

Step 7: Result Interpretation

7.1 Automated Rules (LIMS)

RuleConditionAction
Sexing — two bandsCHD-W + CHD-Z detectedAuto-assign "Female"
Sexing — one bandCHD-Z only detectedAuto-assign "Male"
Sexing — no bandNo PCR productAuto-assign "Inconclusive — re-extract"
qPCR — Ct < 38Target detectedAuto-assign "Positive"
qPCR — Ct 38–40Target detected at low levelAuto-assign "Weak positive — retest recommended"
qPCR — no CtNo targetAuto-assign "Negative"

7.2 Manual Review (Technician)

  • All "Inconclusive" results reviewed by senior technician
  • All "Weak positive" results reviewed by QC officer
  • Discordant duplicates (sexing: male/female from same sample) → repeat with alternate primers
  • Fingerprinting profiles with < 10/12 loci called → re-run

Step 8: QC Review

8.1 Batch QC Review

QC ElementAcceptance Criteria
Positive controlCorrect result (expected species/sex)
Negative controlNo amplification
NTCNo amplification
Extraction blankNo amplification
IC (VIC channel)Ct 25–32 in all samples

8.2 Batch Release

All QC criteria must pass before any results from the batch are released.

Step 9: Reporting

9.1 Report Format

FieldExample
Client IDRoyal Pigeon Loft
Case IDSENO-20260401-1122
Sample IDSENO-20260401-1122-001
SpeciesRacing Homer
TestDNA Sexing
MethodCHD PCR (P2/P8)
ResultFemale (ZW)
Confidence> 99%
Date Tested2026-04-02
QC StatusPassed
Authorized By[SENO QC]

9.2 Delivery Methods

MethodFormatDelivery Time
EmailPDFWithin 1 hour of final QC
Client PortalWeb downloadReal-time
Hard copy (upon request)Printed + signed2–3 business days

Step 10: Sample Storage & Disposal

PhaseConditionDuration
Original sample (feather/swab)Room temperature, dry3 months post-report
Extracted DNA−20°C12 months post-extraction
PCR product (sexing)−20°C1 month (then discarded)
qPCR plate4°C (sealed)1 week (then discarded)
CE data filesLIMS storagePermanent

Estimated Turnaround Times

Test TypeHands-On TimeInstrument TimeTotal Lab Time
DNA Sexing (batch of 48)2.5 hours2.5 hours5 hours
Disease qPCR (96-well plate)3 hours2 hours5 hours
DNA Fingerprinting (48)4 hours3 hours7 hours
Performance Gene (48)3 hours3 hours6 hours

Lab turnaround: 1–2 working days for most tests.Total client turnaround (incl. shipping): 7–10 business days.