Sep 18, 2025
ODGC Genetic Variant Protocol
This protocol is a draft, published without a DOI.
- Cayla Mason1,
- Kathleen Fisch1,
- Kristen Jepsen1
- 1University of California San Diego
Protocol Citation: Cayla Mason, Kathleen Fisch, Kristen Jepsen 2025. ODGC Genetic Variant Protocol. protocols.io https://dx.doi.org/
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: September 09, 2025
Last Modified: September 18, 2025
Protocol Integer ID: 226782
Keywords: genotyping, genetic variants, DNA extraction, predictive biomarkers of chronic pain, chronic pain signature, additional pharmacogenomic, chronic pain, predictive biomarker, clinical research variant, automated genotype screening, genotype screening, genetic variant, genotypes of subject, genotype, custom cluster file generation, including automated dna extraction, automated dna extraction, illumina psych booster, pain
Funders Acknowledgements:
Omics Data Generation Center for the Acute to Chronic Pain Signatures Program
Grant ID: U54DA049115
Abstract
The Acute to Chronic Pain Signatures (A2CPS) Consortium seeks to identify predictive biomarkers of chronic pain using a wide variety of modalities. In this protocol, we describe the methods used to procure genotypes of subjects including automated DNA extraction, automated genotype screening, and, finally, analysis in conjunction with custom cluster file generation.
Genotypes were assessed using GSA Beadchips (Illumina #20068345) plus custom content. The final array consists of > 650,000 multi-ethnic genome-wide content, curated clinical research variants, and quality control (QC) markers plus ~30,000 markers associated with common psychiatric disorders (Illumina Psych Booster), ~500 additional pharmacogenomics (PGx) markers, and 5,000 custom markers selected by the A2CPS Consortium to be of particular interest in the study of chronic pain.
Materials
PaxGene Blood DNA Tube, 2.5 mL (PreAnalytiX #761165)
QIAsymphony DSP DNA Midi Kit (Qiagen #937255)
Qubit dsDNA HS Assay Kit (Invitrogen #Q32854)
Nuclease-free water
GSA Beadchips (Illumina #20068345) with custom content
Infinium HTS Automated Workflow (Illumina)
Safety warnings
For all agents listed below, wear protective gloves, protective clothing, eye protection, and face protection, and dispose of contents/container to an approved waste disposal plant.
Human blood should be handled using universal precautions. Handle within a biosafety cabinet until lysis.
Ethics statement
This protocol was developed and used under the approval of the University of California San Diego Institutional Review Board. Users of this protocol for the purpose of obtaining genomic DNA from human blood should obtain prior approval from their Institutional Review Board (IRB) or equivalent ethics committee(s).
Blood Draw
Draw blood in 2.5 mL PaxGene Blood DNA tube. Store at -80°C until ready to extract.
DNA Extraction
3h 30m
Thaw blood samples at room temperature on bench top.
30m
Prepare QIAsymphony DSP DNA Midi Kit and perform extraction on QIAsymphony instrument as per manufacturer protocol (
EN-QIAsymphony-DNA-Handbook-for-software-version-31-or-lower.pdf651KB , p. 24-26). We use the following parameters:
EN-QIAsymphony-DNA-Handbook-for-software-version-31-or-lower.pdf651KB , p. 24-26). We use the following parameters: - Blood_400_V6_DSP protocolDNABlood400V6DSP-EN.pdf99.9KB
- 400 µL sample volume
- 100 µL elution volume
3h
Illumina Infinium Global Screening Array (GSA)
3d 0h 30m
Dilute DNA 1:10 with nuclease-free water. Quantify DNA with the Qubit dsDNA HS Assay Kit.
Qubit_dsDNA_HS_Assay_UG.pdf265.1KB 15m
Dilute DNA samples to a final DNA concentration of 200 ng per 6 uL with nuclease-free water.
15m
Perform Infinium HTS Automated Workflow following manufacturer instructions beginning at chapter 3, step 2: amplification.
infinium-hts-assay-reference-guide-15045738-04.pdf3.2MB 3d
Create Custom Cluster File
Custom cluster files must be created to process genotype data from Illumina Infinium arrays with custom content. Our custom cluster file was generated following the instructions in the attached technical note using data from Runlists 1-3 in Illumina GenomeStudio v2-0-5-0.
custom-cluster-file-cnv-tech-note-770-2017-017.pdf353.4KB Genotype Analysis
Genetic variant data from Runlists 1-3 were processed in Illumina GenomeStudio v2-0-5-0 (attached). Runlists 4+ were processed with Illumina's DRAGEN Array command line interface v1.0.0 using the custom cluster file previously generated (GitHub).
genomestudio-genotyping-module-v2-user-guide-11319113-01.pdf2.9MB