Single Nucleotide Polymorphism Genotyping Using BeadChip Microarrays

Gilliam Lambert1, Darwin Tsinajinnie1, David Duggan1

1 Genetic Basis of Human Disease Division, Translational Genomics Research Institute (TGen), Phoenix
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 2.9
DOI:  10.1002/0471142905.hg0209s78
Online Posting Date:  July, 2013
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The genotyping of single nucleotide polymorphisms (SNPs) has successfully contributed to the study of complex diseases more than any other technology to date. Genome‐wide association studies (GWAS) using 10,000s to >1,000,000 SNPs have identified 1000s of statistically significant SNPs pertaining to 17 different human disease and trait categories. Post‐GWAS fine‐mapping studies using 10,000s to 100,000s SNPs on a microarray have narrowed the region of interest for many of these GWAS findings; in addition, independent signals within the original GWAS region have been identified. Focused content, SNP‐based microarrays such as the human exome, for example, have too been used successfully to identify novel disease associations. Success has come to studies where 100s to 10,000s (mostly) to >100,000 samples were genotyped. For the time being, SNP‐based microarrays remain cost‐effective especially when studying large numbers of samples compared to other “genotyping” technologies including next generation sequencing. In this unit, protocols for manual (LIMS‐free), semi‐manual, and automated processing of BeadChip microarrays are presented. Lower throughput studies will find value in the manual and semi‐manual protocols, while all types of studies‐‐‐low‐, medium‐, and high‐throughput‐‐‐will find value in the semi‐manual and automated protocols. Curr. Protoc. Hum. Genet. 78:2.9.1‐2.9.34. © 2013 by John Wiley & Sons, Inc.

Keywords: single nucleotide polymorphism (SNP); genotyping; high‐throughput; Infinium assay; genome‐wide association study (GWAS); fine‐mapping; focused microarrays

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Infinium HD Ultra Manual Assay: DNA Amplification, Fragmentation, Precipitation, and Resuspension
  • Basic Protocol 2: Infinium HD Ultra Manual Assay: Single Base Extension, Fluorescent Staining, and Coating
  • Basic Protocol 3: Scanning
  • Alternate Protocol 1: Infinium HD Ultra Automated Assay: DNA Amplification, Fragmentation, Precipitation, and Resuspension
  • Alternate Protocol 2: Infinium HD Ultra Automated Assay: Single Base Extension, Fluorescent Staining, and Coating
  • Basic Protocol 4: Laboratory Data Analysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Infinium HD Ultra Manual Assay: DNA Amplification, Fragmentation, Precipitation, and Resuspension

  • 96‐sample whole‐genomic DNA plate (all samples normalized to 50 ng/µl)
  • Infinium kit (Illumina) containing:
    • Multi‐sample amplification mix 1 (MA1)
    • Multi‐sample amplification mix 2 (MA2)
    • Multi‐sample amplification master mix (MSM)
    • Fragmentation solution (FMS)
    • Precipitation solution (PM1)
    • Resuspension, hybridization, and wash solution (RA1)
    • Infinium HD BeadChips
    • Infinium hybridization chambers
    • Infinium hybridization chamber gaskets
    • Infinium hybridization chamber inserts
    • Multi‐sample BeadChip alignment fixture
    • Te‐Flow Flow‐Through chambers (with black frames, spacers, glass back plates, and clamps)
    • Illumina wash dishes
    • Illumina wash rack
  • 0.1 N sodium hydroxide (NaOH)
  • 100% 2‐propanol
  • Illumina Laboratory Information Management System (LIMS) or equivalent, optional
  • Illumina hybridization oven
  • 96‐well, 0.8‐ml microtiter plate (MIDI)
  • 12‐channel pipettor
  • 96‐well MIDI plate cap mats (Thermo Scientific)
  • High‐speed microplate shaker (Illumina)
  • ABI MicroAmp adhesive film seals
  • Swinging‐bucket refrigerated centrifuge with microtiter plate carriers
  • Hybex microsample incubator
  • Absorbent pads
  • Heat sealer and foil heat seals
  • Speedball brayer or film roller

Basic Protocol 2: Infinium HD Ultra Manual Assay: Single Base Extension, Fluorescent Staining, and Coating

  • Reaction plate(s) (see protocol 1)
  • Infinium Kit containing:
    • Resuspension, hybridization, and wash solution (RA1)
    • Humidifying buffer (PB2)
    • Hybridization preparation buffer (PB1)
    • XStain BeadChip solution 1 (XC1)
    • XStain BeadChip solution 2 (XC2)
    • XStain BeadChip solution 3 (XC3)
    • XStain BeadChip solution 4 (XC4)
    • Two‐color extension master mix (TEM)
    • Anti‐stain two‐color extension master mix (ATM)
    • Superior two‐color master mix (STM)
    • BeadChips
    • Hybridization chambers
    • Hybridization chamber gaskets
    • Hybridization chamber inserts
  • 100% ethanol
  • 95% formamide with 1 mM EDTA (see recipe)
  • Prosat ethanol wipes
  • Illumina hybridization oven
  • Hybex microsample incubator
  • Swinging‐bucket centrifuge with 15‐ml conical inserts
  • Te‐Flow flow‐through chambers (with black frames, spacers, glass back plates, and clamps)
  • Water bath with flow‐through rack
  • Wash dish(es)
  • Multi‐sample BeadChip alignment fixture
  • Wash rack
  • Absorbent pads
  • Small cleaning brush
  • Staining rack
  • Dismantling tool: flat spatula
  • Tube racks
  • Locking tweezers
  • Vacuum desiccator
  • HiScan or iScan system
  • Illumina BeadChip slide storage box, optional

Basic Protocol 3: Scanning

  • Illumina HiScan or iScan system
  • BeadChip carrier(s)
  • Slide storage box(es)

Alternate Protocol 1: Infinium HD Ultra Automated Assay: DNA Amplification, Fragmentation, Precipitation, and Resuspension

  • Illumina LIMS software (optional)
  • Tecan tip alignment guides
  • Tecan BeadChip alignment fixtures
  • Illumina HiScan Autoloader (optional)

Alternate Protocol 2: Infinium HD Ultra Automated Assay: Single Base Extension, Fluorescent Staining, and Coating

  • 1% Alconox solution in water
  • Tecan tip alignment guides
  • Kimwipes
  • Two Tecan liquid handling robots (one located in Pre‐PCR room and one located in post‐PCR/main laboratory/bleach‐safe room)

Basic Protocol 4: Laboratory Data Analysis

  • GenomeStudio software (Illumina)
  • Network/directory where sample sheet is located
  • Network/directory where data repository is located
  • Network/directory where manifest repository is located
  • Network/directory where cluster file is located
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Literature Cited

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