Targeted Sequencing Using Affymetrix CustomSeq Arrays

Polakit Teekakirikul1, Stephanie Cox1, Birgit Funke2, Heidi L. Rehm2

1 Partners HealthCare Center for Personalized Genetic Medicine, Cambridge, Massachusetts, 2 Massachusetts General Hospital, Boston, Massachusetts
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 7.18
DOI:  10.1002/0471142905.hg0718s69
Online Posting Date:  April, 2011
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This unit provides a basic protocol for oligo hybridization‐based sequencing technology and resulting data analysis specific to the Affymetrix GeneChip CustomSeq Resequencing Array platform. All steps and critical aspects related to array design, experimental protocols, data management, and base‐calling algorithms are addressed. This unit is particularly appropriate for sequencing targeted regions of the genome of up to 300 kilobases. The basic technology is most suitable for detecting substitution mutations, unless targeted indel probes are added. Curr. Protoc. Hum. Genet. 69:7.18.1‐7.18.17 © 2011 by John Wiley & Sons, Inc.

Keywords: resequencing microarrays; CustomSeq; base‐calling; sequence‐specific; hybridization; mutation detection

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Table of Contents

  • Introduction
  • Basic Protocol 1: Critical Aspects of Design, Execution, and Analysis of Affymetrix GeneChip CustomSeq Arrays
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Critical Aspects of Design, Execution, and Analysis of Affymetrix GeneChip CustomSeq Arrays

  • AmpliTaq Gold and buffers (Applied Biosystems cat. no. 4311858; store at −20°C)
  • 2.5 mM MgCl 2
  • 5 M Betaine (Sigma cat. no. B0300)
  • Dimethyl sulfoxide (DMSO; Sigma cat. no. D9170)
  • 10 mM dNTP mix: dilute 100 mM PCR‐grade dNTP stock solutions (Roche cat. no. 1969064; store at −20°C), and combine equal volumes of the four 10 mM dNTPs
  • 10 µM synthesized oligonucleotide primer mix: dilute 100 µM stocks solutions (Integrated DNA Technologies; store at –20°C), and combine, e.g., two to three primer pairs per mix
  • 25 ng/µl high‐quality genomic DNA (∼10 µg), e.g., prepared from human peripheral blood samples (unit 14.4)
  • Human genomic DNA (positive control; Clontech cat. no. 6550‐1; store at 4°C)
  • Molecular biology–grade water (HyClone)
  • Low‐range quantitative DNA ladder (Invitrogen cat. no. 12373‐031)
  • QIAquick PCR purification kit (Qiagen cat. no. 28104)
  • GeneChip Resequencing Assay kit (Affymetrix cat. no. 900447; store at −20°C) includes:
    • Fragmentation reagent (DNase I) and buffer
    • DNA labeling reagent
    • Terminal deoxynucleotidyl transferase (TdT) and buffer
    • 130× oligo control reagent
  • 2 and 4% (w/v) agarose gels: general purpose 2% (e.g., E‐gel 96, Invitrogen cat. no. G7008‐02) and high‐resolution 4% (e.g., Invitrogen cat. no. G6200‐04)
  • 5 M tetramethylammonium chloride solution (TMAC; Sigma cat. no. T3411)
  • 1 M Tris⋅Cl, pH 7.8 (Sigma cat. no. T2913; appendix 2D)
  • 1% (v/v) Tween‐20 (Sigma cat. no. P1379)
  • 50 mg/ml acetylated bovine serum albumin (BSA) solution (Invitrogen cat. no. 15561‐020; store at −20°C)
  • 10 mg/ml herring sperm (Promega cat. no. D1811; store at −20°C)
  • GeneChip Hybridization Oven 640 (Affymetrix)
  • GeneChip Fluidics Station 450 (Affymetrix)
  • GeneChip Scanner 3000 7G (Affymetrix)
  • GeneChip operating software (GCOS) version 1.4 (Affymetrix)
  • GeneChip sequence analysis software (GSEQ) version 4.0 (Affymetrix)
  • Library files for custom arrays (Affymetrix)
  • 1.5‐ml microcentrifuge tubes
  • 96‐well semi‐skirted plates (Fisher Scientific cat. no. 08‐408‐250)
  • 384‐well skirted plates (ABGene cat. no. AB‐1111)
  • Adhesive PCR film (ABgene)
  • Benchtop centrifuge with plate adaptors (e.g., Eppendorf 5810R)
  • GeneAmp PCR system 9700 (Applied Biosystems)
  • Gel electrophoresis equipment, e.g., d E‐gel PowerBase, Version 4 (Invitrogen cat. no. G6200‐04) or Agilent 2100 Bioanalyzer
  • Biomek FX Robot (Beckman Coulter), recommended
  • 2‐ml screw cap tubes
  • Rotary vacuum evaporator (SpeedVac DNA 110, Savant)
  • 96‐well semi‐skirted plates (Fisher Scientific cat. no. 08‐408‐250)
  • 37°C, 49°C and 95°C digital dry block heater (e.g., VWR cat. no. 12621084) with thermometer (0°C to 110°C)
  • Conical centrifuge tubes of appropriate sizes (e.g., 15‐ or 50‐ml Falcon tubes, Fisher Scientific)
  • Additional reagents and equipment for carrying out agarose gel electrophoresis (unit 2.7), determining DNA concentration ( appendix 3D), and performing GeneChip hybridization, washing, and staining steps according to Affymetrix protocols (see Internet Resources)
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Literature Cited

   Cutler, D.J., Zwick, M.E., Carrasquillo, M.M., Yohn, C.T., Tobin, K.P., Kashuk, C., Mathews, D.J., Shah, N.A., Eichler, E.E., Warrington, J.A., and Chakravarti, A. 2001. High‐throughput variation detection and genotyping using microarrays. Genome Res. 11:1913‐1925.
   Zimmerman, R.S., Cox, S., Lakdawala, N.K., Cirino, A., Mancini‐DiNardo, D., Clark, E., Leon, A., Duffy, E., White, E., Baxter, S., Alaamery, M., Farwell, L., Weiss, S., Seidman, C.E., Seidman, J.G., Ho, C.Y., Rehm, H.L., and Funke, B.H. 2010. A novel custom resequencing array for dilated cardiomyopathy. Genet. Med. 12:268‐278.
Internet Resources
  CustomSeq array resources provided by Affymetrix.
  GeneChip CustomSeq Resequencing Array Program Data Sheet provided by Affymetrix.
  GeneChip Sequence Analysis Software (GSEQ) User's Guide provided by Affymetrix.
  Technical note on CustomSeq Resequencing Array Base Calling Algorithm Version 2.0 provided by Affymetrix.
  CustomSeq Resequencing Array Design Guide provided by Affymetrix.
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