Targeted Sequencing Using Affymetrix CustomSeq Arrays

互联网2013-12-31

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

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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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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Materials

Basic Protocol 1: Critical Aspects of Design, Execution, and Analysis of Affymetrix GeneChip CustomSeq Arrays

Materials

AmpliTaq Gold and buffers (Applied Biosystems cat. no. 4311858; store at −20°C)
2.5 mM MgCl₂
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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Figures

Figure 7.18.1 Customized pipeline for data analysis. Abbreviations: BRLMM, genotyping software; CEL, cell intensity; DAT, data; GCOS, GeneChip operating software; GSEQ, GeneChip sequence analysis software; SNP, single‐nucleotide polymorphism.

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Figure 7.18.2 DAT file shows an image of the scanned probe array.

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Figure 7.18.3 Minor allele tiling probes include the variant and ±12 base pairs. A total of 49 base pairs is submitted to Affymetrix to create probes to sequence the affected region.

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Figure 7.18.4 Offset tiling uses additional probes with interrogating positions offset to the 5′ or 3′ end of the probe to increase sensitivity and specificity.

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Figure 7.18.5 Probe design strategy for genotyping. Resequencing probes are placed with a minor/mutant allele at different offsets (9 offsets) to interrogate one genotype (A) and sequence 9 contiguous bases at the same time (GTCCTACGG).

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Figure 7.18.6 Sample of a summary report generated from our data analysis pipeline. All positions requiring a confirmatory sequencing method are listed in the Follow Up Positions section. For each position, the gene, exon, and wild‐type base are indicated. In the Call column, triplicate calls are shown, as well as minor allele tiling calls due to the presence of a nearby single‐nucleotide polymorphism (SNP). When tiled, genotyping calls are noted next to the relevant base. The results of confirmatory Sanger sequencing are noted in the final column (Seq Confirmation). Non‐wild‐type base calls that are not followed up are listed in the subsequent sections including benign SNPs, as well as calls filtered out due to identification during test validation as a site of a common false positive or no‐call.

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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
	http://www.affymetrix.com/support/technical/byproduct.affx?product=cseq
	CustomSeq array resources provided by Affymetrix.
	http://www.affymetrix.com/support/technical/datasheets/customseq_datasheet.pdf
	GeneChip CustomSeq Resequencing Array Program Data Sheet provided by Affymetrix.
	https://www.affymetrix.com/support/downloads/manuals/gseq_user_guide.pdf
	GeneChip Sequence Analysis Software (GSEQ) User's Guide provided by Affymetrix.
	http://www.affymetrix.com/support/technical/technotes/customseq_arraybase_technote.pdf
	Technical note on CustomSeq Resequencing Array Base Calling Algorithm Version 2.0 provided by Affymetrix.
	http://www.affymetrix.com/support/technical/other/customseq_design_manual.pdf
	CustomSeq Resequencing Array Design Guide provided by Affymetrix.