Illumina SNP Genotyping or CNV/LOH Analysis

Lab Introduction and Consultation

Illumina's SNP Genotyping and CNV/LOH product line includes whole-genome content, focused content, and custom SNP content. Whole-genome analysis is performed using single-sample or multi-sample Sentrix BeadChips which utilize the Infinium Assay. Focused-content and custom analysis are performed using the 96-sample Sentrix Array Matrix which is supported by the GoldenGate Assay.

The DNA Array laboratory has an Illumina BeadStation System, which includes two BeadArray readers and supporting automation (chip autoloader and a Tecan liquid-handling robot).

The following are instructions on how Fred Hutchinson and other Cancer Consortium members can access Illumina SNP Genotyping and CNV/LOH Analysis services at FHCRC.

Those new to this technology or new to using the Genomics Resource are strongly encouraged to meet with lab personnel prior to planning a project. For non-consortium members interested in services please contact the Genomics Resource at (206) 667-2714.

• For questions regarding experimental design issues, please contact Jeff Delrow at jdelrow@fhcrc.org or (206) 667-2763.



• For questions regarding service fees, scheduling, and protocols please contact Cassie Sather at csather@fhcrc.org or (206) 667-2757.

Reagent and Consumable Purchases

Each investigator is required to purchase Illumina SNP Genotyping and CNV/LOH arrays and reagents directly through Illumina. The Genomics Resource does not stock Illumina products. Please contact Illumina for pricing.

Illumina Sales Contact:

Robin L. Ball
Regional Account Manager
Phone: 206-660-7749
Email: rball@illumina.com

Service Option 1: Genomic DNA for Genome-wide SNP Genotyping or CNV/LOH (Infinium Assay)

Our sample submission requirements for genome-wide SNP Genotyping or CNV/LOH Analysis employing the Infinium protocol are 2ug of genomic DNA at a concentration of 100ng/ul. While the lab will perform QC analysis of all samples submitted for microarray analysis, it is advisable that the investigator perform a preliminary assessment of sample quality before sending samples to the lab. Additionally, it is advised that investigators submit samples for QC prior to authorizing shipment of Illumina reagents. This is due to the limited shelf life of some Illumina reagents. For additional questions regarding sample QC, please contact the Genomics Resource.

Samples must be submitted in the following plate type and the plate should be filled left to right.

PLATES FROM FISHER (NO SUBSTITUTIONS):
(Nunc) 0.45mL Well 96-Well Conical Bottom Polypropylene Plate
Color: Natural
No Lid
Sterile
Fisher Order #: 249946
Cost: $252.64/120plates

TAPE FOR SEALING ABOVE PLATES FROM QIAGEN:
(Qiagen) Adhesive tape sheets for sealing multiwell plates and blocks:
25 sheets per pad
5 pads per pack
Qiagen Order #: 19570
Cost: $40/125 sheets

Please label your DNA plates on the right hand side using either a sticker label or an industrial sharpie. Do not write on the top of the plates.

All samples, Illumina BeadChips, Illumina BeadChip processing reagents, and a fully completed request form should be delivered to the Genomics Resource, located in the Thomas Building, DE-740.

Infinium request forms
If you are using a FHCRC project id for billing, please fill out the Internal Infinium Request Form.

If billing is to be submitted to the University of Washington or other external institution, fill out the External Infinium Request Form.

In addition to a request form, the investigator must complete and submit an Infinium Sample Sheet. The required fields of the sample sheet are highlighted in yellow. Optional fields are highlighted in blue. All other fields will be completed by the Genomics Resource.

Service Option 1: Infinium Sample QC and Processing

Quality control of genomic DNA includes Picogreen quantification. Samples must meet a minimum concentration threshold of 50ng/ul and must not exceed 250ng/ul.

In instances where one or more samples do not pass our QC test, we will not proceed with the project and will immediately contact the submitter with our findings.

The Genomics Resource staff process genomic DNA samples by strictly following the InfiniumII LabSetup & Procedures. Genomic DNA samples undergo a whole-genome amplification (WGA) followed by hybridization to locus-specific 50mers on the BeadChip. Then, the samples undergo a Single-base extension and staining procedure. Single-base extension of the oligos on the BeadChip, using the captured DNA as a template, incorporates detectable labels on the BeadChip and determines the genotype call for the sample. Finally, the BeadChips are imaged using the Illumina BeadArray Reader.

Service Option 2: Genomic DNA for Focused Content SNP Genotyping or CNV/LOH (GoldenGate Assay)

Our sample submission requirements for focused-content SNP Genotyping or CNV/LOH Analysis employing the GoldenGate protocol using a single OPA are 1ug of genomic DNA at a concentration of 100ng/ul. If more than one OPA will be used, please contact the Genomics Resource for adjusted sample requirements.

While the lab will perform QC analysis of all samples submitted for microarray analysis, it is advisable that the investigator perform a preliminary assessment of sample quality before sending samples to the lab. Additionally, it is advised that investigators submit samples for QC prior to authorizing shipment of Illumina reagents. This is due to the limited shelf life of some Illumina reagents. For additional questions regarding sample QC, please contact the Genomics Resource.

Samples must be submitted in the following plate type and the plate should be filled left to right. Additionally, please leave wells A01, A02, B01, and B02 empty. These wells should be completely empty/dry...no buffer or water. These wells will be used for controls which will be added by Genomics Resource staff.

PLATES FROM FISHER (NO SUBSTITUTIONS):
(Nunc) 0.45mL Well 96-Well Conical Bottom Polypropylene Plate
Color: Natural
No Lid
Sterile
Fisher Order #: 249946
Cost: $252.64/120plates

TAPE FOR SEALING ABOVE PLATES FROM QIAGEN:
(Qiagen) Adhesive tape sheets for sealing multiwell plates and blocks:
25 sheets per pad
5 pads per pack
Qiagen Order #: 19570
Cost: $40/125 sheets

Please label your DNA plates on the right hand side using either a sticker label or an industrial sharpie. Do not write on the top of the plates.

All samples, Illumina Sentrix Array Matrices (SAM), Illumina SAM processing reagents, and a fully completed request form should be delivered to the Genomics Resource, located in the Thomas Building, DE-740.

GoldenGate request forms:
If you are using a FHCRC project id for billing, please fill out the Internal GoldenGate Request Form.

If billing is to be submitted to the University of Washington or other external institution, fill out the External GoldenGate Request Form .

In addition to a request form, the investigator must complete and submit a GoldenGate Sample Sheet. The required fields of the sample sheet are highlighted in yellow. Optional fields are highlighted in blue. All other fields will be completed by the Genomics Resource.

Service Option 2: GoldenGate Sample QC and Processing

Quality control of genomic DNA includes Picogreen quantification. Samples must meet a minimum concentration threshold of 50ng/ul and must not exceed 250ng/ul.

In instances where one or more samples do not pass our QC test, we will not proceed with the project and will immediately contact the submitter with our findings.

The Genomics Resource staff process genomic DNA samples by strictly following the Illumina BeadStation System Manual. Genomic DNA samples are first biotinylated and purified from solution using streptavidin conjugated paramagnetic particles. Assay oligos (OPA) are hybridized to the DNA and allele-specific extension and ligation of the hybridized oligos is subsequently performed. The extended and ligated products form a template that is transferred to a PCR mixture. Then the strand containing the fluorescent signal in the PCR products is isolated and hybridized to the Sentrix Array Matrix (SAM). After hybridization, the SAM is washed and imaged on the Illumina BeadArray Reader.

Results

Data generated within the Resource are transferred through automated pipelines to systems supported by the Hutchinson Center's Research Computing Support Shared Resource. Data is transferred to the user's 'fred' account, in the researcher's dnaarray folder.

If you do not currently have an account on the FRED server please fill out the Data Storage- FRED User Account Application prior to completion of your microarray experiment.

Each image is stored as in image data file (.idat extension) in a foldered labeled with the BeadChip or SAM barcode number. Each BeadChip or SAM is accompanied with a disk which contains the Decode Data (.dmap extension) also in a folder labeled with the BeadChip or SAM barcode number. The Bead Set manifest is also included on this disk (.csv extension). For custom OPAs the OPA manifest is included on a separate disk sent with the OPA.

Data Analysis

Researchers may transfer their BeadChip files from the FRED server into the Illumina BeadStudio Application where data can be extracted from the images collected from the BeadArray Reader. Please contact Ryan Basom to gain access to the BeadStudio software.

Additional support is available to those requiring assistance with analyzing and interpreting their data. Please contact one of the following for guidance:

Jeff Delrow, Ph.D.
Director and Staff Scientist, Genomics Resource
voice: 206.667.2763
email: jdelrow@fhcrc.org

Martin Morgan, Ph.D.
Director and Staff Scientist, Computational Biology Shared Resource
voice: 206.667.2793
email: mtmorgan@fhcrc.org