Illumina Methylation Profiling

Lab Introduction and Consultation

Illumina's Methylation Profiling product line includes whole-genome content, focused content, and custom content. Whole-genome profiling is performed using multi-sample Sentrix BeadChips which utilize the Infinium Assay. Focused-content and custom-content profiling are performed using the 96-sample Sentrix Array Matrix which is supported by the GoldenGate Assay.

The DNA Array laboratory is equiped with 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 Methylation Profiling 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 Methylation Profiling 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 Focused Content Methylation Profiling (GoldenGate Assay)

Our sample submission requirements for focused-content Methylation Profiling employing the GoldenGate protocol using a single OMA are 8ul of 500ng input gDNA from the bisufite conversion reaction. The Genomics Resource does not offer bisulfite conversion services. DNA must be converted PRIOR to submission to the Genomics Resource. Submission requirements are based on gDNA that has already been bisulfite converted. If more than one OMA will be used, please contact the Genomics Resource for adjusted sample requirements.

Unlike the GoldenGate genotyping assay, the lab does not perform any QC of the samples after submission. Therefore it is critical that investigators follow the proper upfront (pre-conversion) QC (see manual)and that they use the Illumina-recommended conversion kit (Zymo EZ DNA Methylation kit).

Converted DNA samples are stable at -20°C for only 1 month so it is critical that investigators coordinate a schedule with the Genomics Resource prior to bisulfite converting their genomic DNA.

Bisulfite-converted gDNA 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 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.

Infinium 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 1: GoldenGate Sample Processing

The Genomics Resource staff process genomic DNA samples by strictly following the Illumina protocol. Bisulfite-converted genomic DNA samples are first biotinylated and purified from solution using streptavidin conjugated paramagnetic particles. Assay oligos (OMA) are hybridized to the DNA and allele-specific extension and ligation of the hybridized oligos is subsequently performed. For each CpG site, there are four probes: two allele-specific oligos (ASO) and two locus-specific oligos (LSO). Each ASO-LSO oligo pair corresponds to either the methylated or unmethylated state of the CpG site. 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 BeadArrayer Reader. Each methylation data point is represented by the ratio of fluorescent signals from the methylated and unmethylated alleles.

Service Option 2: Genomic DNA for Genome-wide Methylation Profiling (Infinium Assay)

Our sample submission requirements for genome-wide Methylation Profiling employing the Infinium protocol are 8ul of 500ng input gDNA from the bisufite conversion reaction. The Genomics Resource does not offer bisulfite conversion services. DNA must be converted PRIOR to submission to the Genomics Resource. Submission requirements are based on gDNA that has already been bisulfite converted. Unlike the Infinium genotyping assay, the lab does not perform any QC of the samples after submission. Therefore it is critical that investigators follow the proper upfront (pre-conversion) QC (see manual) and that they use the Illumina-recommended conversion kit (Zymo EZ DNA Methylation kit).

Converted DNA samples are stable at -20°C for only 1 month so it is critical that investigators coordinate a schedule with the Genomics Resource prior to bisulfite converting their genomic DNA.

Bisulfite-converted gDNA 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 2: Infinium Sample Processing

The Genomics Resource staff processes bisulfite-converted genomic DNA samples by strictly following the Illumina protocol. Bisulfite-converted 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 methylation profile of each CpG site. Specifically, two bead types correspond to each CpG locus: one bead type corresponds to methylated, another bead type to unmethylated state of the CpG site. Finally, the BeadChips are imaged using the Illumina BeadArray Reader.

Results\Downstream Analysis

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 OMAs the OMA manifest is included on a separate disk sent with the OMA.

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