Data Sheet: DNA Analysis

Infinium® FFPE DNA Restoration Solution
A complete workflow for genotyping, CNV, and methylation analysis, from sample prep
through data analysis.
Highlights• Integrated Quality Control Kit
Provides primers for a real-time PCR assay to identify
recoverable samples

Figure 1: FFPE QC and Restoration Workflow

Purify DNA from FFPE samples• Novel Restoration Kit
Includes reagents and a simple protocol to restore
degraded FFPE samples• FFPE Sample-Enabled BeadChips
Choose from several arrays providing custom content,
whole-genome genotyping, or methylation sites• High-Quality Data
Robust data for reliable genotyping, copy number variation,and methylation analysis

Evaluate DNA quality withintegrated QC test

Perform FFPE
restoration protocol

Introduction
Millions of formalin-fixed paraffin-embedded (FFPE) archival cancer
tissue samples provide an enormous and invaluable repository of
information for cancer research. The samples hold a wealth of data forthe discovery of biomarkers, drug development, and diagnosis andtreatment of diseases. However, the fixation process and storage of
FFPE samples frequently leads to nucleic acid degradation and basemodification. Because it is inefficiently amplified in the whole-genome
amplification step, degraded DNA from FFPE samples generally
results in poor performance for array-based studies. Such samples
have been traditionally unusable with the Infinium HD Assay. To
overcome this problem, the Infinium FFPE DNA Restore Kit provides
a simple workflow to repair damaged DNA and achieve high-quality
genotyping, copy number variation (CNV), and methylation data using
Infinium HD Assays.

Simple Restoration Workflow
The FFPE DNA restoration workflow allows researchers to easily evaluate sample quality and repair DNA from FFPE samples in preparation
for the Infinium HD Assay (Figure 1). After DNA extraction using one ofmany commercially available kits, the Illumina FFPE QC Kit is used toevaluate the quality of prospective DNA samples and determine if theyare usable. The kit provides primers for a real-time PCR assay thatcan be carried out using standard instrumentation available in most
biological laboratories.
Samples that pass the quality control evaluation are moved on to theDNA restoration step. Using two straightforward enzymatic reactions,the Infinium HD DNA Restore Kit restores degraded FFPE DNA inpreparation for the re-optimized whole-genome amplification stepin the Infinium HD Assay protocol for FFPE samples*. In general,over 80% of FFPE samples that passed the QC assay metric are
successfully restored (Figure 2).

Infinium HD Assay

Analyze data

A streamlined workflow to QC FFPE samples and restore viable DNA
for whole-genome amplification using a modified protocol. Restored
samples can be used in the proven Infinium HD Assay. The assay results
can be easily analyzed using the highly intuitive, powerful DNA analysis
tools in GenomeStudio® and KaryoStudio software.

Proven Infinium Technology
Illumina offers several microarray options for gaining valuable genetic
insight from FFPE samples:• The HumanOmniExpress-FFPE (693,000 markers) andCytoSNP-FFPE (262,000 markers) are 12-sample genotyping
BeadChips that are ideally suited for whole-genome CNV analysis
and provide coverage of regions associated with cancer.
• The HumanMethylation450 BeadChip allows researchers
to interrogate > 485,000 methylation sites per sample atsingle-nucleotide resolution. The content covers 99% ofRefSeq genes and 96% of CpG islands.
• Infinium iSelect® BeadChips allow researchers to create a completely
custom BeadChip, by selecting from 3,072 to 1,000,000 markers,
including content that is highly relevant to cancer studies inFFPE samples such as known somatic mutations.

Data Sheet: DNA Analysis
Figure 2: QC of FFPE Samples

Figure 3: Infinium HD Twelve-Sample BeadChip

4305493023

Researchers with FFPE samples can choose from several Infinium
BeadChips to perform applications ranging from whole-genome
genotyping and methylation studies, to fully custom genotyping.

DNA from 130 FFPE samples of varying quality and control DNA (2 ngeach) were analyzed in triplicate by real-time PCR. The average Cq value
for the control DNA was subtracted from Ct values for FFPE DNA. The
resulting Delta Ct values were plotted against the Infinium HD Assay
call rates for the same samples after restoration. Greater than 80% ofsamples with a QC value below 5 were successfully genotyped (orangebox). Depending on sample origin and age (1–35 years), 35% to 95% ofsamples passed QC.

Each of these BeadChips runs on the proven Infinium HD Assay,
consistently producing high call rates (> 95%), very high reproducibility,and extremely low error rates†. This assay technology has enabledmany revolutionary discoveries in disease research, resulting in a vast
publication record.

Structural Variation Analysis
Structural variation is thought to be a significant contributor to thegenetic basis of many human diseases. Dense marker spacing
on the whole-genome BeadChips coupled with the sensitive

Infinium HD Assay offers a powerful tool for structural variation analysis.
Researchers can profile virtually any chromosomal aberration such as
amplifications, deletions, rearrangements, point mutations, copy number
changes, and copy-neutral loss of heterozygosity (LOH) events. The
assay delivers very high signal-to-noise ratios and low overall noiselevels, which are ideal for precise structural variation analysis.

Exceptional Data Quality
To evaluate the data quality produced from degraded DNA, a seriesof FFPE samples were treated with the Infinium HD DNA Restore
Kit and evaluated on the HumanCytoSNP-FFPE-12 BeadChip. The
assay signal intensities were referenced against canonical genotype
cluster files. The clusters were generated from artificially degraded
Coriell DNA samples that were processed with the Infinium HD
FFPE DNA Restore Kit and evaluated using the Infinium HD Assay.

Table 1: FFPE-Enabled BeadChip Specifications
HumanOmniExpress FFPE
BeadChip

HumanCytoSNP-FFPE
BeadChip

HumanMethylation450
BeadChipInfinium iSelectBeadChips> 693,000> 262,000

450,000

3,072–1,000,000

Number of Samples/BeadChip

1212124–24

DNA Requirement (ng)100100250100Assay

Infinium HD

Infinium HD

Infinium HD

Infinium HD

Instrument SupportHiScanSQ™
or iScan

HiScanSQ, iScan,
BeadArray™ Reader

HiScanSQ, iScan

HiScanSQ, iScan,
BeadArray Reader

Whole-Genome Genotyping
and CNV Analysis

Whole-Genome Genotyping
and CNV Analysis

Epigenetic Studies

Custom Genotyping

Number of Markers

Applications

Data Sheet: DNA Analysis
Kits and Accessories

Figure 4: High Call Rates and Low Standard Deviation
on Restored Samples Compared to Originals

Call RateA

ProductCatalog No.

1.2

Illumina FFPE QC Kit

WG-321-1001

1.0

Infinium HD FFPE DNA Restore Kit(24 samples)

WG-321-1002

0.8

HumanCytoSNP-FFPE (24 samples)

WG-321-1003

0.6

HumanCytoSNP-FFPE-12 (48 samples)

WG-321-1004

0.4

HumanOmniExpress-FFPE BeadChip(24 samples)

WG-321-1005

0.2

HumanOmniExpress-FFPE BeadChip(48 samples)

WG-321-1006

HumanMethylation450 BeadChip (24 samples)

WG-314-1003

01324Samples

StdDev logR Ratio

B
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
012

34Samples
Original

Restored

Restored FFPE samples display high call rates (A) and low variation of thelogR ratio (B) compared to the original, unrestored samples.

As shown in Figure 4A, the highly degraded FFPE samples showed
initial call rates of 40-70%. After restoration, these same samples
produced call rates of > 90%. The associated standard deviation oflog intensity (logR Dev) is < 0.4, indicating high reproducibility andhigh signal-to-noise ratios (Figure 4B). The HapMap concordance forthese samples was > 99.0%, indicating that the assay is capable ofaccurate genotype calls (data not shown).

Data Analysis
Assay data is fed directly into Illumina GenomeStudio DNA analysissoftware. GenomeStudio, along with the integrated CNVPartition
program, provide simplified graphical analysis of SNPs and copy
number variants across many data points.
An open plug-in interface easily integrates third-party applications
for more downstream data analysis options. The Illumina•Connect
program leverages this open architecture and has made numerous

HumanMethylation450 BeadChip (48 samples)

WG-314-1001

HumanMethylation450 BeadChip (96 samples)

WG-314-1002

Human iSelect 3–90K BeadChip (24 samples)

WG-401-1004

Human iSelect 3–90K BeadChip (48 samples)

WG-401-1001

Human iSelect 3–90K BeadChip (288 samples)

WG-401-1002

Human iSelect 3–90K BeadChip (1152 samples)

WG-401-1003

Human iSelect 90–250K BeadChip (24 samples)

WG-400-1004

Human iSelect 90–250K BeadChip (48 samples)

WG-400-1001

Human iSelect 90–250K BeadChip (288 samples)

WG-400-1002

Human iSelect 90–250K BeadChip (1152 samples)

WG-400-1003

Human iSelect 250–1M BeadChip (24 samples)

WG-404-1004

Human iSelect 250–1M BeadChip (48 samples)

WG-404-1001

Human iSelect 250–1M BeadChip (288 samples)WG-404-1002

Human iSelect 250–1M BeadChip (1152 samples)

WG-404-1003

Summary
Archival FFPE samples hold an abundance of invaluable informationfor human cancer studies. Because these samples generally yield
highly degraded DNA, they perform poorly in most array-based
studies. The Infinium HD FFPE DNA Restore Kit can repair thesedegraded DNA samples in preparation for use with the Infinium HD
Assay. Analysis of a series of FFPE samples demonstrated thatafter restoration, these samples produce high-quality genotyping,
CNV, and methylation data.

References
1.

Pokholok DK, Le JM, Steemers FJ, Ronaghi M, Gunderson K. Analysis ofrestored FFPE samples on high density SNP arrays. Presented at American
Association of Cancer Research Meeting, May 2010.

plug-ins available to support genotyping and copy number analysis.

*When using FFPE samples with the HumanMethylation450 BeadChip, a bisulfite conversion step is required between the QC and restoration steps.
Please refer to the Infinium HD FFPE QC Protocol for more information on workflows.
† ue to the variability of FFPE samples, specifications for these BeadChips are calculated at > 80% of the samples passing QC will achieve
Dcall rates of > 95% of CpG sites detected for the HumanMethylation450 BeadChip and logR Dev < 0.4. Specifications are for normal samples only.

Data Sheet: DNA Analysis

Research Field of Use
ILLUMINA’s Infinium HD FFPE DNA Restore Kit (Kit) uses a thermostable ssDNA ligase (Ligase) covered by U.S. or international patents or patent applications (IP) owned by EPICENTRE
Biotechnologies, LLC, Madison, WI (EPIBIO). Purchase of this Kit from ILLUMINA or an Affiliate or distributor of ILLUMINA gives the purchaser a limited, non-exclusive license under the IP
to use the Ligase in the purchased Kit solely for performing methods for restoration of degraded DNA from FFPE or other samples containing degraded DNA for subsequent analysis ofthat restored DNA using an ILLUMINA array-based platform for research and educational purposes, including commercial research-use-only services, but excluding array-based analysis
or sequencing of RNA or array-based analysis or sequencing of DNA that is made by reverse transcription of RNA. No license is granted to use the Ligase for manufacturing a product, formedical, therapeutic or diagnostic use (including as an analyte-specific reagent) in humans or non-humans, or for any other use other than research and educational purposes. For questions
related to licensing, please contact the Intellectual Property Manager at EPIBIO, 726 Post Road, Madison, WI 53713, USA.

Illumina • 1.800.809.4566 toll-free (U.S.) • +1.858.202.4566 tel • techsupport@illumina.com • www.illumina.com
For research use only
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Pub. No. 370-2010-009 Current as of 29 March 2012