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Analyzing FFPE Tissue Samples with the®
Infinium CytoSNP-850K v1.1 BeadChip
A comprehensive workflow for profiling cytogenetic aberrations in DNA isolated from FFPE
tissue samples.
Introduction
Formalin fixation of pathological samples, including tissues biopsiedfrom products of conception (POC) and cancer specimens, preserves
their morphology and characteristics for long-term storage and future
use in clinical research applications.1 Many of these samples harbor
valuable genetic information that has the potential to further theunderstanding of structural and numerical genetic variations and how
these influence phenotype. Unfortunately, the preservation process
frequently leads to nucleic acid degradation and base modification,
making genetic analysis of these formalin-fixed paraffin-embedded(FFPE) tissues challenging. Furthermore, analyzing the data generated
from FFPE tissue samples using standard cluster-fitting methods canprovide a poor representation of the sample, decreasing accuracy ofresults.
Using the Infinium CytoSNP-850K v1.1 BeadChip, researchers haveanalyzed cytogenetic aberrations in both tumor2 and POC 3 FFPE tissue
samples. This technical note describes a comprehensive workflow
from Illumina using the Infinium FFPE DNA Restoration Solution, the®
Infinium CytoSNP-850K v1.1 BeadChip, and BlueFuse Multi Analysis
Software (Figure 1) to profile genetic aberrations in FFPE tissue
samples.

FFPE Tissue Sample QC and Restoration
After DNA extraction using one of many commercially available kits, theIllumina FFPE QC Kit is used to evaluate the quality of prospective DNA
samples. Samples that pass the quality control evaluation areadvanced through the DNA restoration process. Using an optimal
starting amount of 100ng recovered DNA and a proprietary enzymatic
reaction, the Infinium FFPE DNA Restore Kit repairs degraded FFPE
tissue DNA in preparation for the whole-genome amplification step inInfinium Assays.

CytoSNP-850K v1.1 BeadChip Content
The Infinium CytoSNP-850K v1.1 BeadChip offers coverage of nearly
850,000 SNPs with 15× redundancy and enriched coverage for3262 dosage-sensitive genes. The gene list contains content from theClinical Genome Resource (ClinGen) 4 and the Cancer Genomics
Consortium (CGC) 5, providing a comprehensive view of cytogenetic
aberrations. It provides robust performance across a broad range ofsample types, including FFPE tissue samples. Processed arrays®
®
scanned using the iScan or NextSeq 550 Systems produce highquality results (Table 1).

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Figure 1: Workflow for Analyzing FFPE Tissue Samples with the Infinium CytoSNP850K v1.1 BeadChip

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Figure 2: FFPE Tissue Sample–Specific Cluster Files Enable Clear Identification of Variants—DNA was restored from an FFPE tissue sample using the Infinium FFPE DNA
Restoration Solution, processed on the Infinium CytoSNP-850K v1.1 BeadChip, and analyzed with BlueFuse Multi Analysis Software v4.3 using the standard cluster files
and the FFPEtissue sample–specific cluster file. Analysis using the FFPE tissue sample–specific file reduced the noise observed with the standard cluster file and provided
tight profiles from the FFPE tissue sample, enabling clear identification of a ~1 MB mosaic deletion (red circle).

Table 1: CytoSNP-850K v1.1 BeadChip Performance with FFPE
Tissue Samples
FeatureDescription

No. of Samples per BeadChip

8DNA Input Requirement

8 µL restored DNAb

Instruments Support

iScan and NextSeq 550 Systems

No. of SNP Markersa

829,864

DataPerformance

ProductSpecificationiScan System

NextSeq 550
SystemCall Rate

> 95%

99.6%

99.5%

Log R Dev

< 0.30

0.146

0.152

Due tothe variability of FFPE tissue samples,specifications for these BeadChips are calculated at>
80% of the samples passingQC willachieve callrates of > 95%.Specifications are for normalsamples
only.
a. Finalnumber of SNPs postGentrainanalysis for the FFPE tissue sample project.
b. Process modificationis detailed inthe “AnalyzingFFPE Samples withInfinium Microarrays”
technicalnote (www.illumina.com/content/dam/illuminamarketing/documents/products/technotes/technote-infinium-ffpe-sample-analysis.pdf).

Assay Protocol
Optimal assay performance with FFPE tissue samples requires a minor*modification to the standard Infinium HD Super Protocol: when
preparing the MSA1 plate, use 8 µL of the repaired DNA instead of thestandard 4 µL. This change will increase the effectiveness of thewhole-genome amplification step. Following amplification, theremaining protocol steps are performed as recommended.

Data Analysis
Data analysis for Illumina BeadChips relies on GenCall scores made by
the scanner that reflect the distance of a data point from the center of a“cluster” which represents signal intensities that correspond to
genotype calls. Performance differences between normal and FFPE
tissue samples can affect the cluster positions of the assayed SNPs,
leading to indistinct genotyping and copy number calling. To accountfor these clustering differences and increase the accuracy of copynumber visualization using BlueFuse Multi Analysis Software, FFPE
tissue sample–specific manifest and cluster files are available for theCytoSNP-850K v1.1 BeadChip. These files include reduced single
nucleotide polymorphism (SNP) content in comparison to the standard
manifest file, adjusting for the low performance of a small percentageof the SNPs due to poor restoration.
Use of the FFPE tissue sample–specific manifest and cluster files forthe production of genotype call (GTC) files enables generation ofreliable analytical data for copy number analysis from well-restored
FFPE tissue samples (Figure 2). BlueFuse Multi Analysis Software
offers validated algorithms and a suite of features that facilitate
visualization and interpretation of identified copy number variants(CNV).

Accessing FFPE Tissue Sample–Specific
Product Files
Download FFPE tissue sample–specific cluster and manifest files fromthe CytoSNP-850K BeadChip v1.1 support page
(support.illumina.com/array/array_kits/cytosnp-850k_beadchip_kit/downloads.html). Available files include:l

FFPE Tissue Sample–Specific Manifest File (CytoSNP-850Kv1-1_FFPE.bpm)l

FFPE Tissue Sample–Specific Cluster File for the iScan System(CytoSNP-850Kv1-1_FFPE_iScan.egt)l

FFPE Tissue Sample–Specific Cluster File for the NextSeq 550
System (CytoSNP-850Kv1-1_FFPE_NSQ550.egt)* For more informationonthe standard protocol,refer tothe “Infinium HDAssay Super ProtocolGuide” (support.illumina.com/content/dam/illumina-support/documents/myillumina/05340b1f-c179-495d-b790-

fa91ecbb6ff2/inf_hd_super_assay_ug_11322427_revc.pdf).

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Data Quality

References

The performance range of FFPE tissue samples in the Infinium Assay ishighly dependent on the quality of the preserved tissue and restored
DNA. Using the Infinium FFPE Restoration Solution and FFPE tissue
sample–specific cluster file with these sample types provides clear
profiling of chromosomal aberrations, such as duplications, deletions,and copy-neutral absence of heterozygosity (AOH) events, on theCytoSNP-850K v1.1 BeadChip. This high signal-to-noise ratio enables
identification of low-level mosaic abnormalities and reproducible
detection of CNVs.6

1.

Pokholok DK, Le JM, Steemers FJ, et al. Analysis of restored FFPE samples
on high density SNP arrays. Presented at American Association of Cancer
Research Meeting, April 2010.

2.

Mukherjee S, Ma Z, Wheeler S, et al. Chromosomal microarray provides
enhanced targetable gene aberration detection when paired with next
generation sequencing panel in profiling lung and colorectal tumors. Cancer

Genet. 2016; 209(4): 119–129. doi: 10.1016/j.cancergen.2015.12.011.
3.

Sahoo T, Dzidic N, Strecker MN, et al. Comprehensive genetic analysis ofpregnancy loss by chromosomal microarrays: outcomes, benefits, andchallenges. Genet Med. 2016; Jun 23. doi: 10.1038/gim.2016.69. [Epubahead of print].

Summary
Archival FFPE tissue samples are potentially a rich source of genetic
information that could inform many genomic studies, yet they often
perform poorly in array-based assays due to highly degraded DNA. The
Illumina FFPE Sample Restoration Solution for repairing damaged DNA
enables effective and reproducible data analysis from thesechallenging samples. The provision of FFPE tissue sample–specificmanifest and cluster files for the CytoSNP-850Kv1.1 BeadChip
enables high-quality genotyping data for copy number analysis without
the requirement to develop an internal data set for differential analysis.
The streamlined workflow for processing FFPE tissue samples on theCytoSNP-850K v1.1 BeadChip coupled with BlueFuse Multi Analysis
Software provides a reliable method for identifying and databasing
CNV and AOH cytogenomic events.

Learn More
To learn more about the Infinium FFPE DNA Restoration Solution, visit
www.illumina.com/products/infinium_ffpe_dna_restoration_solution.html
To learn more about the Infinium CytoSNP-850K BeadChip, visit
www.illumina.com/products/infinium-cytosnp-850k.html
To learn more about BlueFuse Multi Analysis Software, visit
www.illumina.com/clinical/clinical_informatics/bluefuse.html
Contact Technical Support at techsupport@illumina.com for addiitonal
information.

For R esearch Use On ly. Not f or u se in diagn ost ic procedu res.

4.

Clinical Genome Resource. www.clinicalgenome.org. Accessed July 6,
2016.

5.

Cancer Genomics Consortium. www.cancergenomics.org. Accessed July 6,2016.

6.

Conlin LK, Thiel BD, Bonnemann CG, et al. Mechanisms of mosaicism,chimerism and uniparental disomy identified by single nucleotide
polymorphism array analysis. Hum Mol Genet. 2010; 19(7):1263–1275. doi:
10.1093/hmg/ddq003.

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©2016Illumina,Inc.Allrights reserved.Illumina,BlueFuse,Infinium,iScan,NextSeq,and the pumpkinorange color are trademarks of Illumina,Inc.and/or its affiliate(s)inthe U.S.and/or other countries.Pub.No.1570-2016-018.Currentas of 16September 2016