Assay Technology

DASL Assay

The cDNA-mediated annealing, Selection, extension, and Ligation (DASL) Assay allows for RNA profiling of up to 1536 targets using partially degraded RNA as found in formalin-fixed, paraffin-embedded (FFPE) samples. Using either the Universal-16 BeadChips or the Universal-96 Array Matrix format for sample processing, the DASL Assay offers researchers the Opportunity to analyze hundred to thousands of RNA transcripts derived from newely or previously collected, preserved, samples.

The DASL Assay leverage the proven GoldenGate Assay protocol to provide high-quality data for an RNA-based application. Because the probe groups span only about 50 bases, partially degraded RNA Can Be used in the assay. The availability of additional, unique probe sequences per gene increases the sensitivity of the assay, allowing the quantitation of low-Abundance Transcripts, even in partially degraded samples.

DASL Assay Highlights

Reproducible RNA profiling: Profile NRAs such as partially-degraded Those found in formalin-fixed, paraffin-embedded (FFPE) samples

Proven sensitivity: detect low-Abundance Transcripts

Ideal for clinical sample types: Requires an input of just 100-200 ng total RNA

Flexible formats: Process in groups of 16 or 96 samples

Scalable solution: platform Automatable with low per-sample pricing

Direct Hybridization Assay

Illumina Gene Expression arrays are designed to be fully compatible with the standard reagents, techniques, and tools that are used extensively throughout the community gene expression, from sample labeling methods to analysis software.

Illumina Gene Expression protocols feature a first-and second-strand reverse transcription step, followed by a single in vitro transcription (IVT) Amplification that incorporates biotin-labeled nucleotides. Subsequent steps include array hybridization, washing, blocking, and streptavadin-Cy3 staining.

Direct Hybridization gene expression assays are run on the SCAN system or BeadArray Reader.

Fluorescence emission by Cy3 is quantitatively detected for downstream analysis.GenomeStudio software provides results in standard file formats that CAN be readily processed with commercial grape expression-analysis software programs.

Probe design

Candidate bioinformatics probe sequences undergo extensive screening prior to selection. We concatenate 50-mer gene-specific probe sequences with "address" sequences which are immobilized on a Bead along with hundred of thousands of probes of the same sequence.

Direct Hybridization Assay highlights

• High Reproducibility: r2 value > 0.99

• High Yield: Multiple hybridizations from a single reaction (> 10 µg from just 50 ng of total RNA)

• Low Sample Input Requirements: Just 50–100 ng of total RNA required

• Low Per-Sample Cost: Less than half the price of other commercial arrays

Whole-Genome DASL assay

Our Whole-Genome DASL Assay is an array-based method for expression profiling of partially degraded RNA samples such as those found in FFPE samples.

The Whole-Genome DASL Assay combines the unique PCR and labeling steps of our original DASL Assay with the gene-based hybridization and whole-genome probe set of our HumanRef-8 BeadChip. This BeadChip features up-to-date content covering more than 24,000 annotated genes derived from RefSeq (Build 36.2, Release 22). The Whole-Genome DASL Assay greatly increases the target set of the original DASL Assaywhile retaining the ability to accurately profile partially degraded RNA samples.

Whole-Genome DASL Assay Highlights

• High Sensitivity: start with FFPE samples, brain tissue, blood samples without globin reduction, or as few as 100 cells

• Low-Sample Input: use 10-100 ng total RNA from fresh-frozen, 50-200 ng from FFPE samples

• High Multiplex Capabilities: analyze more than 24,000 annotated probes as once

• High-Quality Results: obtain qPCR-equivalent results with high reproducibility (r2>0.86), as well as excellent correlation with matching fresh/frozen samples (r2>0.98)

Low Per-Sample Cost: pay less than one-third the cost of other methods

GoldenGate Genotyping Assay

The Illumina GoldenGate Genotyping Assay is a flexible, pre-optimized assay that uses a discriminatory DNA polymerase and ligase to interrogate 96, or from 384 to 1,536, SNP loci simultaneously. The protocol can be performed manually or can be easily automated with LIMS and the AutoLoader 2, producing a streamlined workflow that allows a single technician to generate over 300,000 genotypes in just six hands-on hours. This Assay utilizes the iScan system or BeadArray Reader.

GoldenGate Standard Panels include options for human and mouse content. GoldenGate Custom Panels can be designed for many species with user-defined content. GoldenGate Genotyping samples can be conveniently processed on either the Sentrix Universal-16 BeadChip or Universal-96 Array Matrix multi-sample array formats.

GoldenGate assay highlights

• Proven technology: Used in genotyping centers worldwide

• High-quality data: Average call rates > 99%

• Scalable solution: 96, or from 384 to 1,536 SNPs per sample, 16 or 96 samples in parallel

• Streamlined workflow: Manual or automated processing with multiple stopping points

• Flexible content: An expanding selection of standard panels or custom panels with the SNP loci of your choice

Infinium HD Assay

The Infinium HD Assay leverages proven chemistry and a robust BeadChip platform to produce unrivaled data quality, superior call rates, and the most consistent reproducibility. From customized studies on targeted regions to large-scale genome-wide association studies, the flexible Infinium HD design offers a powerful solution for virtually any genetic analysis application.

• Infinium HD BeadChips

The Illumina portfolio of Infinium HD BeadChips feature the most advanced genomic content available for genotyping, and copy number variation (CNV) and Cytogenetic analysis. Profile the human genome with 300,000 to nearly 1.2 million markers strategically-selected to provide dense coverage, few large gaps, and cutting-edge content associated with human disease.

• Infinium HD iSelect BeadChips

Infinium HD iSelect BeadChips allow you to provide your own content for virtually any marker in the genome and any species of interest. Interrogate 3,000 to 200,000 markers per sample on a 12-sample BeadChip for customized, high-throughput SNP genotyping and CNV analyses. Standard BeadChips are also available which feature focused content for agriculturally relevant species.

Convenient protocol

The Infinium HD Assay protocol allows for single tube sample preparation without PCR or ligation steps, significantly reducing labor and sample handling errors. The assay can be scaled to unlimited levels of multiplexing without compromising data quality, unlike many alternative PCR-dependent assays.

Powerful chemistry

Genomic markers are interrogated though a two-step detection process. Carefully designed 50-mer probes selectively hybridize to the loci of interest, stopping one base before the interrogated marker. Marker specificity is conferred by enzymatic single-base extension to incorporate a labeled nucleotide.

Subsequent dual-color florescent staining allows the labeled nucleotide to be detected by Illumina’siScan imagining system, which identifies both color and signal intensity. For genotyping assays, the red and green color signals specify each allele, where homozygotes are indicated by red/red or green/green signals, and heterozyotes are indicated by red/green (yellow) signals.

GoldenGate Methylation Assay

Illumina’s GoldenGate Methylation solution is the first array-based platform that combines high sample throughput, high multiplexing, and single-site CpG resolution for DNA methylation profiling. The GoldenGate Assay can be used to simultaneously analyze the methylation status of up to 1,536 independent CpG sites per assay across 96 samples on either standard or custom content oligo sets.

Several different control types are provided to ensure the highest quality data. Each bead type is represented with an average 30-fold redundancy to further enhance the assay’s accuracy and robustness. Following bisulfite conversion of the sample DNA, researchers can use this high-throughput assay with the standard GoldenGate Methylation Cancer Panel I or design their Custom Methylation Panels targeting CpG loci within a gene or region of interest.

Highlights

• High Multiplex Array Platform: Simultaneously analyze up to1,536 CpG sites

• Streamlined Workflow: 96 samples per Universal Array Matrix with the convenient three-day GoldenGate Assay workflow

• Flexible Content Selection: Standard and custom panels available

Infinium Methylation Assay

DNA methylation plays a critical role in the regulation of gene expression and is known to be an essential mechanism for guiding normal cellular development and maintaining tissue identities. Numerous studies have implicated aberrant methylation in the etiology of many human diseases including cancer. Illumina’s Infinium Methylation Assay provides quantitative methylation measurement at the single-CpG-site level, offering the highest resolution for understanding epigenetic changes.

HumanMethylation27 BeadChip

With the HumanMethylation27 BeadChipresearchers can quantitatively interrogate 27,578 CpG loci covering more than 14,000 genes at single-nucleotide resolution. This high-density panel lets researchers profile up to 12 samples in parallel.

Powerful Infinium chemistry

The Infinium Methylation Assay detects cytosine methylation at CpG islands based on highly multiplexed genotyping of bisulfite-converted genomic DNA (gDNA). Upon treatment with bisulfite, unmethylated cytosine bases are converted to uracil, while methylated cytosine bases remain unchanged.

The assay interrogates these chemically differentiated loci using two site-specific probes, one designed for the methylated locus (M bead type) and another for the unmethylated locus (U bead type). Single-base extension of the probes incorporates a labeled ddNTP, which is subsequently stained with a fluorescence reagent. The level of methylation for the interrogated locus can be determined by calculating the ratio of the fluorescent signals from the methylated vs unmethylated sites.

The Infinium Methylation Assay utilises either theiScan System or the BeadArray Reader.

The Infinium Methylation Assay uses two different bead types to detect CpG methylation. The U bead type matches the unmethylated CpG site; the M bead type matches the methylated site. In the top figure, the unmethylated CpG target site matches with the U probe, enabling single-base extension and detection. It has a single-base mismatch to the M probe, which inhibits extension. If the CpG locus of interest is methylated , the reverse occurs.

VeraCode GoldenGate Genotyping Assay

Illumina’s GoldenGate Assay became the industry standard for higher level multiplexing during the International HapMap Project, which used this assay to call more than 258,000,000 genotypes. Combining the ease of the GoldenGate Assay workflow with the solution-based kinetics ofVeraCode technology provides an accelerated and streamlined method for robust mid-level multiplexed genotyping assays.

Custom VeraCode GoldenGate Genotyping Kits enable you to simultaneously genotype 48, 96, 144, 192 or 384 loci in a single well of a standard 96-well microplate. Every custom kit comes with GoldenGate oligo pools (OPAs) designed with Illumina's proven Assay Design Tool, eliminating the need for time-consuming multiplexed PCR optimization.

The user-friendly VeraScan software interface enables customization of BeadXpress Readerscanning protocols, acquisition and viewing of intensity data, and report generation. Illumina's BeadStudio Genotyping Module provides a forum for simple to complex downstream data analysis that can be easily exported to third-party analysis programs.

VeraCode protein analysis

VeraCode technology provides a simple and flexible solution for multiplexed protein assays. WithVeraCode Carboxyl Bead Sets, any protein or peptide can be covalently attached to the highly stable microbead surface, enabling the development of standard "sandwich" immunoassays. The 48 unique Carboxyl bead types can be pooled in varying combinations to perform up to 48 immunoassays in a single reaction in a standard 96-well microplate.

Analytes can be labeled with standard fluorescent reporters such as phycoerythrin (R-PE), Cy3, Cy5, or AlexaFluor dyes. During analysis, the fluorescence and code inscription for each microbead are detected by the BeadXpress Reader.

It has been demonstrated that the BeadXpress Reader can detect protein concentrations as low as 10 pg/ml in a multiplexed VeraCode Carboxyl bead assay.

The VeraScan Software that comes with theBeadXpress Reader System provides users the flexibility they need to customize scanning protocols. Illumina’s BeadStudio software provides users a forum for simple to complex data analysis that can be easily exported to third-party data analysis programs.

VeraCode DASL Gene Expression Assay

Illumina’s well-established DASL (cDNA-Mediated Annealing, Selection, extension, and Ligation) Assay empowers accurate profiling of intact and difficult samples such as partially degraded or FFPE samples. This robust assay deployed on VeraCode technology delivers an optimal combination of assay accuracy and high sample throughput, offering an ideal solution for low- to mid-plex transcript profiling.

Customizable VeraCode DASL kits allow you to simultaneously profile 32-384 transcripts of choice, and hundreds of samples can be read per day with the BeadXpress Reader. Submit your desired gene list, and Illumina scientists will help you create successful custom content for multiplexed DASL assays.

VeraCode DASL Gene Expression Assay Highlights

• Multiplex transcript profiling: measure expression levels of 32-384 genes per sample

• Highest sample throughput: process hundreds of samples per day

• Proven accuracy: achieve unrivaled reproducibility and precision with VeraCode technology

• Versatile: assay intact, partially degraded, or FFPE samples using as little as 100 ng total RNA (200 ng for FFPE samples)

• Convenient workflow: complete DASL Assay for VeraCode in two days; reduce manual pipetting steps with pre-kitted beads

VeraCode GoldenGate Methylation Assay

DNA methylation is an epigenetic modification known to have widespread effects on gene expression. Dysregulation of methylation has been linked to the etiology of common diseases such as cancer, MS, diabetes, and schizophrenia.

Illumina’s GoldenGate Methylation Assay provides sensitive detection for profiling methylation status at single-site resolution. This robust assay deployed on VeraCode technology offers an economical and high-throughput solution for low- to mid-level multiplexing and presents an ideal platform for focused studies or biomarker validation.

A customized panel of 48-384 CpG regions can be profiled simultaneously, and hundreds of samples can be read per day with the BeadXpress Reader. Individual bead types can be combined in unlimited combinations to support flexible assay design tailored to specific applications. Submit your desired list of CpG regions, and Illumina scientists will help you create successful custom content for multiplexed GoldenGate Methylation assays.

VeraCode GoldenGate Methylation Assay Highlights

• Flexible multiplex: target 48-384 CpG regions in a single reaction

• Specificity: obtain single-site resolution

• High sample throughput: process hundreds of samples per day

• Proven accuracy: achieve unrivaled reproducibility and accuracy with the well-established GoldenGate Assay for Methylation

• Convenient workflow: complete streamlined GoldenGate Assay for VeraCode in two days; reduce manual pipetting steps with pre-kitted beads