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Microarray Processing

Microarray is the first high-throughput genome technology developed in the mid-1990s. Over the years, this technology has seen tremendous growth and maturation. It was originally developed for gene expression profiling, but its application soon diversified into other areas, including genotyping, DNA methylation analysis, and copy number variation analysis with comparative genomic hybridization (CGH). Attested by the large amounts of data deposited in major genomics databases such as Gene Expression Omnibus (GEO), microarray technology has significantly advanced high-throughput genome research for over two decades.

Different from next-gen sequencing, microarray is based on hybridization of nucleic acid molecules present in research samples to pre-designed probes. Signal readouts from the hybridization are then used to analyze gene expression pattern, DNA sequence polymorphism, epigenetic modification, genome instability, etc. Based on how probes are generated and immobilized, there are different microarray platforms. The NUSeq Core provides support to two of the most widely adopted microarray platforms, i.e., the Affymetrix GeneChip and the Illumina BeadArray systems. NUSeq also provides bioinformatics services to data generated on both platforms.

Microarray Platforms

 Affymetrix GeneChip

Affymetrix GeneChip uses DNA probes synthesized in situ using photolithography, a process similar to that used for manufacturing semiconductor chips. Affymetrix uses 25bp oligonucleotide probes, usually in pairs. For each probe that matches perfectly to its designated target, there is a mismatch probe, which differs only at the centre base position, to help determine false signal from non-specific hybridization or background. Affymetrix offers a wide array of GeneChips for different applications. For a list of all Affymetrix GeneChips, please check the vendor website. For transcriptome profiling, the most recent offering from the company is the Clariom arrays. ForGeneChip processing fee, please check the Core Pricing page.

 Illumina BeadArray

Using a different approach invented by Dr. David Walt and colleagues at Tufts University, the Illumina BeadArray technology creates an array using specially prepared microbeads, which self-assemble into microwells etched into a substrate. Each microbead, 3 microns in diameter and spaced 5.7 microns apart, carries on its surface hundreds of thousands of copies of a particular oligonucleotide sequence that act as probes. As each array is formed from random assembly of microbeads into microwells, to identify which bead type is in which microwell requires a decoding process. This process is based on hybridization of oligonucleotides that are complementary to the probes on the beads called decoders. Different sets of these oligonucleotides, labeled with different fluorophores, are used in the decoding process. The ingenuity of this decoding process lies in the fact that only a small number of fluorophores and a small number of steps are needed to achieve the decoding of an extremely large number of beads in the array.

BeadArrays that are currently available from Illumina are mostly for genotyping and methylation profiling. NUSeq provides processing of these arrays for users. Illumina BeadArray processing costs are listed on the Core Pricing page.

Major Applications and Sample Requirements

 Gene Expression Profiling

Affymetrix GeneChips are available for gene expression analysis. Illumina has discontinued their gene expression BeadArrays. All GeneChips available from the vendor (now part of Thermo Fisher) can be processed. The most recent release is the Clariom array series.

Sample requirement: The minimum input is 50ng total RNA in 3ul to use the whole-transcriptome (WT) Plus target prep protocol. For less starting material, the WT Pico target prep protocol is used. The minimum input for WT Pico is 100pg high-quality total RNA, or 500pg for those extracted from FFPE samples, in 3ul. To allow for QC please submit a minimum volume of 12-15ul.

 Whole Genome Genotyping

NUSeq principally processes genotyping arrays available from Illumina. Illumina Infinium Omni, Core, and Multi-Ethnic BeadArray families can be used to analyze from 300,000 to 5,000,000 genome markers, with the option of adding custom content to many of the arrays. Illumina’s Array Selector is a tool to assist in locating the right array to use.

Sample requirement: All samples should be submitted in 96-well plates. The concentration of the samples should be at least 75 ng/ul and we require a minimum volume of 20 ul.

 DNA Methylome Profiling

The Illumina Infinium MethylationEPIC BeadChips target over 850,000 individual potential methylation sites across the genome for epigenome wide association studies (EWAS). These sites encompass CpG islands, RefSeq genes, ENCODE open chromatin, ENCODE transcription factor binding sites, and FANTOM5 enhancers. It is back-compatible and covers >90% of the sites on the earlier HumanMethylation450K array. Each EPIC chip loads 8 samples.

The recently announced Mouse Methylation BeadChips target over 275,000 markers across the mouse methylome for epigenetic analysis of diverse strains. They provide balanced coverage of CpG islands, transcription start sites, enhancers, imprinted loci, gene body regions, repetitive element regions, lamin-attachment domains, CTCF binding sites, and hypermethylated regions in cancer. Each chip loads 12 samples.

Sample requirement: Samples for methylation profiling should be submitted in 96-well plates at a normalized concentration of at least 20 ng/ul with a total of 1.7 ug of genomic DNA. We prefer a normalized concentration by PicoGreen or Qubit of 50ng/ul if possible. FFPE samples can be processed at additional cost.

Frequently Asked Questions

 Since each Illumina BeadArray holds multiple samples, does it matter where to place samples on the arrays?

Yes, where samples are placed on the arrays matters. To avoid introducing unwanted signal variation from array to array, sample placement on BeadArrays need to be randomized. For help with randomizing samples please contact the Core’s bioinformatics team.

 How should I arrange randomized samples for processing on Illumina BeadArrays?

For submission, randomized samples should be arranged in 96-well plate format. Please consult this diagram to determine how the samples should be arranged in 96-well plate. Users should fill out the Infinium HD Methylation Sample Sheet with the sample names and their positions, and bring the sample sheet when dropping off samples.

 I am an external core user. How should I ship my DNA or RNA samples to NUSeq for processing?

Samples should be shipped frozen. Please make sure to use sufficient dry ice in the container to avoid thawing. We cannot accept samples for any study that have thawed or were not shipped frozen. Include a copy of sample list in the shipment and also send an electronic version to NUSeq before sending the samples.

Our mailing address is:

NUSeq Core Facility
300 E. Superior St.
Tarry 2-770
Chicago, IL 60611