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  • Review Article
  • Published:

Highly parallel genomic assays

Key Points

  • Highly parallel genomic assays have two fundamental characteristics: a highly parallel array-based read-out and an intrinsically scalable, multiplexing sample preparation.

  • The power of highly parallel genomic assays is that they tend to follow the principle behind Moore's law: the amount of information extracted from a sample increases linearly with the number of probes on the array, whereas the overall cost of the assay tends to increase at a much slower rate.

  • These general concepts are being applied successfully to an increasing variety of assays, including gene-expression profiling, SNP genotyping, genomic copy-number analysis, measurement of allele-specific expression levels, and methylation status.

  • Early genomic assays, such as gene-expression profiling, relied only on sequence-specific probe hybridization to confer specificity. The next generation of assays have made use of enzymatic discrimination in addition to hybridization to increase specificity and to enable assay designs that extract more information.

  • Data quality, reproducibility and robustness of intrinsically parallel assays that use enzymatic discrimination have been shown to be high, defying the conventional wisdom that increasing sample complexity automatically results in lower data quality.

  • The technology of highly parallel assays is enabling a revolution in genomics that has far-reaching implications for molecular biology and human health. Increasingly, ambitious projects that aim to be more comprehensive in their approach to genomic analysis, such as the International HapMap Project, the ENCODE Project, and the Cancer Genome Atlas, are reliant on new, highly parallel assay technologies.

  • The orders of magnitude decrease in cost and increased speed and accuracy that are provided by highly parallel assays have brought us to the dawn of a potentially revolutionary new era of discovery in human genetics that will be based on comprehensive, high-resolution genetic mapping.

  • Such studies might require about a billion or more genotypes, and were impractical prior to the advent of the assays that are described in this Review. A few years ago, the genotyping costs for such a study would have been in the hundreds of millions of dollars. Today, the costs would be a few million dollars, with far higher data quality and completeness, and genotyping can be carried out in a few weeks instead of many years.

  • Parallel assay systems are assisting a similar revolution in the field of DNA sequencing, and will probably enable powerful new comprehensive studies that are prohibitively costly today.

Abstract

Recent developments in highly parallel genome-wide assays are transforming the study of human health and disease. High-resolution whole-genome association studies of complex diseases are finally being undertaken after much hypothesizing about their merit for finding disease loci. The availability of inexpensive high-density SNP-genotyping arrays has made this feasible. Cancer biology will also be transformed by high-resolution genomic and epigenomic analysis. In the future, most cancers might be staged by high-resolution molecular profiling rather than by gross cytological analysis. Here, we describe the key developments that enable highly parallel genomic assays.

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Figure 1: Multiplex PCR.
Figure 2: Highly parallel genotyping assays.
Figure 3: High-resolution genomic profiling on SNP-CGH arrays.
Figure 4: Highly parallel sequencing on clonal arrays.
Figure 5: Digital analysis of gene expression using serial analysis of gene expression (SAGE).

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FURTHER INFORMATION

Cancer Genome Atlas Pilot Project

ENCODE international consortium

Human Epigenome Project

Glossary

Cancer staging

Classification of cancer types into groups that reflect their localization, metastasis, prognosis, recommended treatment regimen and predicted clinical outcome.

Linkage disequilibrium

The property of two polymorphic loci in a population such that the polymorphic states at the two loci are not independent of one another, and as a result the state of the polymorphism at one locus has a higher probability of being associated with a particular state at the second locus. This association is usually measured with a metric called r2 that ranges between zero (no linkage) and one (complete linkage).

Ligase chain reaction

A cyclic amplification method for amplifying a target sequence that is similar in approach to PCR except that repeated rounds of thermally controlled denaturation, annealing and ligation of a pair of adjacent oligonucleotides are carried out.

Padlock-probe amplification

A ligation-mediated bimolecular assay for a target sequence in which the two query oligonuceotides (5′ and 3′ sequences) are derived from the two ends of a contiguous oligonucleotide. Ligation of the two ends creates a circular structure that is intertwined with the target sequence.

Primer dimer

A parasitic product that is formed during PCR reactions and is caused by multiple primers interacting and extending upon themselves. Appropriate design of primer sequences can reduce this effect.

Universal PCR

A multiplex PCR reaction using a single or pair of universal primer sequences to amplify a broad range of target sequences that all contain common invariant 5′ and 3′ tail sequences

Whole-genome representation

A representation with a sequence complexity that is similar to that of the entire genome from which it was derived.

Reduced-complexity genomic representation

A representation with a sequence complexity that is a fraction of the original sample nucleic-acid complexity. In its simplest version, PCR of adaptor-ligated or restriction-enzyme-digested genomic DNA intrinsically generates a reduced-complexity representation.

DNA-array feature

An individual resolvable element of a DNA array that contains a defined sequence. This element can be created in several ways such as spotting, in situ synthesis or deposition of beads that harbour immobilized DNA sequences.

Tag SNP and tagging SNP

A tag SNP is defined as a SNP that proxies for a set of SNPs in linkage disequilibrium with itself (that is, they are in the same linkage disequilibrium bin). A haplotype tagging SNP, by contrast, is based on the haplotype block concept, in which a set of tagging SNPs are used to uniquely define the variation of all SNPs that reside in the haplotype block.

Uniparental disomy

This rare genetic condition can arise constitutionally through non-disjunction during meiosis that ultimately leads to a duplication of a segment or of the entire maternal or paternal chromosome in the affected individual. A form of apparent uniparental disomy can arise in the course of normal cell division (mitosis) through mitotic recombination (a rare crossover event during mitosis).

Polony

Contraction of 'polymerase colony' that is created by growing DNA colonies from single DNA 'seed' molecules through the use of a PCR reaction on DNA molecules that are diffusely imbedded in a polymer matrix that contains DNA polymerase, primers and appropriate reagents.

BEAMing

A process of cloning on beads in which a library of clones is grown on beads through the use of compartmentalized emulsion PCR. DNA and beads are diluted such that, on average, only a single bead and a single target molecule co-occupy a single compartment. PCR amplification grows a clonal population of molecules on the bead starting from the single target sequence.

Massively parallel signature sequencing

This enables digital transcript counting in a cDNA sample. It is accomplished by cloning a 17–20 base signature sequence tag onto micro-beads that are subsequently fixed in a single layer array in a flow cell. The sequence on the bead is then read out using a ligation-based cycle-sequencing assay.

Type IIS restriction enzyme

Restriction enzymes that primarily exist as monomers and require only Mg2+ as a cofactor. Recognition sites are nonpalindromic, nearly always contiguous and without ambiguities; at least one strand is cleaved outside the recognition sequence.

ChIP-on-chip

Chromatin immunoprecipitation (ChIP) combined with microarray detection. Usually, cells are treated with a crosslinking reagent (for example, formaldehyde), which is used to covalently link protein complexes in situ to DNA. The crosslinked chromatin is then isolated and fragmented. An immunoprecipitation step is used to enrich the protein of interest together with crosslinked DNA fragments. To identify and quantify these DNA fragments, the crosslinks are reversed and the DNA fragments are usually labelled with a fluorescent dye and hybridized to microarrays with probes that correspond to genomic regions of interest.

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Fan, JB., Chee, M. & Gunderson, K. Highly parallel genomic assays. Nat Rev Genet 7, 632–644 (2006). https://doi.org/10.1038/nrg1901

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