by Catherine Shaffer
Copy number analysis is beginning to supplement or replace older forms of genomic analysis such as SNP analysis and microarray analysis. New discoveries in the field of gene copy number have revealed that in many cases the number of copies of a gene may be more significant than the presence or absence of a specific allele or mutation. This new science made a huge splash in 2007 when several high profile papers demonstrated the significance of copy number variation. Since then, a number of vendors have entered the market, offering assays tailored for copy number discovery and for screening known copy number variants for a particular species or set of loci. The most common application for copy number analysis is searching for associations between higher orders of copy number variation and disease states. Autism, schizophrenia, and several autoimmune disorders have been linked to copy number variations. Conventional genomic assays that use PCR amplification are not optimal for counting large numbers of gene copies, so different technologies have been applied to this problem.
Nanostring is a life sciences company founded in 2003 to commercialize a digital molecular bar coding technology invented at the Institute for Systems Biology in Seattle. Nanostring's nCounter copy number variation assay, now available as part of an early access program, uses their digital technology to count DNA target molecules using a fluorescent bar code linked to nucleic acid-specific oligonucleotides. The customer supplies the regions of interest or loci, and Nanostring designs one or more probe sets for the loci. Eight hundred probe sets can be used in a single reaction, so multiplexing of 800 loci at once is possible. Unlike microarrays or PCR, the data the system outputs is digital and thus highly sensitive and precise allowing for distinguishing of higher order copy numbers. “We're counting the number of times we see the target molecule. It's not an analog signal. There's no amplification of the target, no PCR. We're measuring signal directly from the copies of genomic DNA,” says Gary Geiss, PhD, Principle Scientist for Nanostring.
According to Geiss, one of the greatest advantages of the Nanostring system is that the purification and imaging is fully automated. “It's a very simple technology to use. You fragment your DNA, add it to your hybridization mix, and the next day you come in and the rest of the process is automated.” It takes less than 35 minutes of hands on time to set up 12 reactions. The nCounter CNV assay has call accuracy rates of over 99 percent, based on comparison to publicly available HapMap data. NanoString plans to make the CNV assay more widely available in the third quarter of 2010.
Comparative genomic hybridization is another method that has been developed to analyze gene copy number. After Roche purchased NimbleGen in 2007, it launched a high resolution 2.1M feature CGH microarray for the study of copy number variation. High resolution means more probes, which increases the likelihood of finding smaller copy number variants. The 2.1M feature array routinely detects small copy number variations between 5 and 10kb in size. Says Lance Brown, Marketing Manager for Roche, “There are two key advantages to the NimbleGen 2.1M CGH microarray format: high resolution analysis for genome-wide CNV detection and discovery, and multiplex formats that enable more cost-effective analysis.”
Roche offers a variety of microarray formats and designs for CNV discovery and analysis, including the NimbleGen Human CNV 2.1M Array and NimbleGen Human CNV 3x720k Array. Both of these arrays are designed specifically to detect ~41,000 known CNVs from three different CNV databases. While these arrays detect a large number of known CNVs, they also contain a backbone of probes for the discovery of unknown CNVs. Roche also offers custom CGH arrays that are built to customers’ specifications for not only the human genome, but for other important genomes as well.
The strongest selling points of the Roche NimbleGen portfolio are the high resolution arrays and the multiplex formats associated with the 2.1M feature array. To that end, Roche is actively working to increase the number of probes on a single array while providing cost-effective multiplexing options. “In the near future we’ll be looking to increase our resolution yet again,” Brown adds. “We’ll be significantly increasing above our 2.1 million feature arrays.”
Affymetrix has a portfolio of products for copy number analysis specifically designed for cytogenetics applications. Their Genome-Wide Human SNP Array 6.0 platform is designed for the constitutional cytogenetics field, where users need the highest possible resolution of copy number and SNPs across the genome, and where the measurement of uniparental paternal disomy (UPD) and regions identical-by-descent due to consanguinity are important. The high-density SNP Array 6.0 array provides the most comprehensive coverage, with 1.8 million total markers spaced at approximately one marker per 700 bases, with an even distribution of both SNPs and non-polymorphic probes.
"Affymetrix SNP probes are able to measure copy number because they are dose responsive, says Richard Shippy, Director of Product Marketing, Clinical Strategic Marketing. "An increase in chromosomal mass leads to higher signal, which gets translated to copy number. Our competitors have a probe type that is either copy number only and no SNP, or an array that is a SNP only and which results in gaps in coverage."
The 250K Nsp platform is Affymetrix's lower cost option that is part of the GeneChip® Human Mapping 500K Array Set, and is a popular choice among cancer researchers for assessing copy number and loss of heterozygosity (LOH) in leukemia samples such as chronic lymphocytic leukemia, myelodysplastic syndromes, acute myeloid leukemia, multiple myeloma, and acute lymphoblastic leukemia. A third product from Affymetrix for copy number analysis is a formalin-fixed, paraffin-embedded (FFPE) solution that they developed using a proprietary molecular inversion probe (MIP) technology. It enables the assessment of copy number changes in the most degraded of samples, as small as 65 bases in length, which is common for FFPE samples.
"We also have a new MIP Copy Number Service that will offer a more comprehensive view of the cancer genome along with genome-wide coverage-more than 330,000 markers-the highest density for MIP technology," says Shippy. "In the last few years we've witnessed a revolution in the cytogenetics field with the utilization of SNP-based arrays. In less than three years, we went from SNPs being of modest interest within the cytogenetics community to now being absolutely essential."
Copy number analysis is a growing trend in the life sciences, and a number of different tools and technologies are becoming available to help researchers count gene copies. The right choice depends on the degree of resolution and sensitivity required, as well as end user applications. Copy number analysis can enhance another type of genomic screen, such as an SNP array. In some cases, it can replace other types of screens and assays. Copy number analysis technology includes conventional methods such as the microarray, as well as novel technologies such as micro bar codes to digitally tag and count individual genes. The outlook for new and better tools for copy number analysis is bright, as manufacturers are keen to improve and perfect their product offerings.