Utilizing the new high-density, long-oligo NimbleGen 2.1M arrays and an optimized design algorithm, the entire human exome can be captured on a single array. This process offers significant speed and scalability advantages over current PCR-based methods for targeted enrichment.
Targeted exome sequencing enables the discovery of many of the functional variations that are responsible for various diseases. Exome sequencing can also shed light on why diseases occur more often in certain populations, and can help uncover why drugs are effective only in a subset of the patients or population.
The prevailing method for complexity reduction has been the preparation of amplicons by parallel, multiplex, or long-range PCR amplification. These PCR methods have severe cost and performance limitations, when scaled to the level required to take full advantage of the capacity of currently available sequencing systems. As a result, the bottleneck for sequencing projects has shifted to sample preparation.
To address these limitations, Roche NimbleGen has developed the microarray hybridization-based sequence capture technology that utilizes high-density oligonucleotide as a programmable genomic selection device to allow targeted sequencing of genome subsets. These genome subsets can be exons, disease-associated regions, quantitative trait loci, promoters and enhancers, and other targeted regions.
The NimbleGen Sequence Capture 2.1M Human Exome microarrays, launched in January 2009, use an optimized, empirically tested design algorithm (version 2.0) and are built on the new high-density HD2 platform (2.1 million long oligonucleotide probes, >60mer). The new technology enables the capture of nearly all regions of the human genome that code for proteins (~180,000 human coding exons and ~550 miRNA exons) on a single microarray (Table 1). When it is coupled with the Genome Sequencer FLX System, researchers worldwide now can assess genetic variation within the exome of any individual. The array design is based on the 2008 build of the Consensus CDS (CCDS) database project, which represents a collaborative effort to identify a core set of high-quality human and mouse protein coding regions.
The Sequence Capture arrays are optimized for subsequent ultra-high throughput sequencing with the GS FLX Titanium series kits and enable accurate detection of genomic variation. The new GS FLX Titanium chemistry expands upon the previous series by providing researchers with an even greater sequencing power of more than 400-bp sequencing reads and over 1 million reads per run. The system also includes dedicated analysis tools for mapping reads and detecting variants from data generated by the Roche 454 Sequencing System, of captured DNA from NimbleGen arrays, allowing straightforward interpretation of results.
Compared with conventional PCR methods, this array-based process offers the following advantages:
- High performance: capture up to 30 Mb total regions on a single 2.1 M array and up to 5 Mb on a single 385 K array with high coverage and specificity.
- Design expertise: ensure the highest level of specificity and sensitivity with an empirically tested capture design algorithm.
- Embedded quality controls: NimbleGen Sequence Capture arrays incorporate built-in control probes to ensure system performance.
- Maximum flexibility: tailor the array design to capture the genomic regions or thousands of exons in parallel.
- Substantial savings: save time and cost compared with PCR-based methods.
The Sequence Capture protocol represents a novel application of DNA microarray technology to the hybridization-mediated enrichment of target DNA fragments from full-complexity human genomic DNA and consists of eight major steps (Figure 1).
Roche NimbleGen offers two workflow options to fit the schedule, budget and focus of each individual user (Figure 2). Find more details for both options on the web at www.nimblegen.com/seqcap.
Researchers can simply specify target genomic regions and ship the genomic DNA samples to Roche NimbleGen Service Lab to perform the genomic enrichment on the samples, and receive sequencing-ready samples of enriched, amplified genomic fragments.
Labs with experience in DNA microarray hybridization may already have most of the equipment required to perform genomic enrichment. Researchers specify the genomic regions for enrichment, and Roche NimbleGen will design, synthesize, and ship the arrays, along with additional required components including hybridization mixers and elution chambers. Roche NimbleGen also offers a set of kits for genomic enrichment, including a hybridization kit and a wash/elution kit, as well as a customer workshop on-site (3 days) with certified trainers to train researchers on protocols.
This article was originally published in Biochemica 3/2009, pages 13-14. ©Springer Medizin Verlag 2009