Recent technological advances in sequencing systems have rapidly increased raw sequence output. However, these next-generation sequencing systems do not currently have the throughput to sequence the whole human genome cost-effectively. Thus, they require that the complexity of genomic DNA samples be reduced to a manageable subset prior to sequencing. The prevailing method for complexity reduction has been the preparation of amplicons by parallel, multiplex or long range PCR amplification. Such PCR methods have however 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 of these limitations, the bottleneck for sequencing projects has shifted to sample preparation. To address this problem, Roche NimbleGen has developed a microarray hybridisation-based Sequence Capture Technology that utilises high-density oligonucleotide microarrays as a programmable genomic selection device to allow targeted sequencing of subsets of the genome. These genome subsets can be exons, disease-associated regions, quantitative trait loci, promoters and enhancers, and other targeted regions. The revolutionary and simple workflow of NimbleGen Sequence Capture technology enables the isolation of megabase regions in as little as one week and eliminates the cost, labour and infrastructure required for large-scale PCR experiments.  

SEQUENCE CAPTURE PERFORMANCE  
The performance characteristics of the new system can be judged from the first peer-reviewed publications [1,2,3] on the technology.  The currently available NimbleGen Sequence Capture service offers data quality equal or higher than that published. The actual performance depends on the sizes and exact locations of the target regions; pilot projects involving both Sequence Capture and sequencing are recommended to accurately determine how the technology works for the user's region of interest. Updates on the technology’s performance, are available at www.nimblegen.com/seqcap.

• In one demonstration of the power of the new system, a single-array experiment covering 5Mb target regions was carried out, followed by a single Genome Sequencer FLX run producing ~100Mb total sequencing data. The majority of sequencing reads represented selected target regions (typically >70%). While the coverage typically depends on the composition of the target regions, ~8X median coverage can be achieved for exon-sized regions and ~18X median coverage can be achieved for a single 5Mb contiguous genomic region [Figure 2]. The sequence coverage will increase for smaller cumulative target region sizes, if the same amount of sequencing runs is performed. For example, an experiment covering a 500kb contiguous target region followed by a single Genome Sequencer FLX run yielded >90X median coverage. In research requiring small regions, the Sequence Capture technology combined with 454 sequencing using only a portion (1/2 to 1/16) of the picolitre plate will generate sufficient reads for sequencing applications.

REFERENCES
1. Albert TJ, et al. Direct selection of human genomic loci by microarray hybridization. Nature Methods 2007 Nov; 4(11):903-5.  
2. Okou DT, et al. Microarray-based genomic selection for high-throughput resequencing. Nature Methods 2007 Nov; 4(11):907-9.  
3. Hodges E, et al. Genome-wide in situ exon capture for selective resequencing. Nature Genetics 2007 Dec; 39(12):1522-7.

THE AUTHOR
ROCHE NIMBLEGEN
Madison, WI,
USA