Array Capture Technology description

The Array capture system (Nimblegen) now offered by the University of Arizona Genetics Core (UAGC) allows researchers to target specific regions of the genome and analyze them using next-generation sequencing technology. The benefit of sequencing only regions of interest within the genome include smaller and less complex data sets, decreased cost (due to whole genome not being sequenced), and the ability to multi-plex various samples onto one next-gen sequencing run. There are three major steps involved in the array capture process: array design, array synthesis, and sample processing.

Array design (cost $1,300 USD, free with the purchase of 5 or more arrays) – Array capture technology is only possible with organisms for which there is reference sequence for the regions of interest. Researchers submit up to 5MB of unique sequence from the same species they wish capture. This is primarily done through submitting start and stop positions, rs#’s for SNPs, or actual sequence data. These regions of interest are then prioritized and put into a list which is then sent to Nimblegen (parent company Roche). From the submitted list of regions to be captured Nimblegen generates 75-mer overlapping probes. Not all regions are able to be designed for however, any gap in designed probes less than 200 bases will be covered in the sequencing step. This coverage of these gaps is due to the fact that the fragments hybridized to the array are between 500 and 700 bases , so although the probes are only 75mers, the fragments will hang about 200 bases over on either side of the probe. Once regions have been submitted, a design is usually returned within a week and will not move to synthesis until the design is approved by the researcher. On average about 80% of what is requested is able to be designed.

Array Synthesis and capture (cost approx $1,500 USD per array/sample) – Array synthesis takes about 2 weeks to complete. Genomic DNA samples are submitted and fractionated via nebulization to get all fragments to about 500-700 bases. These fragments are washed over the designed array where all complimentary fragments bind to their respective probes. All other fragments are washed away and discarded. The fragments that did hybridize and are left on the array are then eluted off the array using sodium hydroxide buffers and collected. Universal adapters are ligated to the captured fragments and subsequently PCR amplified. Multiple samples may NOT be run on the same array, arrays are single use only and may not be re-used, they are considered expired after hybridizing/capturing one sample.

Next Generation Sequencing (cost depends on number of samples/multiplexing and needed depth of coverage, 1 run/4 samples/500MB = $13,800 ) – Post amplification each sample is run through the regular next gen sequencing work flow. Library construction is done separately for each array/captured sample. After library construction samples are run through emulsion PCR and sequenced on the Roche 454 Genome sequencer. Each sample is aligned to a reference sequence of the regions submitted in the design phase and a separate contig file is built for each region. Multiple samples (if captured using the same design) may also be aligned to the same reference sequence in the same contig for comparative genome analysis. A genome viewer such as Apollo (or equivalent) is often used to visualize all contigs placed in the genome and is useful for depth of coverage analysis, allele calling, ect…

New from Roche NimbleGen Inc, the Sequence Capture Array system is designed to provide an enriched supply of target sequencing regions for the 454 Genome Sequencer instrument.  Custom chips are available, as well as pre-designed arrays to target the human exome (http://www.nimblegen.com/products/lit/SeqCap_HumanExome_2009_01.pdf) Targeted arrays can be designed in 385,000 and 2,100,000 feature sizes, with an average coverage of 10x fold.

Researchers interested in working on the 454 Sequencer platform are encouraged to contact Ryan Sprissler or James Shira to determine whether their projects can be improved through use of the NimbleGen system.