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Life Technologies' Ion Torrent Launches Six-hour AmpliSeq Exome Kit

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Life Technologies' Ion Torrent business has launched an exome capture kit that makes use of its AmpliSeq technology to reduce workflow time to six hours with one hour of hands-on time and enable sample inputs of 50 nanograms.

Early access user Jeremy Stuart, vice president of genomic services at Selah Genomics, told In Sequence that while he is still evaluating data quality of the kit, on an initial check, it "seems to be behaving as it should." And, from a workflow standpoint, the kit is "very simple" and "straightforward."

Stuart added that Selah's researchers are able to make the libraries in one day, as opposed to two to three days with other exome capture kits. "There's no doubt that this is much faster and easier," he said.

He said the next step is to compare the AmpliSeq exome to hybridization-based approaches like Agilent's SureSelect or HaloPlex kits and Ion's TargetSeq exome capture kit, which Life Tech will continue to offer.

Life Tech is not the only firm to address the issues of turnaround time and limited sample input for exome capture. Agilent and Illumina have each developed kits to address these needs. Using Agilent's HaloPlex users have reported a three-day turnaround time, or half that of the SureSelect product, and starting input of 200 ng (IS 3/19/2013). Meantime, Illumina has said its Nextera Rapid Capture Exome kit enables sample prep and exome enrichment for 96 samples in 1.5 days with 50 ng of input DNA.

The AmpliSeq Exome kit comprises 300,000 amplicons, which are multiplexed into 12 pools of 24,000 amplicons. Each of the 12 pools are done as an individual multiplexed PCR reaction and then are combined into a single reaction, where the standard AmpliSeq reactions take place, Andy Felton, Life Tech's senior director for product marketing, told IS.

Felton added that in designing the exome kit the main technical hurdle was being able to multiplex 24,000 amplicons while maintaining uniform coverage. He said that no major changes to the AmpliSeq library preparation had to be made, but the company simply had to optimize the One Touch II machine for library preparation and make some bioinformatics tweaks to ensure that variants were being analyzed correctly.

A key to ensuring uniform coverage, he added, was making sure that the amplicons that are physically close to each other were in separate pools. "When we tile across a particular gene, we want to get the best coverage, so we have to separate amplicons that are physically close together into separate pools," he said. This is the same principle that the company employs in its other AmpliSeq panels, but it "took that to the next level with the AmpliSeq Exome kit by going to 24,000-plex and 300,000 primer pairs," he said.

Each of the 12 multiplexed PCR reactions are done separately and can then be pooled together.

If a user runs two exomes on the Ion Proton PI chip, reagent costs are around $750 per reaction, Felton said, which includes the exome capture, library kit, template kit, sequencing kit, and chip. If the user purchases 48 reactions, it would cost around $400 per reaction, he said.

Felton said that when running a single exome on the PI chip, the kit will cover 98 percent of the bases at 10x and 96 percent of bases at 20x. Additionally, 90 percent of the bases are within 0.2x of the mean coverage, he said.

For a user running two exomes on one chip, 98 percent of the bases are covered at 10x and 94 percent at 20x; and with three exomes, 94 percent of the bases are covered at 10x and 88 percent at 20x.

This will enable users to run "trios in Mendelian research on a single PI chip," or a single exome to very deep coverage, Felton said.

Total workflow from DNA to annotated variants of an exome can be achieved in under two days, he added, including six hours for the exome library preparation and three hours of sequencing time.

Selah Genomics' Stuart said that so far the company has made eight exome libraries using the kit. While he has not yet finished analyzing the data, so far the coverage "looks good," he said, with about 90 percent of the data on target. Additionally, "coverage at 20x or higher is in the 90 percent range," a quality metric he said he looks for in exome data.

Stuart said that the kit's simplicity and speed make it an attractive option.

"One of our limiting factors, in terms of the amount of exome sequencing we'd like to do, is workflow and cost," Stuart said. "In terms of workflow, [with the AmpliSeq exome kit] we can now start processing a lot more samples," he said.

Because Selah Genomics' researchers already have experience using other of Ion's AmpliSeq panels, the transition to the AmpliSeq Exome has been very straightforward, he said.

Compared to hybridization approaches for exome sequencing, one advantage of an amplicon-based approached is that the input amount is small, around 100 nanograms, Stuart said. Additionally, the amplicon approach only requires one round of amplification — although Ion offers an optional second round of amplification — while hybridization approaches typically require two.

Stuart said that he will primarily use the AmpliSeq Exome kit in oncology applications. Initially, he will use the kit to look for germline mutations. But, he said that ultimately he is interested in running a tumor and normal exome on the same chip to look for rare somatic mutations.

He said he plans to use the AmpliSeq Exome kit in conjunction with the Proton PII chip when it is launched, which would "provide the throughput to simultaneously run a tumor and normal exome and detect lower frequency somatic mutations".

Aside from an Ion Proton, Selah Genomics also runs the Ion Torrent PGM, Illumina's MiSeq, the 454 GS FLX, and Pacific Biosciences' RS.

Stuart said Selah Genomics primarily focuses on oncology and virology. It runs the Ion AmpliSeq Comprehensive Cancer panel on the Proton and has launched a laboratory-developed test, dubbed PrecisionPath, based on Ion's AmpliSeq Hotspot panel. The firm has partnered with the Greenville Health System in South Carolina to make the test available for cancer patients (CSN 5/8/2013).

In addition, Stuart said Selah conducts clinical trial support for pharmaceutical companies using companion assays they develop on the PGM and MiSeq.

He said Selah uses the MiSeq for a range of applications, including 16S ribosomal sequencing, metagenomics, and amplicon-based sequencing, while the 454 is used almost solely for 16S sequencing, and the PacBio for de novo sequencing projects and plant projects where long reads are necessary.

Stuart said that the lab has had its Proton since May and routinely generates between 8 and 11 gigabases per run, about on par to what other customers have reported (IS 2/26/2013). Running two exome libraries in one run, it covers around 90 percent of the targets at 20x or higher, he said. "That allows us to run [exome] pairs on a single run and we get enough coverage for germline mutations," he said.

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