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Thermo Fisher Scientific's Ion Torrent Provides Technical Updates, Previews New Products

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NEW YORK (GenomeWeb) – Thermo Fisher Scientific's Ion Torrent gave an update on new developments for its sequencing technology last week at the Advances in Genome Biology and Technology conference in Marco Island, Fla.

During a company workshop held on Saturday, Allan Williams, vice president of research and development for Ion Torrent, gave a preview of products in the works and new applications for the Ion PGM and Ion Proton platforms. Williams discussed topics such as the long-delayed Proton PII chip, improvements for the PI chip, Hi-Q chemistry for the Proton, isothermal amplification for HLA sequencing, AmpliSeq library prep directly from blood, and combining Cynvenio's LiquidBiopsy platform with Ion sequencing for tumor analysis.

Following Thermo Fisher's acquisition of Life Technologies last year, the firm has continued to invest in the Ion Torrent technology. "We continue to invest heavily in improving the throughput, accuracy, and read length," Williams said, with a focus on targeted resequencing and counting applications.

PII, at last?

At last year's AGBT, the company had announced plans for early-access testing and commercialization of the Proton PII chip in 2014. The chip promises to increase the throughput of the Ion Proton platform. However, challenges on the chemistry side – decreasing the size of the wells means the signal is weaker – and with the fast transfer of large amounts of data off the chip meant the firm had to redesign the chip.

Ion Torrent's R&D team has had access to the revised PII chip for about a month now, Williams said, and expects to make it available to early-access customers in the first half of this year, followed by a full commercial launch in the second half.

Internally, company researchers have achieved about 40 gigabases of data and about 350 million reads, with a peak of 115-base pair reads, from a single PII run using a human fragment library with 125-base pair inserts. The commercial chip will likely be able to generate 30 gigabases of data and 200 to 300 million 100-base pair reads.

One application the company has tested the PII chip for is targeted transcriptome sequencing with the Ion AmpliSeq Transcriptome Human Gene Expression kit, which targets about 20,000 transcripts from a variety of samples, including formalin-fixed tissue. It has seen good correlation between data from the PI and the PII chip for this kit as well as for traditional RNA-seq, Williams said.

Andy Felton, Ion Torrent's senior director of product marketing, told GenomeWeb that the read quality for the PII is currently sufficient for counting applications but may not be good enough for exome sequencing yet. "The real advantage is not so much getting better data on PII, but getting four times more throughput" than the PI chip, Williams said.

A PII run will cost less than $1,000, and 24 AmpliSeq transcriptome samples, or six RNA-seq samples, can be multiplexed in a single run.

Exploring another counting application, the firm has tested the PII chip for analyzing cell-free circulating DNA from maternal blood. In a run that used a 120-base pair insert library and multiplexed 21 samples, it was able to identify chromosomal aneuploidies.

Williams said there remains "additional headroom" to improve the read length and accuracy of the PII chip.

PI, updated

The company has also been working on an update for the PI chip that aims to increase read length and data accuracy. Changes to the structure of the chip microwells, including the chip surface chemistry, are enabling runs of 30 gigabases, with 60 to 80 million reads that have a read length peak of 400 base pairs and about 99 percent accuracy.

Ion Torrent researchers were able to use three of the new PI chips to sequence a human genome at 30-fold coverage. The chip, which is expected to be available to early-access customers in the second half of this year, "opens the door to whole-genome sequencing applications on the platform," as well as higher multiplexing of targeted panels with longer reads, Williams said.

Hi-Q for Proton

On the reagent side, Ion Torrent recently launched the Hi-Q sequencing chemistry, which improves read accuracy, for the Proton PI chip. The initial kit is for use with the Ion OneTouch 2 template preparation system, but the firm plans to release Hi-Q for PI for use with the Ion Chef system over the next few weeks. For the PGM, the Hi-Q chemistry has already been available since last year.

For the Proton, the Hi-Q chemistry has reduced the false positive rate of both SNP and indel variants by 75 percent, Williams said, from 35 false-positives per megabase to 8 per megabase. For the PGM, the gains have been similar, with a reduction of false positive indels from 4 to 1 per megabase.

Isothermal amplification for fast HLA typing

Isothermal amplification – an alternative to the current emulsion-PCR that promises greater speed and longer reads – is already available to early-access customers of the PGM with a manual protocol. The plan is to make it compatible with the Ion Chef system and to enable read lengths on the order of 600 base pairs.

With current template amplification technology, an Ion Chef run typically takes about 10 hours, which could be cut down to five hours with isothermal amplification, Williams said.

Ion Torrent has been collaborating with One Lambda, a Thermo Fisher Scientific business that focuses on HLA-typing, to test an HLA sequencing kit that uses the isothermal amplification technology and provides quick turnaround time and long reads on the order of 500 to 600 base pairs.

Last December, One Lambda launched the NXType kit, a research-use-only kit for HLA typing by sequencing on the PGM that enables full-range sequencing of HLA class I and II genes within three days.

According to Felton, another potential application of isothermal amplification, and its greater speed, is in preimplantation genetic screening.

AmpliSeq automation, applications

Ion Torrent has also been working on automating the AmpliSeq library preparation on the Ion Chef. While the run time is a few hours longer than with the manual protocol, the process involves only a few minutes of manual setup. The overall workflow, including sequencing, is 24 hours instead of 21 hours with manual library prep. AmpliSeq automation is already available to early-access customers and slated for commercialization in the second half of this year.

Another area of development has been the generation of AmpliSeq libraries directly from blood or buccal swabs, without upfront DNA extraction. The company has shown good proof of concept for this, Felton said, but there is no commercialization timeline yet.

For existing AmpliSeq kits, the company has been lowering the DNA input requirements further. Current protocols call for 10 nanograms of DNA, but company researchers have shown that with modified protocols, they can still get good performance with as little as 0.1 nanograms of input DNA, the amount of DNA contained in about 20 cells.

Ion Torrent has also been testing the use of the AmpliSeq Cancer Hotspot panel in conjunction with Cynvenios's LiquidBiopsy platform, for which Themo Fisher recently became a distributor. The LiquidBiopsy instrument can isolate circulating tumor cells, cell-free DNA, and cellular DNA from blood.

Along with improvements on the reagent side, Ion Torrent has developed new algorithms that enable the detection of low-frequency variants down to 1 percent, which it has incorporated in version 4.4 of its Ion Reporter software.

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