By Julia Karow
Portuguese diagnostic services lab IPATIMUP Diagnostics is in the process of converting nearly 20 Sanger sequencing-based genetic tests to the Ion Torrent PGM — a move that has already resulted in faster turnaround times and could lead to lower costs in the future.
Last month, the lab started offering its first test on the PGM, a breast cancer susceptibility test that sequences the BRCA1 and BRCA2 genes. The PGM replaces a Sanger-based BRCA1/2 test that the lab has offered for several years. It is currently validating a test for cardiomyopathies on the new platform and by the end of this year, it plans to move all its capillary electrophoresis sequencing tests — most of them for oncology — over to the Ion Torrent platform, including several single-gene tests.
A unit of the Institute of Molecular Pathology and Immunology of the University of Porto, the non-profit lab, which is CAP-accredited and ISO 9001:2008-certified, provides a variety of diagnostic tests in the areas of pathology, molecular genetics, and forensics and genetic identification. Last year, it performed almost 17,000 tests, the vast majority of them in pathology. Almost 4,000 of those were genetic tests, more than half of them tumor mutation screenings, and most were sequencing-based.
About 90 percent of IPATIMUP's diagnostic services are for hospitals and clinics in Portugal, though the lab also provided pathology consultation services for difficult cases from 18 countries last year.
José Luis Costa, who is responsible for genomics technologies at IPATIMUP Diagnostics, told Clinical Sequencing News that the lab started to consider next-gen sequencing early last year.
At that time, Roche 454's GS FLX and GS Junior platforms were well-established in the market and Ion Torrent's PGM was about to be launched in Europe, while no date for the introduction of Illumina's MiSeq in Europe was available yet, Costa recalled.
From discussions with collaborators, the lab knew that the 454 platform still had some problems with homopolymers, and their impression was that "Roche was not really trying to solve them," Costa said. "So we thought, let's try a new thing that has a lot of capacity and a lot of possibilities to grow."
In addition, it was important for the lab to have good user support, which Life Tech was able to provide for the Ion Torrent through its Applied Biosystems unit. "When we buy such a big machine, it's important for us as a user to have support for the machine, someone we can call, and five minutes later, either the person is here or they can give us an answer on the phone," Costa said. Illumina did not offer such support in Portugal, at least not at the time. "We always had to contact someone in the Netherlands or the UK, and that's not practical," he said. Also, the MiSeq "came a bit late" for the lab's needs.
The Ion Torrent PGM arrived at IPATIMUP last August. To validate the BRCA1/2 test on the new machine, it sequenced a number of samples in parallel on Sanger and PGM, including all positive cases from its internal library of cases. To amplify the genes for both platforms, it uses in-house-developed multiplexed PCR.
The PGM was able to call all mutations and polymorphisms that the Sanger platform detected but also produced a number of false-positive results. In addition, Costa said the platform has "some problems" with homopolymers, though he thinks these are less serious than on the 454 platform. Since the lab uses the PGM "as more of a screening tool" and confirms all positive results with Sanger sequencing, neither false positives nor homopolymer errors are big issues. "What we don't want are false negatives," Costa said.
He also noted some "difficult results" that arise from the enzymatic digestion of amplicons, but since those are systematic in nature, the lab can deal with them by using filters during the data analysis.
In February, the lab switched its BRCA1/2 test to the Ion Torrent platform, cutting the turnaround time from two to three months to an average of two to three weeks. In addition, the new test requires less hands-on time, saving labor. Around the same time, the lab became certified by Life Tech as a service provider for the PGM, for which it had to pass a number of tests using control samples and PCR primers provided by the company.
For the new BRCA1/2 test, about three to four days are devoted to sample preparation and sequencing, two to three days to data analysis, and the remainder to validation.
Depending on how many samples need to be analyzed, the lab runs the test on the 314 or the 316 chip, allowing it to multiplex between five and 16 samples per run. Right now, there is no need for the higher-throughput 318 chip, Costa said, though that might change in the future if several different tests can be combined on the same chip.
The cost of the BRCA1/2 test is "more or less the same" as for the Sanger-based test at the moment, but Costa said he expects costs to drop through a combination of price cuts for chips and reagents and increases in chip capacity, which will allow for higher multiplexing.
At the moment, the lab is validating its cardiomyopathy test on the PGM in a similar way to the BRCA1/2 test. That test includes almost 10 genes, of which only those that are ordered by a doctor are sequenced.
By the end of the year, IPATIMUP plans to switch all of its Sanger-based tests — close to 20 in total, many of them single-gene tests — to the PGM. Even for single-gene tests this would make sense, Costa said, "as long as we can multiplex them."
After that, the lab will look into developing additional tests for more complex disorders, such as mitochondrial diseases or X-linked mental retardation, he said, although it has not decided on specific tests yet. For those, it will likely use Ion Torrent's AmpliSeq technology because multiplexed standard PCR will no longer be possible for large gene panels.
Right now, each genetic test needs to be validated individually on the PGM, which Costa believes "does not make a lot of sense nowadays." A better way would be to validate the sequencing platform, independent of what specific genes are sequenced, and he looks forward to new guidelines from CAP later this year that will address this.
Eventually, Costa said, all testing will probably move to exome sequencing, which would require an instrument with higher throughput. "I think the move [to exome sequencing] has started," he said. "As a small country, we always take a bit longer to get there. But I would say that in three to four years, our lab will be there."
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