By Monica Heger
Life Technologies last week said it plans to work with research groups from Stanford University, Addenbrookes Hospital in Cambridge, UK, and the University of British Columbia to develop research assays for human leukocyte antigen typing on the Ion Torrent PGM.
While the collaboration is strictly research-based and Life Tech declined to provide detail on its plans for developing a clinical test, Todd Laird, vice president and general manager of transplant diagnostics for the company, said that the program is being led by Life Tech's Medical Sciences and Molecular Diagnostics groups, which are working with Ion Torrent to provide "greater focus and coordination around our efforts in this space."
Additionally, the company has submitted a separate HLA typing assay for its 3500 Dx capillary electrophoresis sequencer to the US Food and Drug Administration for 510(k) clearance.
Laird said that before bringing a PGM-based HLA typing assay to the FDA it will be important to understand the specific requirements of different types of customers in the market.
"Customers dealing with solid organs, or stem cell transplants, or [HLA typing for] a registry all have different sets of requirements" in terms of coverage, the region of focus, resolution, the ability to do many samples at once, and cost, he said.
Under the research agreement with its academic collaborators, Life Tech is providing each group with a PGM sequencer and 316 chips. They will initially use the 200-base pair read chemistry, but will have access to newer technology, such as 400-base pair reads, as it becomes available, said Laird.
Each of the groups will develop its own unique assay, Laird said, targeting a "novel region of interest" within the major histocompatibility locus depending on the focus of the research, whether it is solid organ transplantation, stem cell transplantation, or HLA typing for registry purposes.
"Rather than the common or well-defined exons," said Laird, the collaborators are working in "shorter areas [of the MHC locus] that are not common and well-defined."
Matthew Anderson, assistant director of the Histocompatibility, Immunogenetics, and Disease Profiling Laboratory at Stanford University and a participant in the program, said that using next-generation sequencing for HLA typing has the potential to yield a greater resolution genotype than other methods like Sanger sequencing.
"Having a high resolution genotype ensures the best possible match between donor and recipient," he told CSN.
While Sanger sequencing is the gold standard, because the HLA region is "the most polymorphic region in the genome," it can be difficult to determine phase with Sanger sequencing, he said.
With Sanger sequencing, when there are heterozygous sequences, "we have to do additional testing to resolve phase ambiguities so that we can tell what HLA type is on each chromosome," he said.
Next-gen offers the potential of doing one test to determine the HLA genotype and phase.
Anderson said that his lab already has a 454 GS Junior and has been developing a protocol for HLA typing on the system that it aims to move into the clinic. He decided to participate in the Ion Torrent collaboration to test the PGM's performance in this application and how it would fit within a clinical workflow.
"One of the things we learned with the 454 is that the workflow is really important, especially in a clinical lab," he said.
He said that when the team first started working with the 454 in 2009, the technology and data quality were up to par, but "the big hurdle for the clinical lab was the workflow," which was very "manual and labor intensive."
The team has since incorporated Fluidigm's Access Array to amplify and barcode many samples simultaneously, which has helped a lot,he said.
Anderson said that when the lab begins its work on the PGM, though, it will stick with Ion Torrent-specific sample-prep.
While 454 has designed its own HLA assays — the GS GType HLA MR and HR — Anderson said that he is not using those assays, but instead has designed his own primers that target the HLA regions his lab is most interested in, for example areas at the intron/exon boundaries.
"When we do clinical genotyping, there are certain areas of the gene that we know are problems, areas that we want to look at to resolve ambiguities," he said.
So far, all of the work on the Junior has been for research purposes, he said, but the plan is to validate it for clinical use. The team will continue to move forward with this effort in parallel with its work on the PGM.
One advantage of the 454 system is that its reads "are long enough to go across an entire exon in phase," he said.
The average length of the exons are around 270 base pairs, he said, so when the team first starts using the PGM with reads of 200 base pairs, it will be just shy of covering an entire exon.
Using "an instrument with shorter read lengths, you have to reconsider your [sequencing] strategy," he said. With the PGM, he said, the researchers will likely use shotgun sequencing as opposed to amplicon sequencing.
Additionally, he said that they may look at more of the gene than what they are currently analyzing with 454. The 454 assay focuses primarily on the exons, but he said they may try and see what other regions of the gene impact transplant outcome.
Different Platforms, Different Roles
Anderson said that he envisions potentially launching HLA typing lab-developed tests on both the GS Junior and Ion PGM as well as continuing to do HLA typing with Sanger sequencing.
"We may find that for certain questions, one platform may give us an advantage," he said. For instance, not every type of clinical HLA typing requires the same level of resolution, he noted. While matching for bone marrow transplantation requires very high resolution HLA genotyping, solid organ transplantation doesn't typically require the same degree of resolution, he said.
"There's a potential place for lots of different assay systems and they may each find their own niche," he said.
Life Tech's Laird agreed, noting that the company is keeping its options open as it looks to develop HLA assays for its different platforms.
Life Tech is currently moving its SeCore HLA typing assay for its 3500 CE sequencer through FDA 510(k) clearance (CSN 8/3/2011) and the company has also said that it will submit the PGM for FDA 510(k) clearance this year.
Laird declined to comment on whether an HLA assay would be submitted with the PGM, though he indicated that while this initial research into HLA typing with the PGM is for research purposes, the aim is to move it into the clinical space.
But, before bringing any HLA assay through clinical development, it will be important to understand the requirements of different customers and potentially develop different assays for these customer subsets, he said.
"We're trying to work with [the customers] to define … the design requirements. What would be the scope and features that each of the unique customer sets would require? Is one assay right, or is it a series of assays?" he said.
Additionally, he said that the HLA assays on the 3500 and the PGM could fulfill different roles. Depending on the application, the researcher or clinician might be dealing with different issues and challenges. "Some could be about resolution alone, some could be about cost and throughput, some could be about total coverage, and some could just be looking at novel and unique areas of the MHC," he said.
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