HLA-typing firm HistoGenetics said this week that it has purchased 20 of Illumina's MiSeq systems that it will incorporate into its HLA typing workflow.
HistoGenetics' CEO Nezih Cereb told Clinical Sequencing News that the MiSeqs will initially complement the firm's 50 Life Technologies 3730xl Sanger instruments for a hybrid typing approach that will utilize the MiSeq to phase variants and provide higher resolution.
HistoGenetics runs its HLA typing services out of a CLIA-certified laboratory. It is also accredited by the American Society for Histocompatibility and Immunogenetics. The laboratory has performed over 10 million sequence-based typing assays as of March 2012. Its clients include donor registries, donor centers, blood stem cell transplant centers, cord blood banks, research projects, and clinical trials.
Earlier this year, the US Food and Drug Administration cleared Life Technologies' SeCore HLA typing assay and its 3500 Dx Genetic Analyzer system (CSN 2/13/2013). Cereb said that HistoGenetics does not use this assay in its lab, nor did the FDA clearance impact its decision to incorporate MiSeq into its workflow, since the company already has its own in-house developed and well-validated assay.
Cereb said that the company decided to move into next-gen sequencing because it had reached a "bottleneck" in its sample processing. "We have a large number of samples so it was a necessity for us to either buy more 3730s or get into [next-gen sequencing].”
He said the company decided on the MiSeq because in tests it found that it had a simple and easy-to-use workflow, high-enough throughput, and fast turnaround time. He said that emulsion PCR, which is used by Roche's 454 GS FLX and GS Junior instruments and Life Technologies' PGM, was "incompatible with the high-throughput setup that we have." Additionally, the company has been collaborating with Pacific Biosciences to use its RS system in research, but he said that the throughput of that system was not yet high enough.
Initially, the MiSeq will be incorporated into the Sanger workflow to analyze the antigen recognition sites on the HLA-related genes that are necessary to determine histocompatibility.
"Sanger will let us know what kind of variation there is and MiSeq will let us know phasing information," Cereb said. "With Sanger sequencing, we need to do additional experiments to get phasing of the exons," he added.
Aside from determining phase, the MiSeq will also add a level of quality assurance, Cereb said.
Cereb said that the company already has well-validated reagents that it uses on its Sanger systems to analyze the HLA antigen recognition sites. These include exons 2 and 3 for HLA-A, -B, and -C genes and exon 2 for DRB1, DRB3/4/5, DQB1, DQA1, DPA1, and DPB1 genes.
He said that the company has already designed a panel for the MiSeq for these gene regions. With the hybrid approach, all samples will be analyzed on both the Sanger system and MiSeq simultaneously, and then the data will be combined to determine variation and phase information, he said.
He anticipated that incorporating the MiSeq into the workflow would speed up turnaround time while also yielding a higher resolution. Cereb said he had not yet determined how the MiSeq introduction will impact cost, but anticipated there would be little change.
The company will likely begin incorporating the MiSeq workflow into its commercial offerings by the end of the summer, Cereb said. Using the MiSeq, he said the goal will be to provide all customers with "Gold Level" typing. Currently, the company offers two different levels of typing: a lower resolution, dubbed Silver, and a higher resolution, dubbed Gold.
The Gold level includes the phase information, which is possible to get without the MiSeq, but is more time consuming and labor intensive. "We have a daily volume of 5,000 to 6,000 samples," Cereb said. "So we're aiming for that kind of throughput. By using MiSeq, we can fasten that process."
Over time, as the MiSeq becomes established in the lab, other applications would be introduced in step-wise manner. For instance, he said, "we may try to look at the whole HLA region, not only exons 2 and 3." Or the company may do extended haplotypes, evaluate other immune response-related genes that may be important for tissue matching or involved in graft versus host disease.
Whether the MiSeq will eventually replace Sanger sequencing completely for HLA typing remains to be seen, said Cereb. "This will be a stress test for MiSeq," he said. "No one has experience doing millions of sequence-based typing using the MiSeq. … It's hard to say whether the accuracy will be good enough." Cereb said that the company aims for 100 percent accuracy, and that 99 percent accuracy is "not good enough."
Even if the MiSeq proves itself, he said, there may still be a role for Sanger sequencing, particularly as a quality control system.