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HistoGenetics to Incorporate PacBio Sequencing into Clinical HLA Typing Workflow

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NEW YORK (GenomeWeb) – HLA typing firm HistoGenetics said it is working to validate the Pacific Biosciences' RS II system for its clinical HLA typing workflow, initially to help resolve ambiguous types and for urgent cases that require a fast turnaround time. The companies announced last week that HistoGenetics had purchased two RS II systems.

HistoGenetics entered the next-generation sequencing space last year when it purchased 20 of Illumina's MiSeq systems in order to alleviate the bottleneck of samples that were building up. Initially, the firm ran samples on the MiSeq in parallel with its previous Sanger-based technique to validate the new technology. Now, the MiSeqs are the firm's main workhorses for HLA typing for donor registries, HistoGenetics CEO Nezih Cereb told Clinical Sequencing News. Sanger sequencing is still used to confirm matches between patients and donors.

HistoGenetics runs a targeted sequencing HLA typing panel that includes 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. It processes between 80,000 and 100,000 samples per month, primarily from donor registries, and has the capacity to process 150,000 samples per month, Cereb said.

Cereb said that the PacBio RS II systems will complement the MiSeq technology. Last year, the company had been collaborating with PacBio for research purposes, but at the time Cereb said that the system lacked the necessary throughput. PacBio has said it plans to increase throughput to 1 gb of sequence data per SMRT cell this year. Recently, it published data from de novo sequencing and assembly of a 54x human genome using its P5-C3 chemistry and achieved 608 mb of data per SMRT cell.

Cereb said that the long read lengths of the PacBio machine, around 8.5 kb on average, will yield greater resolution for phasing, particularly in difficult cases. Additionally, the system has a fast turnaround time, which is critical in some transplant cases. In particular, the long reads will be able to more accurately phase exons 2 and 3 of the HLA-A, -B, and –C genes.

Although HistoGenetics recently upped its orders of MiSeq systems, giving it, at 33 systems, the largest fleet of MiSeqs of any of Illumina's customers, Cereb said that due to the MiSeq's shorter read lengths, there are "limitations with phasing, which could be resolved by the PacBio technology."

Currently, MiSeq offers paired-end reads of 300 bp, while PacBio produces average read lengths up to 8.5 kb with plans to increase average reads to over 10 kb this year.

Right now, HistoGenetics applies bioinformatics techniques to MiSeq sequencing data and also uses Sanger sequencing to confirm the phase and coverage of the exons for clinical purposes — when a patient matches to a potential donor. However, PacBio technology could potentially resolve those cases much more quickly, Cereb said. "We don't want to give typing results based on statistics," he said. "We need to make sure the phasing between exons two and three is accurate and no insertion or deletion is missed." He envisions initially incorporating the RS II for "urgent cases" or for cases that are especially difficult to resolve.

By combining MiSeq's high throughput with PacBio's long reads, the "aim is to eliminate Sanger sequencing."

Cereb said HistoGenetics would likely begin incorporating the PacBio technology into its workflow in about one month. "We're still doing validation," he said.

Additionally, he said the firm is testing in a research setting the ability of the RS II to sequence the entire HLA gene.

PacBio officials declined to comment on HistoGenetics' purchase and use of the RS II for HLA typing, but earlier this month CEO Mike Hunkapiller said during a conference call discussing the company's first quarter results that there is a "growing interest" among its customers in doing HLA typing, adding that it could "become a significant opportunity going forward."

Hunkapiller said that the long reads obtained with PacBio sequencing make it well suited for sequencing the complex HLA region. "A lot of the key opinion leaders within the HLA community have realized that what they really need is to look at the entire gene as a whole and all the elements of the HLA families," Hunkapiller said. "And they realized they can't do that properly with any of the short-read sequencing or probing technologies."

Going forward, Hunkapiller said that he thinks the firm will have a "strong position" in the HLA typing market given that it is "already in the position to demonstrate that even in a production environment, we can do full-length HLA Class I sequencing for a lower cost than people can do the isolated exons."

He added that PacBio would solidify its place first in the HLA market for research and eventually for diagnostics "as we develop [the application] and in the end go through the clearance process for a diagnostic type test."

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