A new publication in PLoS One from authors at Anthony Nolan’s Research Institute describes a feasibility study for HLA typing using SMRT® Sequencing. The research institute, where the world’s first bone marrow registry started in 1973, is part of the UK-based charity dedicated to improving the outcomes of bone marrow transplantation. Scientists at Anthony Nolan are leaders in HLA typing, which is an important step in matching a bone marrow or stem cell donor to a patient in need.
The Anthony Nolan team adopted the PacBio® system last year, and this publication reflects its efforts to test and establish the new standards for HLA typing. In “HLA Typing for the Next Generation,” from lead author Dr Neema Mayor and senior author Professor Steven Marsh, they discuss the sequencing and analysis of various types of representative samples typically seen in their pipeline.
The scientists’ goal in implementing long-read sequencing was to more accurately and comprehensively characterize the complete HLA class I genes of the major histocompatibility complex (MHC) region, a highly polymorphic region of the human genome. More than 13,200 alleles have been identified to date for the six classical HLA genes, with 9,900 being variants of HLA class I genes (full-length class I genes are as long as 3 kb, while some class II genes are longer than 10 kb). “A common problem encountered is the inability to determine the phase of polymorphisms identified in a single individual, a problem that is exacerbated by the extensive genetic diversity seen in HLA genes,” the authors write. “The high workload, cost and time required to generate true allele-level HLA typing using current methods makes it preclusive for most histocompatibility laboratories.”
The scientists note that many HLA typing labs have adopted next-generation sequencing platforms, but that short read lengths are a limiting factor because they require the generation of lots of overlapping sequences that could lead to alignment errors during assembly. “It is possible that in a system as polymorphic as the HLA genes, incorrect phasing of SNPs that are distant to each other across the gene but otherwise show complete homology could result in an incorrect allele being assigned,” the authors caution.
To overcome these problems, the authors tested SMRT Sequencing on seven samples. HLA class I genes for each individual were pooled and samples prepared in SMRTbell format for single molecule sequencing. Each sample generated coverage of at least 150-fold for each allele after adjusting for PCR amplification imbalances. All full-length consensus sequences, derived from continuous long reads using the consensus caller Quiver, showed a mean quality value of at least 70.
In addition to spot-on accuracy (“absolute concordance” between the PacBio sequence and confirmatory sequencing, the authors note), SMRT Sequencing identified novel alleles and even corrected an error in the existing reference sequence database.
“The SMRT DNA sequencing method from Pacific Biosciences has overcome the need to sequence multiple overlapping fragments allowing sequencing of a single fragment in excess of 20 kb in one sequencing reaction,” Mayor et al. write. They also note that the process was completed within three working days using a two-hour movie to collect sequence data — less time than it takes for existing HLA typing technologies.
The scientists say that additional studies will be required to test SMRT Sequencing on a broader range of samples and with higher levels of multiplexing, but the results in this project were very promising. “The implications of this technology in the field of HLA typing could be enormous, allowing for true allelic HLA typing in a single experimental set-up and making redundant the need for multiple experiments on different typing platforms, cross-referencing of results and/or the need for re-sequencing using an allele specific protocol,” they write. “This could ultimately result in considerable improvement in survival rates post transplant.”
For more information on how Anthony Nolan researchers are using SMRT Sequencing, check out this webinar with Neema Mayor.
For more information on Anthony Nolan and its life-saving work, go to www.anthonynolan.org.