Bryan Hoglund

High‐resolution, high‐throughput HLA genotyping by next‐generation sequencing

The human leukocyte antigen (HLA) class I and class II loci are the most polymorphic genes in the human genome. Hematopoietic stem cell transplantation requires allele-level HLA typing at multiple loci to select the best matched unrelated donors for recipient patients. In current methods for HLA typing, both alleles of a heterozygote are amplified and typed or sequenced simultaneously, often making it difficult to unambiguously determine the sequence of the two alleles. Next-generation sequencing methods clonally propagate in parallel millions of single DNA molecules, which are then also sequenced in parallel. Recently, the read lengths obtainable by one such next-generation sequencing method (454 Life Sciences, Inc.) have increased to >250 nucleotides. These clonal read lengths make possible setting the phase of the linked polymorphisms within an exon and thus the unambiguous determination of the sequence of each HLA allele. Here we demonstrate this capacity as well as show that the throughput of the system is sufficiently high to enable a complete, 7-locus HLA class I and II typing for 24 or 48 individual DNAs in a single GS FLX sequencing run. Highly multiplexed amplicon sequencing is facilitated by the use of sample-specific internal sequence tags (multiplex identification tags or MIDs) in the primers that allow pooling of samples yet maintain the ability to assign sequences to specific individuals. We have incorporated an HLA typing software application developed by Conexio Genomics (Freemantle, Australia) that assigns HLA genotypes for these 7 loci (HLA-A, -B, -C, DRB1, DQA1, DQB1, DPB1), as well as for DRB3, DRB4, and DRB5 from 454 sequence data. The potential of this HLA sequencing system to analyze chimeric mixtures is demonstrated here by the detection of a rare HLA-B allele in a mixture of two homozygous cell lines (1/100), as well as by the detection of the rare nontransmitted maternal allele present in the blood of a severe combined immunodeficiency disease syndrome (SCIDS) patient.

A multi-site study using high-resolution HLA genotyping by next generation sequencing

The high degree of polymorphism at human leukocyte antigen (HLA) class I and class II loci makes high-resolution HLA typing challenging. Current typing methods, including Sanger sequencing, yield ambiguous typing results because of incomplete genomic coverage and inability to set phase for HLA allele determination. The 454 Life Sciences Genome Sequencer (GS FLX) next generation sequencing system coupled with conexio atf software can provide very high-resolution HLA genotyping. High-throughput genotyping can be achieved by use of primers with multiplex identifier (MID) tags to allow pooling of the amplicons generated from different individuals prior to sequencing. We have conducted a double-blind study in which eight laboratory sites performed amplicon sequencing using GS FLX standard chemistry and genotyped the same 20 samples for HLA-A, -B, -C, DPB1, DQA1, DQB1, DRB1, DRB3, DRB4, and DRB5 (DRB3/4/5) in a single sequencing run. The average sequence read length was 250 base pairs and the average number of sequence reads per amplicon was 672, providing confidence in the allele assignments. Of the 1280 genotypes considered, assignment was possible in 95% of the cases. Failure to assign genotypes was the result of researcher procedural error or the presence of a novel allele rather than a failure of sequencing technology. Concordance with known genotypes, in cases where assignment was possible, ranged from 95.3% to 99.4% for the eight sites, with overall concordance of 97.2%. We conclude that clonal pyrosequencing using the GS FLX platform and CONEXIO ATF software allows reliable identification of HLA genotypes at high resolution.

Very high resolution single pass HLA genotyping using amplicon sequencing on the 454 next generation DNA sequencers: Comparison with Sanger sequencing.

Compared to Sanger sequencing, next-generation sequencing offers advantages for high resolution HLA genotyping including increased throughput, lower cost, and reduced genotype ambiguity. Here we describe an enhancement of the Roche 454 GS GType HLA genotyping assay to provide very high resolution (VHR) typing, by the addition of 8 primer pairs to the original 14, to genotype 11 HLA loci. These additional amplicons help resolve common and well-documented alleles and exclude commonly found null alleles in genotype ambiguity strings. Simplification of workflow to reduce the initial preparation effort using early pooling of amplicons or the Fluidigm Access Array™ is also described. Performance of the VHR assay was evaluated on 28 well characterized cell lines using Conexio Assign MPS software which uses genomic, rather than cDNA, reference sequence. Concordance was 98.4%; 1.6% had no genotype assignment. Of concordant calls, 53% were unambiguous. To further assess the assay, 59 clinical samples were genotyped and results compared to unambiguous allele assignments obtained by prior sequence-based typing supplemented with SSO and/or SSP. Concordance was 98.7% with 58.2% as unambiguous calls; 1.3% could not be assigned. Our results show that the amplicon-based VHR assay is robust and can replace current Sanger methodology. Together with software enhancements, it has the potential to provide even higher resolution HLA typing.