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Aim The Roche GS GType Assay is an amplicon based NGS approach for high-throughput, high-resolution HLA genotyping. We evaluated the ability of this assay together with Conexio Assign MPS software to define high resolution HLA alleles by sequencing 59 samples and examining the concordance between prior typing information and the GType Assay. Our goal was to determine whether this single method would significantly reduce ambiguity in the assignment of HLA class I and II alleles. Methods PCR amplification was performed using the Very High Resolution HLA GType Assay which consists of three 96-well plates (MR, HR and VHR) containing dried-down exon-specific primers. The MR plate contains 8 primer pairs amplifying exons 2 and 3 of HLA-A, -B, and -C, and exon 2 of DRB1 (plus DRB3, 4, 5) and DQB1. The HR plate contains 6 primer pairs amplifying exon 4 of HLA-A, -B, and -C, exon 2 of DPB1 and DQA1, and exon 3 of DQB1. The VHR plate amplifies 8 exons covering exons 1 and 5 of HLA-A, -B, -C, exon 6 and 7 of HLA-C, exon 2 of DPA1, and exon 3 of DRB1 (plus DRB3, 4, 5). Using 11 unique multiplex identifiers (MIDs) allows pooling 5 samples with 22 amplicons and a negative control on one GS Jr. run. Resulting FNA files were analyzed by Conexio software. Results Sequence analysis of 59 individual samples (55 bone marrow donors/recipients, 1 lung recipient, and 3 controls) including 18 rare alleles demonstrated 100% concordance with genotypes previously high resolution typed by a combination of Sanger sequencing, sequence-specific-primer (SSP), and sequence-specific-oligonucleotide probe (SSOP) methodologies. Increased sequence coverage allowed us to detect additional variants (silent substitutions) providing third field genotyping information. Some residual genotype ambiguity was present, largely due to incomplete exon coverage or incomplete reference sequence in the IMGT/HLA database. No additional methodology was required to obtain the high resolution allele. Conclusions Our results provide support for the use of amplicon based high-throughput sequencing for high-resolution HLA genotyping by NGS. The technique is accurate, and results in reduced genotype ambiguity when compared to standard Sanger sequencing; however, further primer design development and genotyping software are necessary before application for clinical use. [ABSTRACT FROM AUTHOR]

Aim: Genotyping of DRB1 and DRB3/4/5 is performed in our lab using generic DRB PCR primers, followed by sequencing on the 454 GS FLX and use of Conexio Genomics software. During genotyping homozygous cell lines, we detected additional sequences at about 7% of the read depth of the reported true allele. These artifacts appeared to be PCR crossovers between the DRB1 and DRB3/4/5 sequences. The crossovers usually had one or more mismatches with the IMGT database but occasionally matched a very rare allele (e.g. DRB1∗03:42). We performed a study to determine if these sequences were in vitro artifacts and to determine if some rare DRB alleles in the IMGT database might, in fact, be in vitro artifacts that had been submitted. Methods: To test for in vitro artifact formation during late cycles of genomic PCR, we amplified the DRB loci from 21 homozygous cells lines under both our standard PCR conditions (35 cycles) and a reduced number of cycles (28), followed by 454 sequencing. Using the same PCR conditions, we also sequenced the DRB loci of four samples, each of which had previously been identified as having a novel DRB allele that was subsequently submitted to the IMGT database. Results: For all homozygous 21 cell lines, amplification for 35 cycles gave low abundance sequences as possible “second alleles.” In 36% of the cases, these artifactual sequences corresponded to named alleles. Such sequences were not detected with amplification for 28 cycles. In the case of 4 samples with rare alleles previously submitted to the IMGT, we found that 3 were true alleles. The fourth, while not a crossover product, contained a sequencing error which was revealed by our 454 HLA genotyping assay. Conclusions: Formation of in vitro crossover products of the DRB loci can occur in late cycles of PCR. PCR conditions have been identified that minimize generation of these in vitro artifacts. Clonal sequencing on the 454 GS FLX is a valuable method for revealing both these artifacts and traditional sequencing errors. [Copyright &y& Elsevier]

Aim: We have previously reported the development of very high resolution (VHR) HLA genotyping, designed to resolve the majority of both the “common and well documented alleles” and the null alleles using the 454 GS FLX Sequencing System and Conexio genotyping software. Amplicon library construction was performed using 22 PCR fusion primers, containing genome target specific sequence with 454 sequencing adaptors. The VHR assay performed well but required extensive handling of individual amplicons. To simplify the workflow, we prepared the amplicon libraries by three alternative procedures. Methods: PCR amplicons were generated from 10 cell lines for: HLA-A/B exons 1-5, HLA-C exons 1-7 and some intronic class I regions; DPA1, DPB1, DQA1 exon 2; and DQB1, DRB exons 2 and 3. Amplification required 22 pairs of fusion primers (14 primer pairs in the GS GType HLA primers plates from 454, plus 8 amplicons in a third plate) with 10 Multiplex Identifiers (MIDs), or 22 primer pairs each containing, at the 5′end, a universal sequence which matched the 3′ end of a set of primers that contained 10 MIDs and the 454 sequencing adaptors. Amplicons derived from fusion primers were purified, quantified, diluted to a standard concentration, then pooled OR pooled prior to the other operations. Amplicons generated by the four primer system on the Fluidigm Access Array™ were handled by the latter procedure. Pools of amplicons were sequenced on the GS FLX and genotyped with Conexio software. Results: All three procedures resulted in comparable genotyping (concordance up to >98%). Pooling amplicons immediately following the genomic PCR gave a reduction in the number of pipetting steps necessary to produce an amplicon library by ∼80% and ∼90% using fusion primers and the four primer system, respectively. Conclusions: Pooling amplicons early in the 454 library preparation procedure greatly simplifies the workflow of the 22 primer VHR HLA genotyping assay for fusion primers as well as the 4 primer system. Holcomb: Roche: Employee. Hoglund: Roche: Employee. Williams: Roche: Grant Research. Erlich: Roche: Employee. [Copyright &y& Elsevier]