Cherie Holcomb

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.

16(th) IHIW : review of HLA typing by NGS.

Human leucocyte antigen (HLA) genes play an important role in the success of organ transplantation and are associated with autoimmune and infectious diseases. Current DNA‐based genotyping methods, including Sanger sequence‐based typing (SSBT), have identified a high degree of polymorphism. This level of polymorphism makes high‐resolution HLA genotyping challenging, resulting in ambiguous typing results due to an inability to resolve phase and/or defining polymorphisms lying outside the region amplified. Next‐generation sequencing (NGS) may resolve the issue through the combination of clonal amplification, which provides phase information, and the ability to sequence larger regions of genes, including introns, without the additional effort or cost associated with current methods. The NGS HLA sequencing project of the 16IHIW aimed to discuss the different approaches to (i) template preparation including short‐ and long‐range PCR amplicons, exome capture and whole genome; (ii) sequencing platforms, including GS 454 FLX, Ion Torrent PGM, Illumina MiSeq/HiSeq and Pacific Biosciences SMRT; (iii) data analysis, specifically allele‐calling software. The pilot studies presented at the workshop demonstrated that although individual sequencers have very different performance characteristics, all produced sequence data suitable for the resolution of HLA genotyping ambiguities. The developments presented at this workshop clearly highlight the potential benefits of NGS in the HLA laboratory.

Next-Generation DNA Re-Sequencing Identifies Common Variants of TYR and HLA-A that Modulate the Risk of Generalized Vitiligo via Antigen Presentation

TO THE EDITOR Generalized vitiligo (GV) is a common autoimmune disease resulting from the destruction of melanocytes in the involved areas, epidemiologically associated with elevated prevalence of certain other autoimmune diseases (Picardo and Taı̈eb, 2010). In a recent genome-wide association study (GWAS) of GV, carried out in European-derived whites (EUR), we identified 16 loci that contribute to GV risk (Jin et al., 2010a,b; Birlea et al., 2011). Within the major histocompatibility complex (MHC), a major GV association signal localized to HLA-A, in the class I gene region. Outside the MHC, the strongest GV association was with TYR, which encodes tyrosinase (TYR). At HLA-A, the most highly associated single-nucleotide polymorphism (SNP) was rs12206499 (P1⁄41.24 10 , odds ratio (OR)1⁄41.58), which tags HLA-A*02 (r1⁄40.964, D0 1⁄41.0) in the EUR population (Jin et al., 2010a). At TYR, the strongest association was with rs1393350 (P1⁄43.24 10 , OR1⁄4 0.65), which is in linkage disequilibrium (r1⁄40.79. D0 1⁄41) with a common nonsynonymous TYR variant, R402Q (rs1126809; Giebel et al., 1991). TYR is the major GV autoimmune antigen (Song et al., 1994), and TYR peptide antigens are predominantly presented on the melanocyte surface by HLA-A*02:01 (Brichard et al., 1993). The TYR R402Q substitution results in a temperaturesensitive tyrosinase TYR polypeptide (Tripathi et al., 1991) that is retained in the endoplasmic reticulum, is hypoglycosylated, and is preferentially degraded (Toyofuku et al., 2001). We therefore suggested that TYR R402Q might protect from GV by reducing the availability of TYR peptide for antigen presentation by HLA*02:01 (Jin et al., 2010a). To specifically define the HLA-A subtype associated with GV, we performed next-generation DNA re-sequencing of HLA-A exons 2, 3, and 4, which contain the sequence variations that define HLA-A subtypes (http://www.ebi.ac.uk/imgt/hla/), in 20 unrelated EUR GV patients. To maximize information, each patient was selected on the basis of homozygosity for rs12206499-G, which tags the GV-associated HLAA*02 type (r1⁄40.964, D0 1⁄41.0) in the EUR population (Jin et al., 2010a). As shown in Supplementary Table S1 online, of the 20 patients sequenced, 18 were homozygous HLA-A*02:01/ *02:01, 1 was heterozygous HLAA*02:01/*02:20:01, and 1 was HLAA*02:06:01/*02:30. (The allelic typing system does not distinguish between the common HLA-A*02:01:01:01 allele and the very rare HLAA*02:01:01:02L allele; to be conservative, here we represent these alleles using the standard four-digit nomenclature, HLA-A*02:01). This distribution of HLA-A*02 subtypes is similar to that in EUR control populations (http://www. ncbi.nlm.nih.gov/gv/mhc/main.cgi?cmd= init; http://www.allelefrequencies.net/), indicating that the predominant HLAA*02 GV risk subtype is HLA-A*02:01. To specifically identify TYR gene variants associated with GV, we carried out next-generation re-sequencing of 10.4 kb across the TYR locus, including 2.4 kb of promoter, the five exons and adjacent intron sequences, and 3.2 kb downstream, in 114 unrelated EUR GV patients. As shown in Supplementary Table S2 online, we identified 31 SNPs, with no indels or other rearrangements. Bioinformatic analyses predicted that only three of these are functionally significant. One, rs61754388 (T373K), is a known oculocutaneous albinism type 1 (OCA1) mutation (Spritz et al., 1990), seen in a single heterozygote, consistent with the expected carrier frequency of OCA1. The other two, rs1042602 (S192Y) and rs1126809 (R402Q), are common non-synonymous polymorphisms (Giebel and Spritz, 1990; Giebel et al., 1991) that occur almost exclusively in EUR populations (http://www.ncbi. nlm.nih.gov/projects/SNP/). In the GWAS, imputed rs1126809 genotypes demonstrated strongly protective association of the variant A allele with GV (P1⁄41.36 10 , OR1⁄4 0.65), whereas rs1042602 showed no association (P1⁄4 0.611, OR1⁄40.98). However, when both variants were considered simultaneously by logistic regression (Table 1A), rs1042602 was also significantly associated with GV (P1⁄41.23 10 ); when analyzed individually, association of rs1042602 was masked because its protective allele A is in linkage disequilibrium with the risk allele G of rs1126809; r between the two SNPs is 0.15. Furthermore, haplotype analysis of rs1042602 and rs1126809 (Table 1B) showed that, compared with the ancestral reference haplotype C-G, the other three rs1042602-rs1126809 variant haplotypes define a protective haplotypic series, the double-variant A-A haplotype reducing GV risk 3.7-fold: A-G, OR 0.83; C-A, OR 0.61; and A-A, OR 0.27. The overall

Prediction and diagnosis of bladder cancer recurrence based on urinary content of hTERT, SENP1, PPP1CA, and MCM5 transcripts

BackgroundIdentification of urinary biomarkers for detection of bladder cancer recurrence would be beneficial to minimize the frequency of cystoscopy. Our objective was to determine the usability of urine content of mRNA in the detection and prediction of bladder cancer recurrence.MethodsWe analyzed 123 prospectively cross-sectional collected urine samples from 117 patients with bladder cancer (12 incident cancers and 111 control visits). We used biopsies from cystoscopies as diagnostic criteria for recurrence, and followed the patients for a median time of 28.5 months (range 0-44 months). We measured the levels of hTERT, SENP1, PPP1CA, and MCM5 mRNA in urine by q-RT- PCR.ResultsWe found significant differences in urinary content of hTERT (p < 0.001), SENP1 (p < 0.001), MCM5 (p < 0.001), and PPP1CA (p < 0.001) transcripts, when comparing urine samples from patients with and without tumor present in the bladder. We obtained sensitivity and specificity values for hTERT: 63/73, SENP1: 56/78, MCM5: 63/66, and PPP1CA: 69/63, respectively. Including follow-up data resulted in sensitivity and specificity values for hTERT: 62/84, SENP1:53/84, MCM5: 61/73, and PPP1CA: 65/66. Interestingly, at non-tumor visits the urinary content of especially hTERT (p = 0.0001) and MCM5 (p = 0.02) were significantly associated with subsequent tumour recurrence. Combining the markers with cytology improved the detection. The best combination was hTERT and cytology with a sensitivity of 71% and a specificity of 86% after follow-up. Further prospective validation or registration studies needs to be carried out before clinical use.ConclusionsWe could use the urinary content of hTERT, SENP1, PPP1CA, and MCM5 to detect bladder cancer recurrence. All markers showed a higher sensitivity than cytology. The detection rate improved when including cytology results, but also the combination of hTERT and MCM5 increased the detection rate. Furthermore, hTERT and MCM5 levels predicted subsequent tumor recurrences.

Minimum information for reporting next generation sequence genotyping (MIRING): Guidelines for reporting HLA and KIR genotyping via next generation sequencing

The development of next-generation sequencing (NGS) technologies for HLA and KIR genotyping is rapidly advancing knowledge of genetic variation of these highly polymorphic loci. NGS genotyping is poised to replace older methods for clinical use, but standard methods for reporting and exchanging these new, high quality genotype data are needed. The Immunogenomic NGS Consortium, a broad collaboration of histocompatibility and immunogenetics clinicians, researchers, instrument manufacturers and software developers, has developed the Minimum Information for Reporting Immunogenomic NGS Genotyping (MIRING) reporting guidelines. MIRING is a checklist that specifies the content of NGS genotyping results as well as a set of messaging guidelines for reporting the results. A MIRING message includes five categories of structured information - message annotation, reference context, full genotype, consensus sequence and novel polymorphism - and references to three categories of accessory information - NGS platform documentation, read processing documentation and primary data. These eight categories of information ensure the long-term portability and broad application of this NGS data for all current histocompatibility and immunogenetics use cases. In addition, MIRING can be extended to allow the reporting of genotype data generated using pre-NGS technologies. Because genotyping results reported using MIRING are easily updated in accordance with reference and nomenclature databases, MIRING represents a bold departure from previous methods of reporting HLA and KIR genotyping results, which have provided static and less-portable data. More information about MIRING can be found online at miring.immunogenomics.org.

Next-Generation HLA Sequencing Using the 454 GS FLX System

Next-generation sequencing (NGS) of HLA class I and II loci (HLA-A, HLA-B, HLA-C, DRB1, DRB3, DRB4, DRB5, DQA1, DQB1, DPB1) is described here in detail using the 454 Life Sciences GS FLX System and Titanium chemistry. An overview of the protocol with our experience on sequence performance efficiencies, read depth and ambiguity analyses using the GS FLX System are also presented. A total of 14 HLA primer pairs with multiplex identifiers (MIDs) are used in clonal, amplicon-based pyrosequencing of up to 44 samples per plate using the GS FLX. Genotype assignment and ambiguity reduction -analysis is performed using Conexio Assign ATF 454 software. Clonal NGS gives a significant reduction in genotyping ambiguity during analysis of the highly complex HLA system.

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.

16(th) IHIW: immunogenomic data-management methods. report from the immunogenomic data analysis working group (IDAWG).

The goal of the immunogenomic data analysis working group (IDAWG) is to facilitate the consistent analysis of HLA and KIR data, and the sharing of those data among the immunogenomic and larger genomic communities. However, the data management approaches currently applied by immunogenomic researchers are not widely discussed or reported in the literature, and the effect of different approaches on data analyses is not known. With ASHIs support, the IDAWG developed a 45 question survey on HLA and KIR data generation, data management and data analysis practices. Survey questions detailed the loci genotyped, typing systems used, nomenclature versions reported, computer operating systems and software used to manage and transmit data, the approaches applied to resolve HLA ambiguity and the methods used for basic population‐level analyses. Respondents were invited to demonstrate their HLA ambiguity resolution approaches in simulated data sets. By May 2012, 156 respondents from 35 nations had completed the survey. These survey respondents represent a broad sampling of the Immunogenomic community; 52% were European, 30% North American, 10% Asian, 4% South American and 4% from the Pacific. The project will continue in conjunction with the 17th Workshop, with the aim of developing community data sharing standards, ambiguity resolution documentation formats, single‐task data Management tools and novel data analysis methods and applications. While additional project details and plans for the 17th IHIW will be forthcoming, we welcome the input and participation in these projects from the histocompatibility and immunogenetics community.