Deborah Ferriola

Next-generation sequencing: the solution for high-resolution, unambiguous human leukocyte antigen typing

Human leukocyte antigen (HLA) typing has been a challenge for more than 50 years. Current methods (Sanger sequencing, sequence-specific primers [SSP], sequence-specific oligonucleotide probes [SSOP]) continue to generate ambiguities that are time-consuming and expensive to resolve. However, next-generation sequencing (NGS) overcomes ambiguity through the combination of clonal amplification, which provides on-phase sequence and a high level of parallelism, whereby millions of sequencing reads are produced enabling an expansion of the HLA regions sequenced. We explored HLA typing using NGS through a three-step process. First, HLA-A, -B, -C, -DRB1, and -DQB1 were amplified with long-range PCR. Subsequently, amplicons were sequenced using the 454 GS-FLX platform. Finally, sequencing data were analyzed with Assign-NG software. In a single experiment, four individual samples and two mixtures were sequenced producing >75 Mb of sequence from >300,000 individual sequence reads (average length, 244 b). The reads were aligned and covered 100% of the regions amplified. Allele assignment was 100% concordant with the known HLA alleles of our samples. Our results suggest this method can be a useful tool for complete genomic characterization of new HLA alleles and for completion of sequence for existing, partially sequenced alleles. NGS can provide complete, unambiguous, high-resolution HLA typing; however, further evaluation is needed to explore the feasibility of its routine use.

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.

Microtubule Actin Crosslinking Factor 1 Regulates the Balbiani Body and Animal-Vegetal Polarity of the Zebrafish Oocyte

Although of fundamental importance in developmental biology, the genetic basis for the symmetry breaking events that polarize the vertebrate oocyte and egg are largely unknown. In vertebrates, the first morphological asymmetry in the oocyte is the Balbiani body, a highly conserved, transient structure found in vertebrates and invertebrates including Drosophila, Xenopus, human, and mouse. We report the identification of the zebrafish magellan (mgn) mutant, which exhibits a novel enlarged Balbiani body phenotype and a disruption of oocyte polarity. To determine the molecular identity of the mgn gene, we positionally cloned the gene, employing a novel DNA capture method to target region-specific genomic DNA of 600 kb for massively parallel sequencing. Using this technique, we were able to enrich for the genomic region linked to our mutation within one week and then identify the mutation in mgn using massively parallel sequencing. This is one of the first successful uses of genomic DNA enrichment combined with massively parallel sequencing to determine the molecular identity of a gene associated with a mutant phenotype. We anticipate that the combination of these technologies will have wide applicability for the efficient identification of mutant genes in all organisms. We identified the mutation in mgn as a deletion in the coding sequence of the zebrafish microtubule actin crosslinking factor 1 (macf1) gene. macf1 is a member of the highly conserved spectraplakin family of cytoskeletal linker proteins, which play diverse roles in polarized cells such as neurons, muscle cells, and epithelial cells. In mgn mutants, the oocyte nucleus is mislocalized; and the Balbiani body, localized mRNAs, and organelles are absent from the periphery of the oocyte, consistent with a function for macf1 in nuclear anchoring and cortical localization. These data provide the first evidence for a role for spectraplakins in polarization of the vertebrate oocyte and egg.

Determining performance characteristics of an NGS-based HLA typing method for clinical applications

This study presents performance specifications of an in‐house developed human leukocyte antigen (HLA) typing assay using next‐generation sequencing (NGS) on the Illumina MiSeq platform. A total of 253 samples, previously characterized for HLA‐A, ‐B, ‐C, ‐DRB1 and ‐DQB1 were included in this study, which were typed at high‐resolution using a combination of Sanger sequencing, sequence‐specific primer (SSP) and sequence‐specific oligonucleotide probe (SSOP) technologies and recorded at the two‐field level. Samples were selected with alleles that cover a high percentage of HLA specificities in each of five different race/ethnic groups: European, African‐American, Asian Pacific Islander, Hispanic and Native American. Sequencing data were analyzed by two software programs, Omixons target and GenDxs NGSengine. A number of metrics including allele balance, sensitivity, specificity, precision, accuracy and remaining ambiguity were assessed. Data analyzed by the two software systems are shown independently. The majority of alleles were identical in the exonic sequences (third field) with both programs for HLA‐A, ‐B, ‐C and ‐DQB1 in 97.7% of allele determinations. Among the remaining discrepant genotype calls at least one of the analysis programs agreed with the reference typing. Upon additional manual analysis 100% of the 2530 alleles were concordant with the reference HLA genotypes; the remaining ambiguities did not exceed 0.8%. The results demonstrate the feasibility and significant benefit of HLA typing by NGS as this technology is highly accurate, eliminates virtually all ambiguities, provides complete sequencing information for the length of the HLA gene and forms the basis for utilizing a single methodology for HLA typing in the immunogenetics labs.

SNP-specific extraction of haplotype-resolved targeted genomic regions

The availability of genotyping platforms for comprehensive genetic analysis of complex traits has resulted in a plethora of studies reporting the association of specific single-nucleotide polymorphisms (SNPs) with common diseases or drug responses. However, detailed genetic analysis of these associated regions that would correlate particular polymorphisms to phenotypes has lagged. This is primarily due to the lack of technologies that provide additional sequence information about genomic regions surrounding specific SNPs, preferably in haploid form. Enrichment methods for resequencing should have the specificity to provide DNA linked to SNPs of interest with sufficient quality to be used in a cost-effective and high-throughput manner. We describe a simple, automated method of targeting specific sequences of genomic DNA that can directly be used in downstream applications. The method isolates haploid chromosomal regions flanking targeted SNPs by hybridizing and enzymatically elongating oligonucleotides with biotinylated nucleotides based on their selective binding to unique sequence elements that differentiate one allele from any other differing sequence. The targeted genomic region is captured by streptavidin-coated magnetic particles and analyzed by standard genotyping, sequencing or microarray analysis. We applied this technology to determine contiguous molecular haplotypes across a ∼150 kb genomic region of the major histocompatibility complex.

Filling the gaps – The generation of full genomic sequences for 15 common and well-documented HLA class I alleles using next-generation sequencing technology

Many common and well-documented (CWD) HLA alleles have only been partially characterized. The DNA sequence of these incomplete alleles, as published in the IMGT/HLA database, is most often limited to exons that code for the extracellular domains of the mature protein. Here we describe the application of next-generation sequencing technology to obtain full length genomic sequence from a single long-range PCR amplicon for 15 common and well-documented HLA Class I alleles. This technology is well suited to fill in the gaps of the current HLA allele sequence database which is largely incomplete. A more comprehensive catalog of HLA allele sequences would be beneficial in the evaluation of mismatches in transplantation, studies of population genetics, the evolution of HLAs, regulatory mechanisms and HLA expression, and issues related to the genomic organization of the MHC.

Towards allele-level human leucocyte antigens genotyping – assessing two next-generation sequencing platforms: Ion Torrent Personal Genome Machine and Illumina MiSeq

Human leucocyte antigens (HLA) typing has been a challenge due to extreme polymorphism of the HLA genes and limitations of the current technologies and protocols used for their characterization. Recently, next‐generation sequencing techniques have been shown to be a well‐suited technology for the complete characterization of the HLA genes. However, a comprehensive assessment of the different platforms for HLA typing, describing the limitations and advantages of each of them, has not been presented. We have compared the Ion Torrent Personal Genome Machine (PGM) and Illumina MiSeq, currently the two most frequently used platforms for diagnostic applications, for a number of metrics including total output, quality score per position across the reads and error rates after alignment which can all affect the accuracy of HLA genotyping. For this purpose, we have used one homozygous and three heterozygous well‐characterized samples, at HLA‐A, HLA‐B, HLA‐C, HLA‐DRB1 and HLA‐DQB1. The total output of bases produced by the MiSeq was higher, and they have higher quality scores and a lower overall error rate than the PGM. The MiSeq also has a higher fidelity when sequencing through homopolymer regions up to 9 bp in length. The need to set phase between distant polymorphic sites was more readily achieved with MiSeq using paired‐end sequencing of fragments that are longer than those obtained with PGM. Additionally, we have assessed the workflows of the different platforms for complexity of sample preparation, sequencer operation and turnaround time. The effects of data quality and quantity can impact the genotyping results; having an adequate amount of good quality data to analyse will be imperative for confident HLA genotyping. The overall turnaround time can be very comparable between the two platforms; however, the complexity of sample preparation is higher with PGM, while the actual sequencing time is longer with MiSeq.

Targeted Next-Generation Sequencing for Human Leukocyte Antigen Typing in a Clinical Laboratory: Metrics of Relevance and Considerations for Its Successful Implementation

CONTEXT - Numerous feasibility studies to type human leukocyte antigens (HLAs) by next-generation sequencing (NGS) have led to the development of vendor-supported kits for HLA typing by NGS. Some clinical laboratories have introduced HLA-NGS, and many are investigating the introduction. Standards from accrediting agencies form the regulatory framework for introducing this test into clinical laboratories. OBJECTIVES - To provide an assessment of metrics and considerations relevant to the successful implementation of clinical HLA-NGS typing, and to provide as a reference a validated HLA-NGS protocol used clinically since December 2013 at the Childrens Hospital of Philadelphia (Philadelphia, Pennsylvania). DATA SOURCES - The HLA-NGS has been performed on 2532 samples. The initial 1046 and all homozygous samples were also typed by an alternate method. The HLA-NGS demonstrated 99.7% concordance with the alternate method. Ambiguous results were most common at the DPB1 locus because of a lack of phasing between exons 2 and 3 or the unsequenced exon 1 (533 of 2954 alleles; 18.04%) and the DRB1 locus because of not sequencing exon 1 (75 of 3972 alleles; 1.89%). No ambiguities were detected among the other loci. Except for 2 false homozygous samples, all homozygous samples (1891) demonstrated concordance with the alternate method. The article is organized to address the critical elements in the preanalytic, analytic, and postanalytic phases of introducing this assay into the clinical laboratory. CONCLUSIONS - The results demonstrate that HLA typing by NGS is a highly accurate, reproducible, efficient method that provides more-complete sequencing information for the length of the HLA gene and can be the single methodology for HLA typing in clinical immunogenetics laboratories.

Identification of a novel HLA-DQB1*02 variant allele, DQB1*02:01:06.

The new HLA-DQB1 allele most closely resembles DQB1*02:01:01, differing at a single position 141 (exon 2, codon 15.3).

Identification of a novel HLA-A*23 variant allele, A*23:50.

HLA-A*23:50 differs from HLA-A*23:01:01 by one nucleotide at position 112 resulting in an amino acid change, Arginine to Tryptophan, at codon 14 of exon 2.