Pyrosequencing™ : A one-step method for high resolution HLA typing
© Ramon et al; licensee BioMed Central Ltd. 2003
Received: 17 October 2003
Accepted: 26 November 2003
Published: 26 November 2003
While the use of high-resolution molecular typing in routine matching of h uman l eukocyte a ntigens (HLA) is expected to improve unrelated donor selection and transplant outcome, the genetic complexity of HLA still makes the current methodology limited and laborious. Pyrosequencing™ is a gel-free, sequencing-by-synthesis method. In a Pyrosequencing reaction, nucleotide incorporation proceeds sequentially along each DNA template at a given n ucleotide d ispensation o rder (NDO) that is programmed into a pyrosequencer. Here we describe the design of a NDO that generates a pyrogram unique for any given allele or combination of alleles. We present examples of unique pyrograms generated from each of two heterozygous HLA templates, which would otherwise remain cis/trans ambiguous using standard s equencing b ased t yping (SBT) method. In addition, we display representative data that demonstrate long read and linear signal generation. These features are prerequisite of high-resolution typing and automated data analysis. In conclusion Pyrosequencing is a one-step method for high resolution DNA typing.
Solid organ transplantation and allogeneic stem cell transplantation currently represent a common treatment for end-stage organ failure and several hematological and non-hematological malignances. Matching of patient and unrelated donor for h uman l eukocyte a ntigen (HLA) molecules significantly decreases the probability of graft rejection, graft vs. host disease and transplant-related mortality . However, the extensive diversity of the HLA genes makes the identification of matched donors extremely challenging. Although, in several instances it might not be feasible to identify perfect matches, algorithms have been developed that allow identification of likely histocompatibility based on the molecular definition of individual alleles [2, 3]. This algorithm grades mismatches according to the number of variant epitopes present between donor and recipient. As histocompatibility is inversely correlated with number of mismatches it is likely that sequence-based information that provides the definitive information about HLA allele identity will become increasingly important in the future. High-resolution information about HLA alleles identity is best achieved using sequencing-based methodology that could be performed using high-throughput automated systems . Although significant advancement has been made in resolution, automation, throughput and data analysis in DNA sequencing and other polymorphism analysis techniques, the search continues for more efficient methods that could resolve cis/trans ambiguities in highly polymorphic genetic systems such as HLA genes. Currently, commonly used HLA molecular typing methods include s equence s pecific o ligonucleotide p robes (SSOP), p olymerase c hain r eaction (PCR) using s equence s pecific p rimers (SSP) and sequence based typing (SBT) . Among them, SSOP solely exploits DNA hybridization and, therefore, results in the most cis/trans ambiguities. SSP can solve ambiguous combinations if primers are designed to cover the geneomic region where the ambiguity is present. In this case, amplification of the genomic region framed by two primers assures the occurrence in cis of these two regions. This strategy, however, requires a large number of primers to reach a desired resolution and cover various combinations of ambiguous sites within HLA loci. SBT provides by far the highest resolution and currently represents the golden standard for high resolution DNA typing and novel allele discovery. In addition, recent advances made possible to perform SBT at a high throughput level in routine HLA typing laboratories . The biggest challenge that SBT of HLA alleles incurs is the resolution of intrinsic cis/trans ambiguities that cannot be solved by SBT unless time consuming cloning of individual genes is performed . This is because nucleotide incorporation proceeds simultaneously along all DNA templates in a SBT reaction .
Pyrosequencing™ [9–11] is a real-time, sequencing by synthesis method catalyzed by four kinetically well-balanced enzymes, DNA polymerase, ATP sulfurylase, luciferase, and apyrase. It fundamentally differs from Sanger's sequencing method in the order of nucleotide incorporation. Each nucleotide is dispensed and tested individually for its incorporation into a nascent DNA template. Each incorporation event is accompanied by release of pyrophosphate (PPi) in a quantity equimolar to the amount of nucleotide incorporated. ATP sulfurylase quantitatively converts PPi to ATP in the presence of adenosine 5' phosphosulfate. ATP then drives the luciferase-mediated conversion of luciferin to oxyluciferin that generates visible light in amounts that are proportional to the amount of ATP. The light is detected by a charge coupled device (CCD) camera and displayed as a peak in a pyrogram™. Each peak height is proportional to the number of nucleotides incorporated. Unincorporated dNTP and excess ATP are continuously degraded by Apyrase. After the degradation is completed, the next dNTP is added and a new Pyrosequencing cycle is started. As the process continues, the complementary DNA strand is built up. To pyrosequence an unknown DNA sequence, a cyclic n ucleotide d ispensation o rder (NDO) is generally used. As a result of each cycle of dATP, dGTP, dCTP and dTTP dispensation, one of the four dNTPs is incorporated into the DNA template while the other dNTPs are degraded by Apyrase. When a DNA sequence is known, non-cyclic NDOs can be programmed with predictable pyrograms. Nucleotide sequence is determined from the order of nucleotide dispensation and peak height in the pyrogram.
Based on the programmable nucleotide incorporation feature of Pyrosequencing, we set out to optimize Pyrosequencing for high resolution HLA DNA typing. Here we describe the design of NDO that generates a pyrogram that is unique for any given allele or combination of alleles. We present unique pyrograms generated from each of the heterozygous HLA templates that would otherwise be cis/trans ambiguous using s equencing b ased t yping (SBT) methods. We also present representative data that demonstrate long read and linear signal generation. These features are prerequisite of high-resolution typing and automated data analysis. In conclusion, Pyrosequencing can be used as a one-step method for high resolution DNA typing and could be applied in several settings spanning from HLA typing in support of donor/recipient selection to become a complement to comprehensive immunogenetic profiling in several clinical setting where other aspects of immune polymorphism need to be explored .
Design of n ucleotide d ispensation o rder (NDO) that generates unique pyrogram for any allele or combination of alleles
Pyrosequencing resolves intrinsic s equencing b ased t yping (SBT) cis/trans ambiguity
The general principles for the design of NDO can be summarized as follows: a primer is usually placed in proximity upstream of the reference polymorphic site chosen to be the one closest to the ambiguous polymorphic site to be investigated. The first nucleotide dispensation is usually out-of-phase. As a result, SBT ambiguity at one position is generally magnified into pyrograms differences at multiple peaks. This greatly enhances sensitivity and accuracy in detection of peak height differences. In our experience, ambiguities that cannot be solved by SBT within the HLA-DRB1 locus can be consistently solved by unique Pyrosequencing NDO (Wang et al, unpublished results).
Long read and linear signal generation facilitates automated data analysis
Pyrosequencing offers a new approach to data acquisition, analysis and identification of known and unknown (new) alleles, in particular in heterozygous conditions. This method may represent a useful tool to the screening and characterization of polymorphic genetic markers in several clinical or experimental settings [12–24]. In addition, Pyrosequencing has been applied for the study of gene expression  and could be a usefull complement to high throughput single nucleotide polymorphism identification system as a substitute to SBT [8, 24]. Here we propose that Pyrosequencing may confront the most challenging task of solving ambiguities in HLA typing by SBT in heterozygous conditions. Although its reading length is currently shorter than that routinely covered by SBT, automated dNTP dispensation could compensate for this limitation by controling simultaneous reactions in multiple wells using primers that anneal to different locations of the template DNA. In fact, a reading length of 70 to 100 nucleotides allows the high-resolution genotyping of Exon II of HLA-DRB1 (Wang et al, unpublished results). NDOs can also be designed to achieve higher throughput and lower genotyping resolution by introducing fewer numbers of out-of-phase dispensations (Wang et al, unpublished results). Without automatization, it is possible to process 96 to 384 wells PCR product by Pyrosequencing within 4 hours. Constant improvements in the chemistry for sample preparation for Pyrosequencing and Pyrosequencing [25–34] and the implementation of automation devices http://www.pyrosequencing.com it may be possible in the future to apply this technology directly for routine typing of HLA and other immune related genes characterized by extensive polymorphisms .
Materials and Methods
Genomic DNA samples were locally available or obtained from the International Histocompatibility Workshops (IHW) cell lines panel, UCLA interchange panel and samples.
Each PCR amplification mixture of 50 μl contains 1 × PCR buffer (made in house), 2 mM MgCl2, 0.2 mM of each dNTP (purchased from Amersham Biosciences Inc.), 0.2 mM PCR primers, 2 U Taq DNA polymerase, and 250 ng genomic DNA. Either forward or reverse primer is biotinylated. PCR reaction starts with a 95°C denaturation for 5 minutes. This is followed with a 50-cycle thermal cycling. Each cycle is programmed to include 30 seconds denaturation at 95°C, 60 seconds annealing at appropriate temperature, and a 10 seconds final extention at 72°C. The PCR amplicon produced is enough for more 8 pyrosequencing reactions. The PCR amplicons used in this work is 286 bp containing Exon II and the flanking intron sequences.
Biotinylated PCR products are immobilized on streptavidin-coated Sepharose beads (Amersham Biosciences). 50 ul of Binding buffer (PyrosequencingAB) was added to the 50 ul of PCR product. Then 4 ul of streptavidin-coated Sepharose beads was added and the mixture was vigorously mixed at room temperature for 10 minutes. The streptavidin-coated Sepharose bead and PCR mixture is transferred to a filter plate (Amersham Biosciences) and the Binding buffer is removed by vacuum. The biotinylated DNA attached to the streptavidin-coated Sepharose beads was denaturated in 50 ul of Denaturation buffer (PyrosequencingAB) for 1 minute. The Denaturation buffer was removed by vacuum and DNA was washed twice in 150 ul of Wash Buffer (PyrosequencingAB). The DNA is resuspended in 50 ul of Annealing buffer (PyrosequencingAB).
40 ul of well mixed DNA was transferred to a 96-well PSQ96 plate (PyrosequencingAB). The appropriate sequencing primer was added in a volume of 5 ul using a 3 uM stock solution, resulting in 45 ul reaction volume. The sequencing primer is allowed to anneal on a heat plate set for 80°C for 2 minutes. Samples are allowed to cool for 5 minutes at room temperature. Once samples have cooled down the plate in placed on the Pyrosequencer and the PSQ96 reagents are added to the SQA cartridge (PyrosequencingAB). NDO is automatically designed using software developed at Pel-Freez Clinical Systems. Pyrosequencing data output is quantified using Peak Height Determination Software v1.1 (PyrosequencingAB).
We wish to acknowledge PyrosequencingAB for their outstanding technical support. We are grateful to Mostafa Ronaghi for the valuable discussions. We thank Yunxia Wang, Joel Shi, Dina Berchanskiy and Xiang Jun Liu for their assistance and helpful discussions. Daniel Ramon is a PhD candidate in the field of Biochemistry at Universidad Nacional de San Luis (Argentina).
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