Pharmacogenetics of Asthma Treatment
 
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Pharmacogentics of Asthma Treatment Project (PhAT).

Introduction

The Genotyping Facility in Boston is headed by Director Dr David Kwiatkowski, and run on a day to day basis by the Technical Director, Alison Brown. At present, we have four full time technical research assistants, Katie Weiland, Dennis O'Brien, Mel Hernandez and Kerri Bourgeois, who have all been involved in the Genotyping aspect of this PGA program. Also involved in the SNP Genotyping are Jody Senter and Dr Benjamin Raby.

Protocol for choosing SNPs for gene interrogation

Genotyping of all SNPs identified through our sequencing efforts would be costly and inefficient. In this light, a subset of identified SNPs were chosen for large-scale genoptyping in the PGA cohorts after detailed analysis. A major factor influencing SNP selection was to identify those that permitted haplotype identification. For this purpose, the sequence-derived genotype data was used to infer haplotypes using PHASE software (http://www.stats.ox.ac.uk/mathgen/software.html). In addition, the extent of linkage disequilibrium between SNPs was determined by analysis of the sequence-based data using Arlequin (http://lgb.unige.ch/arlequin/).

Based upon the output from these two programs as well as careful manual scrutiny of the sequence-based genotyping data for each gene, SNPs were chosen that would permit resolution of the largest number of haplotypes, considering only those haplotypes that were found in at least one of the four ethnic groups at a frequency of 5% or greater. In many cases, strong to complete association (linkage disequilibrium) was found between pairs of SNPs, meaning that either SNP provided the same discriminant value for haplotype identification.

Second, rarer SNPs (rare allele frequency < 5%) were considered for genotyping when the SNP caused a non-conservative amino acid change, implying potential functional significance.

Last, SNPs which demonstrated marked variation in allele frequencies between the Caucasian control group and the Asthmatic cohort were also considered for genotyping.

Therefore, SNPs chosen initially for genotyping were a compilation of those meeting the criteria listed above. However, approximately 10% of SNPs failed to perform well in genotyping assays despite expenditure of moderate effort, and were therefore discarded from further analysis.

SNaPshot Genotyping

Until recently our SNP genotyping effort has been done using the SNaPshot primer extension kit from Applied Biosystems. Briefly, multiplexed PCR followed by a minisequencing reaction are performed, then reaction products are sized by separation on an ABI 3100 capillary electrophoresis system. Genotyping analysis is done using ABI Prism Genotyper software.

Summary (see Snapshot Methods for a detailed description)

DNA from 96 well DNA master plates is plated out into 96 well PCR plates using a Robbin Hydra. The DNA is dried in the plate overnight, then stored in a sealed container at room temperature. PCR is carried out in multiplex on MJ Research DNA Engine tetrads. After checking for product using agarose electrophoresis, the PCR product was cleaned using EXOSAP-IT (USB Corp) to digest unincorporated dNTPs and dephosphorylate excess PCR primer so that they could not take part in the SNaPshot reaction. The SNaPshot reaction was carried out in multiplex using the SNaPshot Multiplex Kit (Applied Biosystems). The reaction involves the extension of an oligonucleotide probe designed to lie adjacent to the SNP of interest by one of four fluorescently labelled dideoxynucleotides complementary to the base found at the SNP site. The oligonucleotide probes within one multiplex reaction are designed to be of different lengths by the addition of neutral sequence so that when they are separated by capillary electrophoresis only one product will be seen within a size range. The !SNaPshot reaction is carried out essentially as described by the manufacturer except that the SNaPshot mix is diluted 1:1 with HalfBD BigDye sequencing dilution buffer (Genpak). Following the SNaPshot reaction, the mixture is digested with shrimp alkaline phosphatase to prevent any further primer extension. The fluorescently extended probes are then separated on an ABI 3100 capillary electrophoresis system in the presence of a fluorescently labelled size standard. Allele determination is done using ABI Prism Genotyper software.

Sequenom MassARRAY SNP Genotyping

We are now in the process of moving our SNP Genotyping assays to Sequenom MassARRAY system. In this method, genotype data is generated by analysis of minisequencing reaction products by mass spectrometry. PCR is performed in multi- or uniplex format, followed by a ThermoSequenase sequencing reaction using an oligonucleotide bound directly adjacent to each SNP. Extension of the oligonucleotide occurs by either one dideoxynucleotide, or by one deoxynucleotide and one dideoxynucleotide, depending on genotype. Each of the extended products is visualized directly without labeling by MALDI-TOF mass spectrometry. The Extension cocktail is spotted onto a chip containing 384 matrix pads. This matrix aids in desorption and ionization of the DNA. Once inside the mass spectrometer, a laser is fired onto the pads and an electrical charge is applied, attracting the extended oligonucleotide to a detector. The time taken to reach the detector (TOF for time of flight) is solely based upon the mass of the oligonucleotide. The resulting spectra is converted into genotype using an intelligent software system called the SpectroTYPER-RT (RT for real-time) from Sequenom. Through the assay design process, the software is informed of the expected mass of all possible extension products. If a peak is seen where expected the software calls the allele, but if no peak is seen, the laser is fired again until a spectra is achieved containing the expected peaks. Genotypes of up to 5 SNPS can be generated in one reaction by multiplexing the PCR reaction and extension reaction. Lower levels of multiplexing are anticipated, as the level of multiplexing is dependant on the ability of PCR products to be generated within the multiplex.

Due to the higher throughput of this system it has become necessary to incorporate a higher level of laboratory automation into the laboratory. Equipment includes a Tomtec Quadra for pre-PCR procedures such as plating out DNA into 384 well plates, a 96-head Multimek robot (SpectroPREP) for dispensing in the clean up and extension reactions and a nanoliter plotting robot (SpectroPLOTTER) for spotting the extension products from 384 well plates onto 384 pad SpectroCHIPS.

Primer Design

External amplification primers and internal interrogation primers were both designed using Primer3 (http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi). Target primer parameters were fixed to achieve maximal probability of successful multiplex reactions, based on recommendations from Wang and collegues (Wang 1998, Lindblad-Toh 2000). Specifically, external primers were designed according to the following specifications:

   
        Primer length           15-30 nt
        GC content              20-80%
        Melting Temperature     57-63°C;C
        Amplicon Length         50-120 bp
        3' GC clamp             1-3bp

Internal interrogation primers were designed to end immediately 5' to the target SNP. Primers were rejected if they annealed to sequence containing adjacent SNPs, or if they were predicted to undergo self-dimerization or to form 3' hairpin structures. Primers were also required to have melting temperatures between 50-60°C; (target 55°C) and annealing length of 15-30nt. When design of an interrogation primer failed to meet these requirements, design was performed using the complementary strand as template. Following primer design, compatibility of primers for multiplex reactions was assessed using NetPrimer (http://www.premierbiosoft.com/netprimer/netprimer.html). Multiplexing incompatibilities (e.g. cross-dimerization of primers) led to PCR reactions being performed in separate reactions, or to the redesign of the amplification primers. To achieve separation of SNP extension products during electrophoresis, variable length tails were added to the 5' ends of interrogation primers (5'-AACTGACTAAACTAGGTG CCACGTCGT GAAAGTCTGACAA-3' or 5'-ATGCTCAGACACAATTAGCGCGACCCTTAATCC TTAGGTA-3').

Initially, interrogation primers were designed to have lengths of 20bp, 24bp, 28bp, 32bp, 36bp, 40bp, 44bp, and/or 48bp. However, empirical adjustment of primer lengths to achieve separation of extension products was often necessary. In addition, empirical adjustment of primer concentrations was performed for both the amplification primers and the SNP interrogation primers to partially normalize the SNP read quality for each SNP in a multiplex reaction.

References

  1. Lindblad-Toh, K. et al. Large-scale discovery and genotyping of single-nucleotide polymorphisms in the mouse. Nat Genet 24, 381-6. (2000).

  2. Wang, D.G. et al. Large-scale identification, mapping, and genotyping of single- nucleotide polymorphisms in the human genome. Science 280, 1077-82. (1998).

Tracking of DNA samples

All DNA samples are identified by a barcode linking them to sample information within our in-house database Generations. In the production of 96 well DNA master plates, DNA samples are assigned to the wells of the plate by scanning barcode information from that individual into a template DNA plate. The master plates are then assigned a unique barcode. On production of PCR replica plates from the Master plate further barcodes are produced which are appended to the replica plates. This system also allows tracking of sample volume within the master plate. During electrophoresis of the samples, each sample is run individually within a capillary so at this point it is essential to link each sample back to their individual barcode. This is done through the production of a sample sheet by the Generations Database, which is then imported into the ABI 3100 or MassARRAY system and linked to the plate to be run.

Quality Control

Quality control of the Genotyping analysis is performed by several steps. First, during the assay design phase, DNA samples from the same individuals used in the Sequencing SNP discovery phase are used. Thus, results from genotyping and sequencing can be compared. Second, during genotyping, each plate contains a negative control well (water), and a positive control, CEPH individual 1347-02. Results from these wells are checked on every plate that is run to ensure that there is no contamination and that the orientation of the plates have not been confused during handling. Third, genotype analysis for SNaPshot assays is performed using ABI Prism Genotyper software (v3.7). The automated genotyping calls were confirmed by manual check by one member of the facility, with a second independent check performed by a second individual. Finally, for any genotyping reaction performed, at least 5% of samples are repeated in a blinded manner. Conflicts in genotyping calls between the two runs are scrutinized. If persistent conflicts above a level of 2% are present, then all genotyping reactions are repeated for more extensive confirmation.