Key words

1 Introduction

Single Nucleotide Polymorphism s (SNPs) are single-base changes at specific genetic loci of a DNA sequence that distinguish between members of a species. SNPs in human occur in a significant proportion (more than 1 %) of a large population [1]. SNPs can be located in both coding and noncoding DNA sequences, and therefore for those located in noncoding regions, the majority of the SNPs are of no biological consequence, and only a fraction of the SNPs located in the coding regions have functional significance [2]. SNPs are common, great in number, and widely distributed throughout the entire human genome with an estimated frequency of about 1/1000 base pairs [3, 4]. All these properties of SNPs make them suitable genetic markers for high-throughput array-based approaches and support their clinical applications since SNPs might impact disease diagnosis and predisposition. More importantly, the presence of genetic variations strongly impact drug therapies and responses, by contributing to the efficacy and/or side effects for any given drug [5, 6]. Furthermore, it is important to conduct the investigation of the genetic variations related to the absorption, distribution, metabolism, and excretion (ADME) of drugs, which is called pharmacogenomics. The main intent of pharmacogenomics is to identify personalized therapies in order to reduce the inter-individual variability and to improve efficacy and safety of drug therapy [5]. The Drug Metabolizing Enzyme s and Transporters (DMET) assay by Affymetrix is an array-based genotyping platform that interrogates genetic variants in 231 ADME-related genes simultaneously. Among these genes, there are genes that are related to drug-metabolizing enzymes (thiopurine S-methyltransferase or TPMT, dihydropyrimidine dehydrogenase or DYPD, and members of the cytochrome P450 family), the UDP glucuronosyltransferases (primarily UGT1A1 and UGT1A9), the ATP-binding cassette (ABC) transporters (ABCB1 or P-glycoprotein, and ABCG2 or breast cancer resistance protein), other novel drug transporters (e.g., sulfotransferase), and transcription regulators (e.g., NR1I2), and also genes known to induce other ADME genes (e.g., PPARD) [7]. The DMET genetic markers are collectively 1936, and they are classified into genotyping markers (biallelic SNPs, triallelic SNPs, and insertions/deletions of varying lengths) and five regions of copy number variation (CNV ) [8]. The molecular inversion probes (MIP) amplification technology can genotype in a single reaction all the interrogated genomic sites [9]. Indeed, DMET array-based genotyping interrogates a sufficient number of polymorphisms related to the ADME of drugs, allowing the identification of clinically significant metabolic profiles for the patient-specific therapy. Moreover, the collected SNPs data are then converted into known haplotypes based on standardized nomenclature (i.e., star alleles nomenclature), routinely used in pharmacogenomic studies [10]. This conversion is essential for describing and interpreting known haplotypes. In recent times, several research groups have successfully used the DMET platform to identify genetic profiles that influence the sensitivity to specific therapies [6, 11–15].

2 Materials

2.1 Laboratory Configuration

During the DMET protocol it is essential to use two separate rooms to avoid contamination of the samples with amplified PCR products. The pre-Amp Lab is an area free of PCR products (can do PCR, but not cleanup of PCR products). In this Lab a fume hood is required to perform the multiplex PCR (mPCR), and this is called the mPCR Staging Area in the pre-Amp Lab. mPCR is a pre-amplification step to amplifiy some genetic markers before they join with other markers in the PCR amplification using the highly selective MIP technology. The post-Amp Lab is a separate room where the steps following PCR are performed. Each of these areas and Labs requires dedicated equipment, including pipettes and 96-well iceless coolers.

2.2 Affymetrix Reagents, Equipment, and Software

There are two types of DMET assay packs (see Note 1 ) and they contain the DMET Plus Reagent kit which consists of the Pre-Amp Kit, Labeling Kit, Hyb-Stain Kit, and Panel Kit.

  1. 1.

    Pre-Amp Kit consists of pre-amp water, Buffer A, Enzyme A, Gap Fill Mix 1 and 2, Exo Mix, Cleavage Enzyme, Universal Amp Mix and dNTP mix. Part of these reagents is used to make the Anneal Master Mix, see Table 1.

    Table 1 Preparation of the anneal master mix
    Full size table
  2. 2.

    Labeling Kit consists of PCR cleanup mix, post-amp water, Frag buffer, Frag reagent, DNA labeling reagent, 5× TdT buffer, and TdT enzyme. Part of these reagents is used to make the Labeling Master Mix, see Table 2.

    Table 2 Preparation of the labeling master mix
    Full size table
  3. 3.

    Hyb-Stain Kit consists of hybridization solution, oligo control reagent, stain buffer, and hold buffer.

  4. 4.

    Panel Kit consists of MIP panel, mPCR primer mix, 1× TE buffer, gDNA control 2, and PCR dilution buffer.

  5. 5.

    Wash Solution: Wash Solution A and Wash Solution B.

  6. 6.

    DMET Plus Array.

  7. 7.

    GeneChip® Fluidics Station 450.

  8. 8.

    GeneChip® Hybridization Oven 640.

  9. 9.

    GeneChip® Scanner.

  10. 10.

    Affymetric GeneChip® Command Console (AGCC) Software.

  11. 11.

    DMET™ Console Analysis Software: provides a fast genetic analysis of the data obtained by the DMET™ Plus Reagent kit in order to discover the involvement of metabolic pathways in drug metabolism.

2.3 Other Reagents and Equipment

  1. 1.

    AccuGENE® Water (Lonza Group LTD).

  2. 2.

    Molecular biology grade water.

  3. 3.

    Strip tubes.

  4. 4.

    DNA Blood Mini Kit (Qiagen).

  5. 5.

    Quant-iT™ PicoGreen® dsDNA Assay Kit (Life Technologies), containing λ DNA (100 μg/mL). Several λ DNA standard solutions are made according to Table 3.

    Table 3 Serial dilution of λ DNA standard solutions for quantitation of gDNA concentration
    Full size table
  6. 6.

    QIAGEN® Multiplex PCR Kit consists of multiplex PCR master mix, Q solution 5×, RNase-free water. The multiplex PCR master mix, which consists of HotStar Taq polymerase, dNTPs, MgCl2, and multiplex PCR buffer, is used to make the mPCR Master Mix as shown in Table 4.

    Table 4 Preparation of the mPCR mix
    Full size table
  7. 7.

    Streptavidin, R-phycoerythrin conjugate (SAPE).

  8. 8.

    Taq Polymerase (TITANIUM™, Clontech).

  9. 9.

    TE Buffer 1×, pH 8.0.

  10. 10.

    TECAN plate reader (Infinite 200 PRO).

  11. 11.

    Thermal cyclers.

  12. 12.

    96-well iceless cooler.

3 Methods

3.1 Genomic DNA Preparation

To genotype patients affected by hematological malignancies, genomic DNAs (gDNAs) are required, and they will be obtained from remission samples (the “normal” counterpart). Blood samples are the major source of gDNA in pharmacogenomic studies. However, alternative and valuable gDNA sources might be saliva samples [16].

  1. 1.

    Extract gDNA from blood samples using the DNA Blood Mini Kit (Qiagen).

  2. 2.

    gDNA concentration should be determined using the Quant-iT™ PicoGreen® assay, which is an ultrasensitive fluorescent nucleic acid stain for the detection of double-stranded DNA (dsDNA). Prepare an aqueous working solution by making a 200-fold dilution of the concentrated PicoGreen® reagent in 1× TE buffer in a plastic container. For 6 mL of reagent solution, add 30 μL of PicoGreen® reagent to 5970 μL 1× TE buffer. Protect the solution from light.

  3. 3.

    Prepare the λ DNA standard solutions by adding 1470 μL of 1× TE buffer to 30 μL of the λ DNA standard to obtain a final concentration of 2 ng/μL. Proceed with serial 1:2 dilution in 1× TE buffer to obtain the λ DNA concentration of 1 ng/μL, 0.5 ng/μL, 0.25 ng/μL, see Table 3.

  4. 4.

    Dilute the extracted gDNA by ten times before quantification (i.e., 3 μL gDNA + 27 μL 1× TE buffer). It is because of the high sensitivity of PicoGreen assay and the good yield of gDNA from the blood samples.

  5. 5.

    Prepare the microplate for gDNA quantification. For λ DNA standards, mix 50 μL of each standard solution with 50 μL of the PicoGreen working solution. The final λ DNA concentration in the assay will be 1 ng/μL, 0.5 ng/μL, 0.25 ng/μL, and 0.125 ng/μL (see Table 3). Ensure to insert the reagent blank, i.e., mixing 50 μL of 1× TE buffer to 50 μL of the PicoGreen working solution.

  6. 6.

    For each gDNA sample, mix 10 μL of diluted gDNA with 40 μL of 1× TE buffer and 50 μL of the PicoGreen working solution.

  7. 7.

    For both λ DNA standards and gDNA samples, they should be quantified at least in duplicate. Incubate for 2–5 min at room temperature. Protect the solutions from light.

  8. 8.

    Measure the fluorescence using a Tecan plate reader. Subtract the fluorescence value of the reagent blank from that of the λ DNA standard and gDNA samples. Plot the calibration curve using the λ DNA standards, and determine the gDNA concentration from the curve.

  9. 9.

    The gDNA samples should be normalized to a single concentration of 60 ng/μL. With the quantified gDNA concentrations, dilute the samples in 1× TE buffer to give a final concentration of 60 ng/μL.

3.2 Day 1 Operation: Pre-amplification and Annealing (Pre-Amp Lab)

  1. 1.

    Start the operation in the pre-Amp Lab at noon. Pay attention in working with enzymes because they are temperature-sensitive. Keep them at −20 °C until use. Spin down at 13,400 rcf for 30 s the enzyme solutions in the tubes to uniformly mix the content, and do not vortex. All other reagents should be vortexed after thawing and then spun down.

  2. 2.

    Thaw and then place on ice the reagents from the DMET Plus Reagent Kit, i.e., mPCR Primer Mix, gDNA Controls, 1× TE Buffer, and PCR Dilution Buffer.

  3. 3.

    Thaw and then place on ice the reagents from the QIAGEN Multiplex PCR Kit, i.e., Multiplex PCR Master Mix, Q-Solution, and RNase-free water. The Q-Solution contains some PCR additives that facilitate amplification of difficult templates.

  4. 4.

    In the pre-Amp Lab, prepare the Genomic Plate 1 (GP1 plate), which consists of 17 μL of each gDNA sample (at a concentration of 60 ng/μL) and 17 μL of each gDNA control. The gDNA controls in the Kit are already normalized to a working concentration of 60 ng/μL. The same sample scheme must be maintained in each step to facilitate the liquid transfer using the multichannel pipettes and to minimize pipetting errors. Seal the plate and spin it down at 800 rcf for 60 s. Maintain the plate at 4 °C using the 96-well iceless cooler.

  5. 5.

    Prepare the Genomic Plate 2 (GP2 plate). Using a multichannel P20 pipette, aliquot 10 μL of 1× TE Buffer and then transfer 2 μL of each sample or gDNA control from GP1 plate to the corresponding well of GP2 plate. Vortex and spin down GP2 plate. Seal GP1 plate and store it on ice or at 4 °C until use.

  6. 6.

    Prepare the mPCR Mix by adding reagents in the order shown in Table 4.

  7. 7.

    Transfer the mPCR Mix to a reagent reservoir. Aliquot 45 μL of the Mix to each well of a new 96-multiwall plate (mPCR plate) using a multichannel P200 pipette. Follow the same sample scheme of GP1/GP2 plates. Using a multichannel P20 pipette, transfer 5 μL of each sample/control from GP2 plate to the corresponding well of mPCR plate. Seal GP2 plate and keep it on ice until the pre-amplification stage is successfully completed. Seal, vortex, and spin down mPCR plate, then place it on a thermal cycler and run the temperature program shown in Table 5.

    Table 5 Temperature program used for pre-amplification of some genetic markers
    Full size table
  8. 8.

    The mPCR products will be diluted twice. Two new plates are used for the dilution. After the temperature program is finished, spin down mPCR plate. In the mPCR Staging Area (i.e., fume hood), prepare Dilution Plate 1 (DP1 plate) and Dilution Plate 2 (DP2 plate). Using a multichannel P200 pipette, aliquot in DP1 and DP2 plates 153 μL PCR Dilution Buffer to the corresponding well of each sample/control in mPCR plate. Transfer 5 μL of each sample/control from mPCR plate to the corresponding well of DP1 plate and mix slowly by pipetting up and down ten times. Then, transfer 5 μL of each sample/control from DP1 plate to the corresponding well of DP2 plate, and mix slowly by pipetting up and down ten times (see Note 2 ).

  9. 9.

    In the Pre-Amp Lab, prepare the Anneal Master Mix by adding reagents from the Pre-Amp Kit in the order shown in Table 1. Pipette up and down the Anneal Mix five times using a P1000 pipette set to 900 μL and do not vortex.

  10. 10.

    Prepare the Anneal Plate (ANN plate) by adding 21.7 μL Anneal Master Mix per well. Transfer 13.4 μL of each sample from GP1 plate to ANN plate (final volume = 35.1 μL). Seal and transfer ANN plate to the mPCR Staging Area.

  11. 11.

    Add 5 μL diluted mPCR products from DP2 plate to the corresponding wells of ANN plate (final volume = 40.1 μL). Seal and transfer ANN plate to the Pre-Amp Lab, vortex, and spin. Put the ANN plate on the thermal cycler and start the temperature program shown in Table 6 (see Note 3 ):

    Table 6 Temperature program for annealing
    Full size table
  12. 12.

    At the end of the first 95 °C hold, press “pause” on the thermal cycler, remove the ANN plate and put it on an iceless cooler for 2 min. Then, spin the ANN plate, remove the seal and add 5 μL MIP Panel from the Panel Kit to each reaction well (final volume = 45.1 μL). Seal again the ANN plate, vortex, spin down and place it back on the thermal cycler and resume the temperature program (Table 6). Incubate the samples at 58 °C for 16–18 h.

3.3 Day 2 Operation: Cleanup, Fragmentation, Labeling, Denaturation, and Hybridization

Start these steps in the morning, immediately after the Anneal stage (do not incubate samples for more than 18 h for annealing). The day 2 operation starts in the pre-Amp Lab. The first stage (Gap Fill Through Amplification ) requires adding reagents during the thermal cycler reaction and stopping the program at specific time points. The following stages must be located in the post-Amp Lab. Before starting, thaw, spin down, and then place on ice until use the dNTP Mix and the Universal Amp Mix, whereas leave the Exo Mix, Cleavage Enzyme, Gap Fill Mixes 1 and 2, and the TITANIUM Taq Polymerase at −20 °C until ready to use.

  1. 1.

    In the pre-Amp Lab, prepare the Gap Fill Mix by mixing 190 μL Gap Fill Mix 2 and 10 μL Gap Fill Mix 1 in an Eppendorf tube. Mix well by pipetting slowly up and down and aliquot 14 μL of Gap Fill Mix to each 0.2 mL PCR tube of an 8-strip tube. Cap and spin down the strip tubes and place them in an iceless cooler.

  2. 2.

    Remove ANN plate from the thermal cycler, place the plate in an iceless cooler for 2 min and then spin down at 800 rcf for 60 s.

  3. 3.

    Using a multichannel P10 pipette, add 2.5 μL Gap Fill Mix to each reaction well of ANN plate. Seal, vortex, and spin.

  4. 4.

    Prepare the Assay plate (ASY plate) by transferring 12 μL each reaction from the ANN plate. Seal and spin the ASY plate.

  5. 5.

    Place the ASY plate in the thermal cycler and run the temperature program shown in Table 7 (see Note 3 ).

    Table 7 Temperature program for PCR
    Full size table
  6. 6.

    After the first 11 min at 58 °C, pause the thermal cycler, place the plate in an iceless cooler for 2 min, and spin down. Remove the seal and add 5 μL dNTP Mix to each reaction. Reseal the plate, vortex, spin down, and place the plate back on the thermal cycler. Resume the temperature program (Table 7).

  7. 7.

    When thermal cycler reaches 37 °C, pause the thermal cycler, place the plate in an iceless cooler for 2 min, and spin down. Remove the seal and add 5 μL Exo Mix to each reaction. Reseal the plate, vortex, spin down, and place the plate back on the thermal cycler. Resume the temperature program (Table 7).

  8. 8.

    In the pre-Amp Lab, prepare the Universal Amp Mix by directly adding 25 μL of Cleavage Enzyme and 70 μL of Taq Polymerase into the Universal Amp Mix Tube. Mix by pipetting up and down ten times.

  9. 9.

    When thermal cycler reaches 60 °C, pause the thermal cycler, place the plate in an iceless cooler for 2 min, and spin down. Remove the seal and add 30 μL Universal Amp Mix to each reaction. Reseal, vortex, spin down the plate, and place it back on the thermal cycler. Resume the temperature program (Table 7).

  10. 10.

    When the program has ended, transfer the sealed ASY Plate to the Post-Amp Lab and place it on ice.

  11. 11.

    In the post-Amp Lab, aliquot 2.5 μL PCR Cleanup Mix to each reaction of the ASY plate. Seal, vortex, and spin down the plate, then place it in a thermal cycler and run the temperature program shown in Table 8 (see Note 4 ).

    Table 8 Temperature program for PCR cleanup
    Full size table
  12. 12.

    Prepare the first QC gel (3 % agarose gel) to identify if the gDNA samples have amplified by PCR .

  13. 13.

    After the cleanup step, spin down the plate, remove the seal, and take 2 μL from each reaction well. Reseal the plate and place at 4 °C. For each reaction, prepare a loading sample by mixing 2 μL of PCR products, 2 μL of 2× loading buffer, and 8 μL water.

  14. 14.

    Load 10 μL of the loading sample onto the agarose gel and run gel electrophoresis at 120 V for 20 min. It should be detected for each sample a PCR product between 100 and 150 bp to ensure good-quality PCR.

  15. 15.

    In the post-Amp Lab, transfer 25 μL of each reaction to a new plate for fragmentation and labeling (Frag/Label plate).

  16. 16.

    Thaw and then place on ice the Post-Amp Water and the Fragmentation Buffer. Prepare the fragmentation mix by mixing 8.9 μL Post-Amp Water and 1 μL of Fragmentation Buffer per sample, and cool the mixture on ice for 5 min. Then, add 0.0675 μL/sample of Fragmentation Reagent to the mixture, then vortex, spin, and place on ice. Add 10 μL fragmentation mix to each reaction well (see Note 5 ). Seal, vortex, spin, and place the plate to thermal cycler to run the temperature program shown in Table 9 (see Note 6 ):

    Table 9 Temperature program for fragmentation
    Full size table
  17. 17.

    Prepare a second QC gel (3 % agarose gel) to check the fragmentation reaction to confirm acceptable fragment size. For each reaction well, take 10 μL of sample (from the Frag/Label plate) and mix it with 2 μL of 2× loading buffer. Load 10 μL of the loading sample onto the agarose gel and run gel electrophoresis at 120 V for 24 min. A gel that consists of fragments of less than 120 bp with the smear centered on around 50 bp shows the good quality in fragmentation.

  18. 18.

    In the post-Amp Lab, prepare the Labeling Master Mix by adding reagents in the order shown in Table 2.

  19. 19.

    Aliquot the Labeling Master Mix into strip tubes (45 μL/tube) and then add 10 μL of the mix to each sample of the Frag/Label plate by using a multichannel P20 pipette. Seal, vortex, and spin down the plate, then place it in a thermal cycler and run the temperature program shown in Table 10.

    Table 10 Temperature program for labeling
    Full size table
  20. 20.

    Before the hybridization step, preheat the Hybridization Oven to 49 °C with rotation on (35 rpm) (see Note 7 ).

  21. 21.

    Equilibrate the DMET Plus arrays to room temperature. Mark each array. For each array, insert a 200 μL pipette tip into the upper right septum.

  22. 22.

    Thaw the Hybridization Solution and Oligo Control Reagent, then prepare the Hybridization Master Mix by adding the Oligo Control Reagent directly to the Hybridization Solution tube. Pour the mix into a reagent reservoir on ice.

  23. 23.

    Prepare the Hybridization plate. With a multichannel (P200 and P20) pipette, aliquot 92 μL of Hybridization Master Mix and 8 μL of each sample from the Frag/Label plate (see Note 8 ). Seal, vortex, and spin the plate, then place the plate on a thermal cycler prewarmed at 95 °C. Leave the plate at 95 °C for 10 min to denature the samples before loading the arrays. Then, place the plate in an iceless cooler for 2 min and spin the plate.

  24. 24.

    Generate a sample batch registration file using the predefined template provided with the DMET Console (DMET.TEMPLATE) or using your own created template. The Batch Registration File could be a TSV or Excel file type and must contain sample information, such as sample file name, probe array type, sample name, source plate, source well, and sample type. Scan the array barcodes into this file.

  25. 25.

    Upload the Batch Registration File to Affymetrix GeneChip® Command Console™ (AGCC) software and click “save”. The message “Batch Array Registration is complete” is displayed.

  26. 26.

    In each array, inject 95 μL of denatured sample via the pipette tip. Remove it from the upper right septum of each array and cover both the array septa with adhesive spots.

  27. 27.

    Place the array in the hybridization oven and allow to hybridize at 49 °C and 35 rpm for 16–18 h (see Note 9 ).

  28. 28.

    Thaw the Stain Buffer and Hold Buffer at 4 °C overnight.

3.4 Day 3 Operation: Stain, Wash and Scan

  1. 1.

    Prime the Fluidics Station, install the Wash Solution A and B bottles and fill the dH2O container. Then, run the PRIME_450 script in the AGCC software.

  2. 2.

    Prepare SAPE Stain Solution by adding 90 μL of SAPE to the Stain Buffer tube. Mix by inverting the tube. Keep the mix at room temperature and protect it from light.

  3. 3.

    Setup the Software and the Fluidics Station according to the appropriate DMET protocol to wash and stain DMET Plus Arrays.

  4. 4.

    Wash no more than eight arrays in the Fluidics Station at any time, leave remaining arrays in the hybridization oven. Before washing, remove the adhesive spots from each array. Wash and stain arrays.

  5. 5.

    Warm up scanner for 10 min, then remove arrays from fluidics station and cover again both septa with adhesive spots (do not cover the window). Load arrays onto scanner and scan (see Note 10 ).

3.5 Analysis of DMET Data by the Affymetrix DMET™ Console Analysis Software

  1. 1.

    After scanning the DMET Plus Arrays, the files containing the data of hybridization intensity (CEL files) are generated by the AGCC software. Analysis files can thereafter be downloaded within DMET Console.

  2. 2.

    To start analyzing data in DMET™ Console, first create a workspace and add one or more datasets to the workspace. A dataset points to a collection of sample files (ARR), intensity files (CEL), and genotyping files (CHP). The CEL files are loaded in a new workspace and dataset are then converted to genotype calls (CHP files).

  3. 3.

    The CHP files get through the automatic Quality Control (QC), where the console divides the results in two groups: “In Bounds” and “Out of Bounds” depending on the default QC call rate threshold of 98 %. This means that samples with a QC call rate >98 % are “In Bounds” and samples with a call rate <98 % are “Out of Bounds.”

  4. 4.

    Once the selected CHP files pass sample QC, the user can use a predefined marker list, or create one, to adapt the signal boundaries to the behavior of the samples.

  5. 5.

    The genotypes are translated into haplotypes alleles (i.e., SULT2B1_40556C>T) for many genes using the star allele nomenclature and then into a metabolizer condition which reveals the level of metabolic activity of the related enzyme.

  6. 6.

    The DMET™ Console software presents the genotyping results in the CHP Summary, Marker Summary, and Copy Number Summary tables [17]. The Marker Summary cluster graph is an X − Y scatter plot of the signal data for the selected marker across all CHP files in the user-selected results group (Fig. 1). Prior to genotyping, the DMET™ Plus array summarized signals are transformed using the “Minus vs. Average” signal transformation.

    Fig. 1
    figure 1

    Marker Summary cluster graph. The cluster graph displays the signal data for the marker selected by the user in the Marker Summary Table (SULT2B1_40556C>T). The genotype calls (AA, AB, BB, and noCall) are identified by color and shape (see Table 11). The graph legend uses abstract allele names, which is how the calls are stored in the genotype result files (CHP). In order to interpret these codes, the graph also displays the actual allele name: SULT2B1_40556C>T(L213L) on the reported strand (which is intended to be the gene coding strand)

    Full size image
  7. 7.

    The marker summary cluster is summarized by the call codes listed in Table 11. The data in Fig. 1 include numerous AA, AB, BB calls, and one noCall, in which A represents the T allele and B represents the C allele of the SULT2B1_40556C>T mutation of the sulfotransferase SULT2B1 gene.

    Table 11 Call codes used in the marker summary cluster graph
    Full size table
  8. 8.

    A concordance check is used to compare the genotype results (CHP) with the reference data and help the user to evaluate data quality. The reference data can be text files (TXT) or genotype results (CHP). The genotypes that are not found in both files, as well as genotypes reporting “NoCall” or “NotAvailable” for at least one of the two compared files, are excluded from the comparison.

  9. 9.

    The software shows the results in tables and graphs [17], so the user can:

    • Quickly identify possible rare alleles or missing data.

    • Identify haplotype and probe-level sequence variation in the test samples relative to a standard reference sequence.

    • Identify variation at other genes in the panel that impart functional or structural changes to the gene product.

    • Annotate the reported genotypes across probes to indicate genomic, mRNA, or peptide changes resulting from any observed variation in the analyzed samples.

    • Extract known functional or structural variants in a DMET profile to identify and summarize genes of potential altered activity.

    • Predict general gene activity based on detected diplotypes.

    • Integrate missing data into final study summary reports with the use of an Override Report.

3.6 Analysis of DMET Files by DMET Analyzer Software

This software was developed at the Department of Medical and Surgical Sciences, Magna Graecia University of Catanzaro, Italy. The software is freely available for academic purposes at https://sourceforge.net/projects/dmetanalyzer/files/. DMET-Analyzer lacks the quality control capabilities available in DMET Console and the possibility to manage Affymetrix binary files directly. The tool allows the following operations.

  1. 1.

    Automate the workflow of analysis of DMET-SNP data avoiding the use of multiple tools [18]. The user can prepare his/her own dataset in both excel file and delimited table files. The software is able to find the class-labels directly from the input files. DMET Analyzer automatically selects the relevant SNPs. In fact, the software includes several statistical software, such as the Fisher’s test to check the association among the presence of SNP and the classes already determined, the Bonferroni test and False Discovery Rate to get better statistical significance of results, the Hardy-Weinberg calculator to analyze the linkage disequilibrium, and the Pearson’s chi-square test to calculate the deviation from the Hardy-Weinberg equilibrium and to determine the significance of the deviation.

  2. 2.

    Automate the annotation of DMET-SNP data and to search in existing databases for SNPs (e.g., dbSNP) [19].

  3. 3.

    Associate SNP with pathways through the search in PharmaGKB, a major knowledgebase for pharmacogenomic studies.

4 Notes

  1. 1.

    There are two DMET assay packs and they are the DMET™ Plus Premier Pack and the DMET™ Plus Starter Pack. They contain the same types of reagents: the DMET™ Plus Starter Pack contains sufficient reagents for eight reactions (7 samples and 1 control), but the DMET™ Plus Premier Pack contains sufficient reagents for 48 reactions (45 samples and 3 controls).

  2. 2.

    Dilution Plate 1 (DP1) and Dilution Plate 2 (DP2) should be maintained at 4 °C in a 96-well iceless cooler.

  3. 3.

    Pay attention to the time when the program in the thermal cycler must be paused to add specific reagents.

  4. 4.

    The thermal cycler for the Cleanup Mix must be located in the post-Amp Lab. Do not return the PCR products to the pre-Amp Lab to avoid contamination with mPCR products and samples.

  5. 5.

    During the fragmentation step, it is essential to work on ice to maintain the reagents, mix and samples (on Frag/Label plate) cool, and work quickly to avoid the reaction to start prematurely.

  6. 6.

    Place the Frag/Label plate onto the thermal cycler only when the temperature is exactly 37 °C.

  7. 7.

    Accurate hybridization temperature (49 ° C) is critical for this assay.

  8. 8.

    The Frag/Label plate can be stored at –20 °C for up to 1 week, if hybridizations need to be repeated.

  9. 9.

    When working with a large number of samples load multiple arrays and place them in the hybridization oven at the same time to reduce the effect of oven temperature changes on the hybridization results on different arrays.

  10. 10.

    Before scanning, inspect each array for the presence of bubbles and for blemishes/dirt/marks on the array window.