High-Throughput, Multiplex Genotyping Directly from Blood or Dried Blood Spot without DNA Extraction for the Screening of Multiple G6PD Gene Variants at Risk for Drug- Induced Hemolysis  Xiaoyi Tian, Jun Zhou, Ye Zhao, Zhibin Cheng, Wenqi Song, Yu Sun, Xiaodong Sun, Zhi Zheng  The Journal of Molecular Diagnostics  Volume 19, Issue 5, Pages 638-650 (September 2017) DOI: 10.1016/j.jmoldx.2017.05.007 Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Schematic diagram of multiplex extension and ligation-based probe amplification (MELPA) assay. Target DNA of lysed samples are captured on a 96-well plate by tailed capture probes (CP) and ligation probes (LP) in an overnight hybridization reaction, followed by removal of unbound probes by washing. Subsequent enzymatic extension and ligation reactions form single-stranded templates spanning target single-nucleotide polymorphism (SNP) sites. All templates then are PCR-amplified using universal primers targeting the tail sequences of the ligation probes. An unextended primer (UEP) specific for each SNP binds 5′ adjacent to the SNP site and extends a single base at the SNP site in an enzymatic reaction, yielding a product with a unique mass that forms a characteristic peak in subsequent matrix-assisted laser desorption/ionization time-of-flight mass spectrum. Multiplexed genotyping is achieved by analyzing well-separated multiple product peaks in one spectrum. The bold lines indicate the universal primer tag. P, phosphate group. The Journal of Molecular Diagnostics 2017 19, 638-650DOI: (10.1016/j.jmoldx.2017.05.007) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Comparison of genotype call quality, reproducibility, and accuracy by multiplex extension and ligation-based probe amplification (MELPA) and iPLEX for genotyping 23 single-nucleotide polymorphism (SNP) sites on archival blood samples. Four archival blood samples from malaria patients were genotyped in triplicate on 3 separate days for the 23 glucose-6-phosphate dehydrogenase (G6PD) single-nucleotide polymorphism (SNP) sites by MELPA and iPLEX. A: Call quality distribution. The qualities of SNP genotype calls were classified by the mass spectrometry (MS) genotyping call software into four categories according to decreasing signal quality and level of confidence, as follows: A, conservative; B, moderate; C, aggressive; and D, low probability. Examples of the four quality levels can be found in Supplemental Figure S1. B: Consistency rates of the genotype results for each assay on each day are shown. A consistent genotype was defined as a genotyping result with three identical calls other than no alleles from triplicates. The consistency rate was the percentage of consistent genotypes among the total of 92 genotypes assayed that day. C: Genotyping call accuracy for the 23 G6PD SNP sites. An accuracy rate was calculated for each genotype result as the percentage of correct calls (ie, identical to the sequencing result) among triplicates. The mean accuracy rate was the average of the accuracy rates of the four samples. **P < 0.01 (Fisher exact probability test); †††P < 0.001 versus iPLEX (paired χ2 test). The Journal of Molecular Diagnostics 2017 19, 638-650DOI: (10.1016/j.jmoldx.2017.05.007) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Mass spectra examples of discrepant genotyping results from the two assays. The site, genotype call, and its quality ranking (in parentheses) are indicated above the spectra. Blue vertical lines indicate peaks of unextended primer (UEP) and the expected 1-base extension products for the SNP site of interest. For each site the two assays used the same UEP. Gray vertical lines indicate the UEP or extended products for other SNP sites. The iPLEX provided clear, but wrong, heterozygous genotype calls, suggesting the assay generated some significant nonspecific primer extension products. The UEP of site 159 targeted the sense strand whereas the UEP of site 825 targeted the antisense strand. MELPA, multiplex extension and ligation-based probe amplification. The Journal of Molecular Diagnostics 2017 19, 638-650DOI: (10.1016/j.jmoldx.2017.05.007) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Genotyping of pooled samples. One dried blood sample with heterozygous glucose-6-phosphate dehydrogenase (G6PD) 392G->T mutation was pooled with five wild-type samples and genotyped with or without a specific wild-type blocker. The addition of a blocker prevents the relative dilution of the mutant T signal that resulted from pooling. Dashed lines indicate peaks of unextended primer (UEP) and extended products. The Journal of Molecular Diagnostics 2017 19, 638-650DOI: (10.1016/j.jmoldx.2017.05.007) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S1 Examples of four quality levels of a genotyping call by the mass spectrometry analysis software. The single-nucleotide variant [single-nucleotide polymorphism (SNP)] and the call are shown above the spectrum. A. Conservative through D. Low Probability represent decreasing reliability of the genotype call. The peak positions of the unextended primer and the expected 1-base extension products for the SNP site are indicated by blue letters. Gray dotted lines relate to other SNP sites. Factors affecting quality ranking include signal-over-noise of the expected extension peaks and (in the case of heterozygotes) the peak ratios (ideally 1:1). The Journal of Molecular Diagnostics 2017 19, 638-650DOI: (10.1016/j.jmoldx.2017.05.007) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S2 DNA sequencing results of two discrepant genotype calls of four blood samples. The arrows indicate the targeted single-nucleotide variant sites; red arrows, DNA sequencing of the sense strand; black arrows, sequencing of the antisense strand. The Journal of Molecular Diagnostics 2017 19, 638-650DOI: (10.1016/j.jmoldx.2017.05.007) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S3 Distribution of genotype call qualities by multiplex extension and ligation-based probe amplification (MELPA) on blood and dried blood spot (DBS) samples. A. Conservative through I. Bad Spectrum were the quality rankings of mass spectrometry results with a decreasing level of confidence. The call rate was defined as the percentage of calls ranked C or above. The Journal of Molecular Diagnostics 2017 19, 638-650DOI: (10.1016/j.jmoldx.2017.05.007) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S4 DNA sequencing results of 10 mutations detected by multiplex extension and ligation-based probe amplification (MELPA). The red arrows indicate hemizygous mutations; black arrows, heterozygous mutations; yellow boxes, the sample identification numbers; asterisks, results by reverse DNA sequencing primers. The Journal of Molecular Diagnostics 2017 19, 638-650DOI: (10.1016/j.jmoldx.2017.05.007) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S5 The multiplex extension and ligation-based probe amplification (MELPA) cluster plot for 23 G6PDd single-nucleotide polymorphism (SNP) sites with a total of 106 whole-blood samples and 438 dried blood spot (DBS) samples. The x axis shows low mass peak height, and the y axis shows high mass peak height. The triangles indicate homozygous calls; rectangles, heterozygous calls. The numbers above the plots indicate cDNA nucleotide substitution of the glucose-6-phosphate dehydrogenase (G6PD) SNP variants. Mahidol (cDNA. 487G->A) is shown in the red box. The Journal of Molecular Diagnostics 2017 19, 638-650DOI: (10.1016/j.jmoldx.2017.05.007) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions