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Interferon λ 3 and 4 Genotyping Using High-Resolution Melt Curve Analysis Suitable for Multiple Clinical Sample Types  François M.J. Lamoury, Sofia Bartlett,

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Presentation on theme: "Interferon λ 3 and 4 Genotyping Using High-Resolution Melt Curve Analysis Suitable for Multiple Clinical Sample Types  François M.J. Lamoury, Sofia Bartlett,"— Presentation transcript:

1 Interferon λ 3 and 4 Genotyping Using High-Resolution Melt Curve Analysis Suitable for Multiple Clinical Sample Types  François M.J. Lamoury, Sofia Bartlett, Brendan Jacka, Behzad Hajarizadeh, Jason Grebely, Gail V. Matthews, Gregory J. Dore, Tanya L. Applegate  The Journal of Molecular Diagnostics  Volume 17, Issue 5, Pages (September 2015) DOI: /j.jmoldx Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

2 Figure 1 High-resolution melting (HRM) analysis curves. HRM analysis enables homogeneous genotyping without probes, even when the sequence change is only a single base. With saturation dyes, the PCR product is labelled along its entire length so that all melting domains are detected. Single-base genotyping is identified by the difference of melting curves as shown by the normalized melting curve (left graphs) and temperature shift (right graphs) for rs , rs , and rs , with favorable homozygotes as green curves (TT, CC, and TT/TT, respectively), heterozygotes in blue curves (GT, CT, and ΔG/TT, respectively), and unfavorable genotype as red curves (GG, TT, and ΔG/ΔG, respectively). The assay was performed using the Roche LightCycler 480 System and the HRM analysis performed with the Roche Gene Scanning software. The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx ) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

3 Figure 2 The number of cycles (Cp) values and genotype success rate in clinical samples for single-nucleotide polymorphisms (SNPs) rs , rs , and rs Every sample type high-resolution melting analysis (HRM) result for genotyping were compared to the result from sequencing using buffy coat samples, and the calculated success rate is shown on top of each sample measurement distribution. The y axis shows the Cp needed for PCR amplification. Cp per sample type is inversely proportional to DNA concentration. A threshold of amplification has been estimated for every SNP for which every cohort samples would need to be amplified under this threshold for 100% success rate genotyping. The threshold has been estimated in our condition at 37.2 cycles for rs , 38.4 cycles for rs , and 39.7 cycles for rs DBS, dried blood spots. The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx ) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

4 Figure 3 Characteristics of Australian Trial in Acute Hepatitis C (ATAHC) participants among those with available genotypes for IFNL single-nucleotide polymorphisms (SNPs) rs , rs , and rs Data for IFNL rs , rs , and rs polymorphisms were available for 109, 106, and 109 individuals, respectively. The genotype frequency is shown for each SNP. The ethnicity distribution of the ATAHC participants tested is 93% Caucasian (102 participants of 110), 4% Asian (4 of 110), 2% indigenous (2 of 110), 1% African ancestry (1 of 110), and 1% defined as mixed or other (1 of 110). PBMCs, peripheral blood mononucleocytes. The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx ) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

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