Sanger sequencing detection limit and single-cell MYD88 PCR analysis. (A) MYD88 PCR was conducted to amplify both MYD88^L265P and MYD88^WT alleles. Amplified sequences were verified to encode either CCG or CTG corresponding to the mutant MYD88^L265P and MYD88^WT alleles, respectively. (B) The PCR amplicons were mixed in titrating proportions to determine the Sanger sequencing detection limit. The lowest detection limit of Sanger sequencing in detecting MYD88^L265P and MYD88^WT MYD88 alleles was ∼30%. (C) Single-cell MYD88 PCR was conducted on a clinical DLBCL sample with 15 single cells (1-15); 14 of 15 cells gave a PCR band of ∼200 bp. (D) Multiple sequence alignment of the excised and sequenced PCR amplicon showed a T → C point mutation in 5 representative cells from clinical samples but not in Pfeiffer cells (a germinal center B cell–like DLBCL exhibiting the MYD88^WT allele). (E) Electropherogram showed clean and well-resolved sequencing peaks for MYD88^L265P and MYD88^WT. The black arrows throughout indicate the location of the point mutation. NTC, no template control; WT, wild type.