title: WGS of human PBMC: P2 clonal
design: For the two patients with an IGHV1-69 B cell clone (P1 and P4), the G6 antibody was used to sort the expanded B cell clone (G6+ IgK+ B cells) and the control polyclonal B cell population (G6 B cells) (Figure 2B). For the other two patient B cell clones encoded by heavy chains for which no anti-idiotypic antibody exists (P2; IGHV4-59 and P3; IGHV3-7), the B cell clone was identified and sorted from the IgM+ kappa light chain restricted memory B cell population (CD27+ IgM+ IgK+ B cells) and the control polyclonal B cell population consisted of all remaining B cells that did not co-express IgM and IgK (Figure 2B). These gating strategies were predicted to result in purity >90% (>95% for the IGHV1-69 B cell clones). Approximately 30,000 cells (minimum 8,000) of either the B cell clone or polyclonal B cells of the patients were directly FACS sorted into 100L ALT lysis buffer (Qiagen MicroKit). gDNA was extracted as per manufacturers instructions (Qiagen MicroKit). Approximately 2/3 of gDNA was taken to generate DNA libraries (minimum 10ng), with remaining gDNA to be stored for variant confirmation. DNA libraries for whole genome sequencing were generated using the Kapa Hyper Plus Kit (Roche) for low input DNA. DNA libraries were generated according to manufacturer instructions, with the following modifications: fragmentation for 17 minutes, adapter ligation for 1 hour and PCR amplification with 3-5 cycles depending on starting DNA amount (5 cycles for 10ng DNA). DNA libraries were sequenced on a HiSeqX instrument (Illumina) with 150bp paired end reads at 30X coverage for the polyclonal population and at 60X coverage for the B cell clone. WGS sequencing quality was verified by calculating the coverage (unique reads) per nucleotide base over 180 genes at exonic locations totalling 1,430kb, using the BAM files generated from somatic variant analysis and BEDtools.