Kapa2g multiplex dna polymerase

Mutations interrogated by SCG are in Supplementary Table 3. Single cells were flow-sorted into 96 well plates containing 2μl of phosphate buffered saline. WGA was carried out using Single Cell RepliG kit (Qiagen, Crawley UK). Briefly, following cell lysis, alkali denaturation and neutralisation, a master mix containing Phi29 polymerase, dNTPs and random oligonucleotide primers was added. WGA was carried out at 30°C for 8 hours followed by heat inactivation. Diluted (1:20) amplified DNA was used in single or multiplex PCR using primers relevant to the sample and high Fidelity Phusion Taq polymerase (NEB, UK) or KAPA2G Multiplex DNA Polymerase (KAPA Biosystems, UK). Barcoding and sequencing oligonucleotides were added by PCR and sequencing performed on Illumina MiSeq (Illumina, Saffron Walden, UK). ~94% of reads had Phred scores of >30. A threshold of 50 reads was set for analysis inclusion. VAF thresholds for determining detection of mutations were determined by genotyping 48 single cells derived from normal bone marrow, and set at the 95% confidence level (mean ± 1.96 x standard error of mean (SEM); i.e. <5% chance of false positive, Supplementary Table 6).

Mutations detected by targeted re-sequencing in hematopoietic cell populations are in Supplementary Table 3. Average and range of read depths for each mutation is shown in Supplementary Table 4. DNA was extracted (DNeasy Blood and Tissue extraction kit, #69506 Qiagen Manchester UK) from bulk and flow-sorted cells from patient samples. Where material was limiting, whole genome amplification (WGA, RepliG, Qiagen, UK)) was performed. Targeted PCR was performed using high Fidelity Phusion Taq polymerase (NEB, UK) or KAPA2G Multiplex DNA Polymerase (KAPA Biosystems, UK) with 10ng of gDNA. Primers used are in Supplementary Table 7. A second PCR reaction added Illumina barcodes and sequencing oligonucleotides prior to sample purification, quantitation, pooling and library preparation for sequencing on Illumina MiSeq (Illumina, Saffron Walden, UK). Raw data (average depth ~996x) was aligned using Stampy (v1.0.20)^{53 (link)}. A minimum sequencing depth of 100 was set as a threshold for inclusion of data for analysis. >94% of reads had Phred scores of >30. VAF was obtained using the Unix ‘grep’ (globally search regular expression and print) command line.