Seqcap adapter kit a and b

A high sensitivity TP53 assay was established based on serial 10-fold dilutions of DNA from patient samples with donor DNA. By including a dilution step for each sequenced sample, background noise was filtered and an LOD was established at 0.2% VAF (Online Supplementary Methods, Online Suppplementary Table S1 and Online Suppplementary Figure S2). For each patient, DNA extracted from PBMCs was analyzed undiluted and diluted 20% (dilution factor 5) in DNA derived from the SU-DHL4 cell line. A known TP53mut (p.Arg273Cys) harbored in the cell line DNA acted as internal control of dilution grade. Using 100 ng gDNA per reaction, TP53 exons 2–10 incl. 2 bp intronic overlap for splice sites were amplified with 30 cycles of PCR using Phusion® HSII High-Fidelity DNA polymerase (Life Technologies, Waltham, MA, USA). A list of the primers used is provided in Online Supplementary Table S2. In brief, library preparation was performed following manufacturer protocol KAPA DNA Library Preparation (Nimblegen). Using SeqCap Adapter Kit A and B (Roche NimbleGen) or NEXTflex™ DNA Barcodes 96 (Bioo Scientific, Austin, TX, USA), libraries were pooled (24 or 96 samples per lane) and sequenced as paired-end on a HiSeq2500 using HiSeq® SBS Kit v.4 (2x125 base PE, Illumina) to obtain a minimum target read depth of 20,000x.