Sample preparation and next generation sequencing

DNA was isolated with QIAamp DNA Mini Kit (Qiagen, Netherlands) from either deep‐frozen BM mononuclear cells or serum‐free PB cells. The custom panel based on the Illumina TruSeq Custom Amplicon Low Input assay including 594 amplicons in 56 genes and the next generation sequencing were applied as previously reported. ⁹ The median coverage across all samples was 4354× before unique molecular identifier (UMI) family clustering and 675× with inclusion of UMIs.

Variant calling was performed as previously reported. ⁹ Variants with a VAF of 0.45 to 0.55 and ≥0.95 were not considered to exclude potential germline variants. The identified variants were processed and filtered using the r programming language (Ver. 3.3.1). Common SNPs with a minor allele frequency ≥ 5% in either the 1000 Genome Project, Exome Variant Server or ExAC databases were excluded. In addition, variants with a low mapping quality (<20) and variants occurring in 5% or more of the subjects in the studied cohort were considered as technical artefacts and excluded. Furthermore, variants covered with less than 100 reads in at least one set of amplicons (CAT A or CAT B), variants called with only one of the set of amplicons (CAT A or CAT B), SNPs identified as common in the dbSNP database (≥1% in the human population), and variants with sequence ontology terms ‘LOW’ or ‘MODIFIER’ were filtered out.