Fig 1: Detected mutations in the analyzed samples. (A) The detected mutations in the relapse-phase samples and their relation to the given tyrosine kinase inhibitor treatment. Timeline starting from the diagnosis. For the T315I digital droplet polymerase chain reaction (ddPCR) assay, RNA was extracted using a QIAamp RNA Blood Mini kit (Qiagen, Hilden, Germany) and 2 mg was converted to cDNA using a SuperScript VILO cDNA Synthesis Kit (ThermoFisher, Waltham, MA) according to the manufacturer’s protocol. A 40 cycle PCR amplification was performed with a forward primer located in BCR exon 1 and reverse primer in ABL1 exon 10,2 using Q5 High Fidelity DNA polymerase (New England Biolabs, Ipswich, MA) according to the manufacturer’s protocol. ddPCR was performed on the 4 dilutions (105 to 108) using ddPCR Supermix for Probes on a QX200 ddPCR system (Bio-Rad, Hercules, CA) with forward primer: GGTCTGCACCCGGGAG, reverse primer: AGGTAGTCCAGGAGGTTC, wild-type probe: HEX-CCGTTCTATATCATCACTGAGTTCATGACCTAGAACG-BHQ1 and T315I probe: FAM-CCGTTCTATATCATCAtTGAGTTCATGACCTAGAACGG-BHQ1. Cycling conditions were 95°C for 10 minutes, followed by 40 cycles of 94°C for 30 seconds and 60°C for 60 seconds. (B) The detected mutations in the diagnosis-phase samples. Copy-number alterations in IKZF1, CDKN2A/B, PAX5, EBF1, ETV6, BTG1, and RB1 genes were detected with SALSA MLPA Probemix P335 ALL-IKZF1 kit (MRC Holland, Amsterdam, the Netherlands). The assay was performed according to the manufacturer’s protocol and the data were analyzed with Coffalyser.Net software (MRC Holland, Amsterdam, the Netherlands). Both diagnosis and relapse-phase samples were analyzed with a targeted next-generation sequencing gene panel consisting of 75 leukemia-associated genes. 150 ng of genomic DNA was processed according to SeqCap EZ HyperCap Workflow User’s Guide, v2.1 Dec 2017 Enzymatic Fragmentation (Kapa Biosystems, Inc., Wilmington, MA, USA) using Unique Dual Index adapters by IDT (Integrated DNA Technologies, Coralville, IA, USA). Library quality check was performed using LabChip GX Touch HT High Sensitivity assay (PerkinElmer, USA). 7 cycles were used for precapture amplification. SeqCap custom captures (170621_HG38_ALL-75G_EZ_HX3) were performed in 6-7 samples multiplexed DNA Sample Library Pools using 600 µg of each library. 10 cycles were used for post capture amplification. The captured library pools were quantified for sequencing using KAPA Library Quantification Kit (KAPA Biosystems, Wilmington, MA, USA) and 2100 Bioanalyzer High sensitivity kit. The samples were sequenced in 3 batches. The first batch was sequenced with Illumina HiSeq2500 system in HiSeq high output mode using v4 kits (Illumina, San Diego, CA, USA). Read length for the paired-end run was 2x101 bp. The following batches were sequenced with Illumina NovaSeq system using S4 flow cell with lane divider (Illumina, San Diego, CA, USA) and v1.0 chemistry. Read length for the paired-end run was 2x101.