GSM4826572: TS139_02_165; Homo sapiens; OTHER
Genomic DNA was extracted from 5x106 cells using a QIAamp DNA mini kit (Qiagen, Manchester, UK). 1μg of genomic DNA was sheared with a g-TUBETM (Covaris, Inc., 520079) using 50μl sample volume and a 30s spin at 11,000rpm followed by inversion and a further 30s at 11,000rpm. Barcoded libraries were prepared from 45μl of fragmented DNA using a Ligation Sequencing Kit (Oxford Nanopore Technologies, SQK‐LSK108) according to a 1D Sequence Capture protocol with the following modifications: the pre-hybridisation PCR was replaced by a custom PCR with 6 cycles using Herculase II fusion DNA polymerase (Agilent). The subsequent AMPure XP purification was performed using a 0.6x bead to sample volume ratio. All purified product was used for the hybridization, which was performed using a SureSelect Target Enrichment System following the manufacturer's protocol (https://www.agilent.com). The oligonucleotide library was designed using the online Agilent SureDesign service (https://earray.chem.agilent.com/suredesign/home.htm). The library targeted a 500kb region that included ABCB1 and upstream neighbouring genes (RUNDC3B, SLC25A40, DBF4 and ADAM22; chr7: 87,132,949-87,632,948 hg19). Following pull-down, the post-capture amplification was replaced by a custom PCR with 14 cycles as described above. Amplified libraries were quantified by Qubit (Invitrogen) and TapeStation 2200 (Agilent Technologies). An additional amplification was performed using 10 cycles of PCR and the product was quantified. Amplified libraries were again quantified by Qubit and 1μg of each library used for the End-prep. AMPure XP purification was performed using a 0.47x bead to sample ratio. Four end-prepped libraries were pooled into one adapter ligation reaction using 125ng of each, as measured by Tapestation. A 0.47x bead to sample ratio was used for the AMPure XP bead binding. Sequencing was performed on the MinION using the R9.4 Flowcell (Oxford Nanopore Technologies, FLO-MIN106) according to the manufacturer's instructions. Nanopore sequencing of THP-1_R generated 0.92M reads with a mean read length of 1901bp and a maximum read length of 224.8kb. The coverage of the targeted region was 58.1x. Nanopore sequencing of relapsed AML generated an average of 1.28M reads per sample with a mean read length of 1484bp. Mean coverage of the targeted region was 45.3x.