Overview and evaluation ofBCR-ABL1mutation detection using the PacBio sequencing. A) Schematic overview of the workflow. Total RNA was used to generate a 1578 bp long BCR-ABL1 fusion transcript cDNA amplicon. PacBio adaptors were ligated to the amplicon and the resulting library was sequenced on a PacBio SMRT cell. The data analysis detected BCR-ABL1 mutations down to a frequency of at least 1%, as well as the different clones present in the sample. B) Alignment of reads to the BCR-ABL1 reference sequence. The grey area shows reads for a CML sample (patient P3,49 months) produced from one SMRT cell on the PacBio RSII instrument. The sequencing generates a uniform coverage of about 10,000X over the entire reference sequence. The red vertical line indicates the presence of a T315I mutation, present in 88.9% of the reads. The mutation F359C was also detected in this sample at a frequency of 4.2% and can be seen as a faint vertical line. C) Results of a dilution experiment of the CML sample in panel B) (P3, 49 m). The leftmost bars show mutation rates of T315I (red) and F359C (blue) for the undiluted sample. To the right are observed mutation frequencies for a dilution series where the expected T315I frequency reached 50%, 10%, 1% and 0.5%. The expected frequencies of T315I and F359C are shown in red and blue letters, respectively. Positions marked with ‘X’ indicate mutations not detected by the PacBio sequencing.