Next-generation Quantitative sequencing (NQ-seq) assay procedure
For cDNA synthesis, 1 µg of total RNA was used with SuperScript IV VILO Master Mix RNA (Invitrogen, CA, USA). The cDNA (200 ng equivalent of total RNA) was amplified with primers targeting BCR::ABL1 and ABL1. The PCR conditions consisted of an initial denaturation step of 95℃ for 5 minutes, followed by 26 cycles of 98℃ for 45 seconds, 60℃ for 1 minute, 72℃ for 1 minute, and a final elongation step at 72℃ for 5 minutes. The libraries were cleaned with Agencourt AMPure XP beads (Beckman Coulter, CA, USA). These target primers for multiplex amplicon had the end tagged with index (p5 and p7) and universal sequencing adapter. The amplicons were clonally amplified and sequenced. Libraries were quantified using Qubit Fluorometric Quantification (Invitrogen), normalized, and processed for sequencing on a Nextseq 550Dx (Illumina, CA, USA) with a 75 bp, dual-indexed, paired-end according to the manufacturers' instructions.
Data Processing And Fusion Read Normalization
Raw demultiplexed NGS data were mapped to the reference genome of GRCh38 using Spliced Transcripts Alignment to a Reference (STAR) aligner tool[6]. The BCR::ABL1 fusion junctions were called using STAR 2.7.3a, and final fusion copy numbers were determined through international scale (IS) conversion[7] with fusion and ABL1 control reads. The test results used IS values by determining and maintaining conversion factors (CF) and molecular response (MR) to reduce variation and improve accuracy. BCR::ABL1 transcripts types were visualized by the Arriba[8].
BCR::ABL1 standard materials
We used the BCR::ABL1 b3a2 RNA Dilution Set (Invivoscribe, San Diego, CA) to evaluate analytical performance. This standard material consists of RNA that has been extracted from confirmed BCR::ABL1 b3a2 positive and BCR::ABL1 negative cell lines. Also, levels not contained in this reagent (copy numbers 10− 6 and 10− 7) were manually mixed by the serial dilution method from b3a2 RNA (10− 5 copy numbers) positive and negative materials.
Assay Performance Evaluation With Positive Standard Materials
Precision, linearity, limit of detection (LOD), and limit of blank (LOB) were evaluated with varying levels of BCR::ABL1 standard materials. Precision and linearity were assessed using five levels of standard materials (10− 1, 10− 2, 10− 3, 10− 4, and 10− 5). To evaluate assay precision, each material was measured seven times, including two or three replicates per single run on three separate days. Each dilution was measured three times to evaluate linearity. The LOD was estimated based on measurements at seven levels of dilution (10− 1, 10− 2, 10− 3, 10− 4, 10− 5, 10− 6, and 10− 7 dilutions) at five replicates for each. Additionally, the LOB was assessed by repeating a total of 24 times for four days using two BCR::ABL1 negative RNA specimens consisting of subtypes b2a2 and b3a2.
Patient Sample And Rna Extraction
A total of 145 clinical samples from 75 CML and 2 B-lymphoblastic leukaemia patients was included for comparison with RQ-PCR (Table 1). The samples were referred to our laboratory to detect and quantify BCR::ABL1 transcripts at diagnosis or follow-up. This study was approved by the Institutional Review Board of Severance Hospital, Yonsei University Health System (4-2019-0277). Informed consent was waived for this retrospective study that evaluated anonymized samples and data and involved no potential risk to patients. Total RNA was extracted from bone marrow aspirate or whole blood with a QIAamp RNA Blood Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s protocol. RNA quality control and quantification were performed with an Agilent 4200 Tapestation (Agilent Technologies, CA, USA).
Real-time Quantitative Polymerase Chain Reaction (Rq-pcr)
RQ-PCR analysis for BCR::ABL1 expression was performed using the Ipsogen kit and protocol (Ipsogen, Marseille, France). This protocol quantifies BCR::ABL1 copy numbers relative to a total ABL1 copy number using a real-time TaqMan method. We used 5 µL of cDNA (200 ng equivalent of total RNA) as a template in a 25 µL PCR reaction. Any BCR::ABL1 or ABL1 real-time PCR for individual samples was performed in duplicate.
Statistical analysis
Statistical analysis was carried out using R version 4.02 (R Foundation for Statistical Computing, Vienna, Austria). LOD was determined by probit analysis (95% detection rate). The correlation was analyzed based on Pearson’s correlation coefficient, and regression analysis was performed by the least-squares method. A p-value < 0.05 was considered statistically significant.