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Simultaneous Detecting Methylation and Genetic Variations of BCR-ABL1 Fusion Gene by Nanopore Cas9-Targeted Sequencing
30 Pages Posted: 22 Jun 2023
More...Abstract
Background: The structure variations (SVs) of BCR-ABL1 fusion gene are important companion diagnostic biomarkers for leukemia patient, and its kinase domain’s single nucleotide variations (SNVs) and promoter aberrant methylations (5mC) are also associated with drug response. However, there is no technique that can detect these events simultaneously.
Methods: Based on nanopore Cas9-targeted sequencing (nCATS) protocol, we designed 4 crRNAs to target the interest of regions, including most breakpoints, ABL1 kinase domain and BCR promoter, of BCR-ABL1gene for library construction. The sequencing data was obtained from R9.4 flow cell with running on GridION sequencer, and analyzed by Cas9-nanopore pipeline.
Findings: We found that coverages of the targeted regions were at 300×~500× and characterized a b3a2 subtype of BCR-ABL1 fusion gene. Furthermore, we evaluated Bcftools, Clair3 and Freebayes software for SNVs identification and Megalodon software for 5mC methylation. Our result showed that Bcftools had the best performance for SNVs detection in ABL1 kinase domain with 5 annotated SNPs identified. We also observed hypomethylated status in upstream half and hypermethylated status in the downstream half of the CpG island in BCR promoter region. Additionally, we developed a pipeline, named as Cas9-nanopore, to simplify the analysis of SVs, SNVs, and 5mC for nCATS data and confirmed its performance with applications of the nCATS data of HTT gene from K562 cells.
Interpretation: This study established an nCATS technology specifically for detecting BCR-ABL1 fusion gene, its KD region mutations and promoter 5mC modification, providing a more comprehensive and efficient detection for hematological tumors with high incidence of BCR-ABL1 fusion gene, which has potential clinical application value and cutting-edge technological innovation.
Funding: The Jiangxi Province's “Double Thousand Plan” Innovation Leading Talent (Grant#: jxsq2019101060), the Youth Jinggang Scholars Program in Jiangxi Province and the National Natural Science Foundation of China (Grant No. 82200653).
Declaration of Interest: The authors declare no competing interests.
Keywords: BCR-ABL1 fusion gene, nanopore Cas9-targeted sequencing, genetic variation, BCR promotor methylation, companion diagnostics biomarker
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