Pb1709: a complete digital karyotype of the b-cell leukemia reh cell line resolved by long-read sequencing

Topic: 1. Acute lymphoblastic leukemia - Biology & Translational Research Background: The B-cell acute lymphoblastic leukemia (ALL) cell line REH, with the t(12;21) ETV6-RUNX1 translocation, is known to have a complex karyotype defined by a series of large-scale chromosomal rearrangements. Taken from a 15-year-old at relapse, the cell line offers a valuable model for the study of high-risk pediatric B-ALL patients. However, traditional karyotyping techniques and short-read next-generation sequencing alone have been unable to fully resolve the aberrations in this widely used genome. Aims: We explore the use of long-read PacBio and Oxford Nanopore whole genome sequencing (WGS) and IsoSeq RNA-sequencing alongside short-read sequencing to create a comprehensive digital karyotype of the REH cell line. Methods: DNA and RNA were extracted from the human pre B-cell ALL cell line REH and subjected to long- and short-read sequencing alongside traditional g-banding. In total, we generated three WGS datasets (Illumina PCR-free on HiSeq X; Oxford Nanopore Ultralong on PromethION24; PacBio HiFi on Sequel II) and three RNA-seq datasets (TruSeq stranded total RNA on NovaSeq 6000; 2x PacBio IsoSeq RNA-seq on Sequel II, with standard- and full-length transcripts). The short-read datasets were processed using nf-core pipelines (sarek with TIDDIT; rnafusion). The long-read datasets were mapped to GRCh38 using Minimap2, with structural variant (SV) calling using Sniffles2 and fusion gene calling using cDNA_Cupcake and JAFFAL. The SV and fusion gene candidates were programmatically filtered on quality, read support and size criteria (SVs > 100 kbp), and high-confidence candidates were confirmed manually in IGV and Ribbon. Results: WGS refined the breakpoints of known and expected aberrations in the REH cell line and clarified the molecular traits of disrupted ALL-associated genes BTG1 and TBL1XR1, as well as the glucocorticoid receptor NR3C1. Several previously underreported structural variants were also uncovered, including deletions affecting the ALL-associated genes VPREB1 and NFATC1. In total, the consensus callset (intersection of all three WGS callsets) found 2100 SVs, and 23 SVs > 100 kbp were manually confirmed. Meanwhile, transcriptome sequencing identified seven fusion genes within the genomic breakpoints, including two in-frame fusions (ETV6-RUNX1 and RUNX1-PRDM7), two fusions resulting from the balanced translocation t(5;12), and the GC receptor-disrupting NR3C1-ARHGAP26. Summary/Conclusion: Our findings highlight the need for comprehensive genomic analysis of commonly used and perturbed cell lines. Further, we underscored the importance of the increased informational context of long reads and how they can identify critical biologically important SVs. Finally, we have cataloged multiple aberrations in REH that are frequently observed in high-risk ALL patients, making this cell line a highly relevant model for researchers investigating the biological underpinnings of relapse and GC resistance. Together, our extensive whole-genome investigation makes high-quality open-source data available to the leukemia genomics community. Keywords: B cell acute lymphoblastic leukemia, Cell line, Complex aberrant karyotype, OMICS