Anna Dolnik from the Charite University Medical Center in Berlin, Germany, presented at London Calling 2019 on “Genomic profiling in acute myeloid leukemia with complex karyotype.” They presented data on the survival of over 5,100 patients with acute myeloid leukemia (AML). The WHO in 2016 revised the classification of AML. A newly diagnosed AML patient, Dolnik explained, would undergo molecular screening at a reference lab. Then, genotype-adapted treatment would be determined. Rapid screening is critical to diagnosing high-risk AML. The model cell line K-562 with Philadelphia chromosome t(9;22) is used for karyotyping. First, cells need to be cultivated, then fixed, and then stained. Dolnik noted this process is tedious and often challenging. With genomic DNA sequencing, a ligation-based library preparation takes one hour, and sequencing ~18 hours. The throughput of one flow cell they obtained with the LSK 109 kit is between 10-14 Gb, obtaining 2.5-3.6 coverage. Two tools are used for the bioinformatics workflow: ngmlr aligner and IGB Tools. The ACE package was used to estimate absolute copy number variations from low-coverage sequencing. Dolnik shared examples of complex karyotypes based on nanopore sequencing. The major chromosome aberrations are evident. A transcriptome workflow was used to detect translocations. Seventy-five ng of mRNA were obtained from Dynabeads. The SQK-RNA002 protocol and kit were then used. The minimap2 aligner and BLASTn were used after filtering for low complexity. The length of alignment was set to at least 250 bp. With this pipeline, they discovered translocation events and fusion transcripts. Dolnik concluded that nanopore sequencing of genomes at low coverage can provide important copy number data within 24 hours. Structural variants, including translocations and inversions, are challenging, and direct transcriptomics allows for the discovery of known translocations. This session complicated and highlighted how AML has challenging genomics that can be analyzed with nanopore sequencing techniques. Data can then be used to inform patient treatments.
