Stephanie Chrysanthou from the Memorial Sloan Kettering Cancer Center in the US spoke at the Nanopore Community Meeting about “Detection of germline alterations in homologous recombination repair genes by adaptive sampling.” They mentioned that pathology comes from the Greek word that is the study of suffering! The Memorial Sloan Kettering Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) is a high-throughput targeted humor-sequencing test, noted Chrysanthou. It has been approved by the NY State Department and the FDA. They use the approach for research purposes and clinical trial matching. The DNA is obtained from consenting patients and used for Illumina sequencing library prep. Chrysanthou explained that the detection of pathogenic germline variants is important for cancer surveillance, genetic counseling, clinical trials, and target therapies. For the pilot, the team wanted to use adaptive sampling to use a .bed file with genomic target sequences, a reference fasta file, and DNA. They used HMW DNA and the LSK-114 kit with 1 ug of DNA. Chrysanthou explained that they created a target bed file with the MSK-IMPACT 505+3 housekeeping genes with 20 kb buffer on either side! They load 80 fmol with one sample per PromethION flow cell. The pod5 files are filtered, on-instrument SUP basecalling is used followed by alignment to GRCh38 reference using Minimap2. EPIE2ME wf-human-variation is used to identify single nucleotide variants (SNV) and structural variants (SVs). Chrysanthou shared examples of patient sequencing results from this pipeline. THe team tested several variant callers and found that Severus worked best for their samples. Fifteen solid tumor blood samples have been sequenced with ~30X coverage. Currently, 100% consensus has been achieved with the Illumina data. Chrysanthou said that the on-instrument analysis and ONT tools were the best options. The team has now processed 25 samples and continues to improve the analyses. They want to optimize the extraction and library prep to take one day for a three-day turnaround with sequencing and analysis.
