Analysis of simple and complex variants and biomarkers for comprehensive genomic profiling using a single NGS workflow for FFPE and cfDNA samples.
Introduction: Current comprehensive genomic profiling (CGP) approaches are challenging as they require two separate eluates and a minimum of two workflows, one for DNA and another for RNA. To overcome these challenges, we developed the QIAseq Pan Cancer Multimodal Panel, a streamlined, single workflow and integrated bioinformatics for the analysis of a wide range of variants and biomarkers for oncology research.
Methods: Panel performance was verified using FFPE and cfDNA reference samples from SeraCare. Tumor nucleic acid (TNA) from FFPE samples were extracted using modified QIAGEN protocols. TNA (20–50 ng) was used as input for library construction. Libraries were constructed using the Pan Cancer Panel workflow, quantified and sequenced either on a MiSeq® or NextSeq® instrument. FASTQ files were processed with preconfigured analysis pipelines using the QIAGEN® CLC Genomics Workbench.
Results: All variants in reference samples were detected.
DNA: Single-nucleotide variants (SNVs) and Insertions-Deletions (InDels) were detected in cfDNA samples at variant allele frequencies (VAF) <1% and in FFPE samples down to 1% VAF. Complex variants such as the CALR type-1 deletion (52-bp deletion) and FLT3 ITDs were also detected. The panel called two insertions in CEBPA, a GC-rich gene. Analysis of copy number variations (CNVs) showed that the panel can call CNVs at both the gene and exon levels by accurately calling six additional copies of the EGFR, MET and MYCN genes. Tumor mutational burden (TMB) scores were accurately called as ‘low’ or ‘high’ in the respective TMB reference samples.
RNA: All fusions, exon skipping (ES), and alternatively spliced variants (ASVs) covered by the panel were correctly called, including, but not limited to, NTRK1, NTRK2, NTRK3, BCR, ALK and RET fusions. The panel was designed to cover only one partner of all detected fusions.
Conclusion: These results provide proof-of-principle evidence that the QIAseq Pan Cancer Panel enables CGP from FFPE and cfDNA samples using a single workflow with TNA or DNA as input for both solid tumors and hematologic malignancies.
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