DNA Marker seqencing of human HPV associated head and neck squamous cell carcinoma pair normal patient tissue
We developed a custom Ion Torrent AmpliSeq targeted DNA sequencing panel specifically designed to interrogate key genes involved in the NF-kappaB signaling pathway, including TRAF3, CYLD, TRAF2, NF-kappaBIA, TNFAIP3, TRAF6, BIRC2, BIRC3, MYD88, and MAP3K14. The panel comprises 250 overlapping amplicons arranged in a 2-primer pool format, achieving an overall coverage of 93.65 percent across the 24.86 kb target region. Genomic DNA was isolated from formalin-fixed paraffin-embedded (FFPE) macro-dissected tumor and matched normal (non-cancerous) tissues at the UNC Lineberger Comprehensive Cancer Center (LCCC) Translational Genomics Lab (TGL) using the Maxwell 16 MDx Instrument (Promega AS3000). DNA concentration was quantified using a Qubit 3.0 fluorometer (Life Technologies Q33216). Library preparation and template generation were carried out on the Ion Chef system, followed by sequencing on the Ion GeneStudio S5 Prime platform at Mako Genomics. Samples were multiplexed across Ion 510, 520, and 530 chips to optimize throughput. Subsequent data analyses were conducted using the raw sequencing data files provided by Mako Genomics.