Gong, BinshengLi, DanKusko, RebeccaNovoradovskaya, NataliaZhang, YifanWang, ShangziPabon-Peña, CarlosZhang, ZhihongLai, KevinCai, WanshiLoCoco, Jennifer SLader, EricRichmond, Todd AMittal, Vinay KLiu, Liang-ChunJohann, Donald JWilley, James CBushel, Pierre RYu, YingXu, ChangChen, GuangchunBurgess, DanielCawley, SimonGiorda, KristinaHaseley, NathanQiu, FujunWilkins, KatherineArib, HananeAttwooll, ClaireBabson, KevinBao, LonglongBao, WenjunLucas, Anne BergstromBest, HunterBhandari, AmbicaBisgin, HalilBlackburn, JamesBlomquist, Thomas MBoardman, LisaBurgher, BlakeButler, Daniel JChang, Chia-JungChaubey, AlkaChen, TaoChierici, MarcoChin, Christopher RClose, DevinConroy, JeffreyCooley Coleman, JessicaCraig, Daniel JCrawford, Erindel Pozo, AngelaDeveson, Ira WDuncan, DanielEterovic, Agda KarinaFan, XiaohuiFoox, JonathanFurlanello, CesareGhosal, AbhisekGlenn, SeanGuan, MeijianHaag, ChristineHang, XinyiHappe, ScottHennigan, BrittanyHipp, JenniferHong, HuixiaoHorvath, KyleHu, JianhongHung, Li-YuanJarosz, MirnaKerkhof, JenniferKipp, BenjaminKreil, David PhilipŁabaj, PawełLapunzina, PabloLi, PengLi, Quan-ZhenLi, WeihuaLi, ZhiguangLiang, YuLiu, ShaoqingLiu, ZhichaoMa, CharlesMarella, NarasimhaMartín-Arenas, RubénMegherbi, Dalila BMeng, QingchangMieczkowski, Piotr AMorrison, TomMuzny, DonnaNing, BaitangParsons, Barbara LPaweletz, Cloud PPirooznia, MehdiQu, WubinRaymond, AmeliaRindler, PaulRingler, RebeccaSadikovic, BekimScherer, AndreasSchulze, EgbertSebra, RobertShaknovich, RitaShi, QiangShi, TieliuSilla-Castro, Juan CarlosSmith, MelissaSolis-Lopez, MarioSong, PingStetson, DanielStrahl, MayaStuart, AlanSupplee, JuliannaSzankasi, PhilippeTan, HaowenTang, Lin-YaTao, YonghuiThakkar, ShraddhaThierry-Mieg, DanielleThierry-Mieg, JeanThodima, Venkat JThomas, DavidTichy, BorisTom, NikolaVallespin-Garcia, ElenaVerma, SumanWalker, KimbleyWang, CharlesWang, JunwenWang, YexunWen, ZhiningWirta, ValtteriWu, LeihongXiao, ChunlinXiao, WenzhongXu, ShibeiYang, MaryYing, JianmingYip, Shun HZhang, GuangliangZhang, SaZhao, MeiruZheng, YuantingZhou, XiaoyanMason, Christopher EMercer, TimothyTong, WeidaShi, LemingJones, WendellXu, Joshua2021-10-042021-10-042021-04Genome Biol. 2021; 22(1):1091474-760Xhttp://hdl.handle.net/20.500.12105/13424Targeted sequencing using oncopanels requires comprehensive assessments of accuracy and detection sensitivity to ensure analytical validity. By employing reference materials characterized by the U.S. Food and Drug Administration-led SEquence Quality Control project phase2 (SEQC2) effort, we perform a cross-platform multi-lab evaluation of eight Pan-Cancer panels to assess best practices for oncopanel sequencing. All panels demonstrate high sensitivity across targeted high-confidence coding regions and variant types for the variants previously verified to have variant allele frequency (VAF) in the 5-20% range. Sensitivity is reduced by utilizing VAF thresholds due to inherent variability in VAF measurements. Enforcing a VAF threshold for reporting has a positive impact on reducing false positive calls. Importantly, the false positive rate is found to be significantly higher outside the high-confidence coding regions, resulting in lower reproducibility. Thus, region restriction and VAF thresholds lead to low relative technical variability in estimating promising biomarkers and tumor mutational burden. This comprehensive study provides actionable guidelines for oncopanel sequencing and clear evidence that supports a simplified approach to assess the analytical performance of oncopanels. It will facilitate the rapid implementation, validation, and quality control of oncopanels in clinical use.engVoRhttp://creativecommons.org/licenses/by/4.0/Atribución 4.0 Internacional3386334422110910.1186/s13059-021-02315-0Genome biologyopen access