Good Enough to Sequence: Fluorometry-Guided Salvage of Low-Volume and Compromised Paediatric DNA in Custom Amplicon Next-Generation Sequencing

Introduction: Amplicon-based next-generation sequencing (NGS) is widely used in clinical practice, yet few studies address the impact of fluorometric quality control and of low-volume or compromised paediatric samples on library quantification and sequencing metrics. We reframe this question around a practical goal: whether sub-optimal paediatric DNA can be reliably salvaged for custom-panel amplicon NGS. Materials and Methods: Data from 314 paediatric samples sequenced on 16 custom-designed amplicon panels (Ion Torrent S5) were analysed retrospectively by Qubit input quantity (Group A ≥ 10 ng/µL; Group B < 10 ng/µL) and Nanoquant A260/280 quality (Group C 1.7–1.9; Group D < 1.7 or >1.9), and compared against post-library quantification, mean depth, and ten coverage QC parameters. Method agreement (Bland–Altman, proportional-bias regression), effect sizes (Cliff’s δ), panel-adjusted mixed-effects models and ROC/logistic prediction of library failure were added. Results: Median input was 59.75 ng/µL by Nanoquant and 28.25 ng/µL by Qubit (p < 0.0001); median A260/280 was 1.86. Nanoquant overestimated DNA ~2.1-fold, but Bland–Altman analysis revealed wide, concentration-dependent limits of agreement (mean ratio 2.17×; 95% LoA 0.57–8.33×; proportional-bias p < 0.0001), indicating that no single correction factor is reliable. Group A comprised 91.7% (288/314) and Group B 8.3% (26/314); Group C 75.2% (236/314) and Group D 24.8% (78/314). Median post-library Qubit and mean depth were 1254 vs. 552 ng/mL (p < 0.0001) and 848× vs. 1352× (p = 0.025) for A vs. B, and 1254 vs. 1026 ng/mL (p = 0.049) and 745× vs. 1297× (p < 0.0001) for C vs. D. The higher depth in quantity Group B lost significance after adjusting for panel (mixed-model p = 0.083), identifying it as confounding. Input DNA predicted library failure (<300 ng/mL), with an ROC AUC of 0.79 and an exploratory empirical optimal threshold of ≈21 ng/µL (derived and tested in the same cohort; 14 failure events). None of the ten coverage QC parameters differed across groups. Discussion and Conclusions: Qubit quality control is strongly recommended because Nanoquant inflates readings ~2.1-fold, with no dependable correction factor. Low-volume or compromised paediatric DNA (<10 ng/µL and/or A260/280 outside 1.7–1.9) can be salvaged for amplicon NGS with defined protocol adaptations, with only ~19–23% of the worst-case samples failing library QC and comparable coverage in a small, concentration-matched representative subset (n = 5 per group) that was underpowered for formal equivalence testing. An empirical input threshold of ≈20 ng/µL, roughly double the manufacturer’s minimum, best protects against library failure.