FFPE-derived DNA breaks the usual relationship between input mass and amplifiable template, which is exactly the scenario fixed-cycle PCR handles worst. In this n6/NEB study, FFPE colon and liver DNA plus a high-quality NA12878 control were prepared at 1, 10, and 100 ng using the NEBNext UltraShear FFPE DNA Library Prep Kit and NEBNext Multiplex Oligos for Illumina, then amplified with either standard fixed-cycle PCR or iconPCR with AutoNorm. Standard PCR required three separate thermocycler runs (14, 12, and 8 cycles by input level); iconPCR ran every sample simultaneously on a single icon96, with AutoNorm dynamically assigning each well its own stop cycle — accounting for the reality that even samples at the same input mass need different cycle counts depending on degradation. The result: markedly reduced concentration variability across libraries and comparable data-quality metrics (duplication, adapter dimer, chimera, mismatch rates) to standard PCR, with far less hands-on time and no batch segregation required.

Built on the same production-scale story as n6's Ultima Genomics collaboration, this app note shows how AutoNorm's real-time, per-well control eliminates the post-PCR quantification and normalization steps that become the real cost bottleneck once sequencing itself gets cheap. Instead of fixed-cycle amplification followed by individual SPRI cleanup, quantification, and manual pooling for every sample, AutoNorm continuously tracks fluorescence in each well and stops amplification once it reaches an empirically defined optimal endpoint — so high-input samples finish early and low-input samples keep cycling until they catch up, and the whole plate comes off already balanced. The approach cuts hands-on time by up to 60%, minimizes failed libraries, and maintains balanced read distribution for whole genome sequencing, all without extra reagents or a liquid-handling robot.