PCR amplification introduces taxon-specific distortions in 16S rRNA gene amplicon sequencing, but these distortions can be quantified and partially corrected by running a multi–cycle calibration on a representative pooled community. A practical question, however, is whether a calibration estimated once can be reused across later sequencing runs, different thermocyclers, or new primer lots. In this study we replicated the standard four-step calibration procedure (pooled DNA → multiple PCR cycle numbers → sequencing → log-ratio linear fit) across multiple PCR runs, two thermocycler models, and two lots of the same 16S V4 primer pair, all using the same master DNA pool. We modeled taxon-level amplification slopes with a hierarchical compositional model that decomposed variation into global (taxon-intrinsic), run-level, instrument-level, and primer-lot components. Slopes were highly reproducible within runs, and run-to-run as well as instrument-to-instrument differences were modest, indicating that calibrations are generally portable under unchanged reagents. Primer-lot changes, however, produced the largest and most systematic shifts, especially for low-abundance or mismatch-prone taxa, often pushing leave-one-condition-out predictions above a practical Aitchison-distance threshold. We therefore propose a lightweight QC gate: include one pooled-community sample on each run, predict its composition from the archived calibration, and reuse the calibration only if the compositional error is below the threshold. This provides an operational recipe for labs to treat PCR-bias calibration as a reusable asset while still detecting non-portable situations triggered by reagent changes.
PCR amplification introduces taxon-specific distortions in 16S rRNA gene amplicon sequencing, but these distortions can be quantified and partially corrected by running a multi–cycle calibration on a representative pooled community. A practical question, however, is whether a calibration estimated once can be reused across later sequencing runs, different thermocyclers, or new primer lots. In this study we replicated the standard four-step calibration procedure (pooled DNA → multiple PCR cycle numbers → sequencing → log-ratio linear fit) across multiple PCR runs, two thermocycler models, and two lots of the same 16S V4 primer pair, all using the same master DNA pool. We modeled taxon-level amplification slopes with a hierarchical compositional model that decomposed variation into global (taxon-intrinsic), run-level, instrument-level, and primer-lot components. Slopes were highly reproducible within runs, and run-to-run as well as instrument-to-instrument differences were modest, indicating that calibrations are generally portable under unchanged reagents. Primer-lot changes, however, produced the largest and most systematic shifts, especially for low-abundance or mismatch-prone taxa, often pushing leave-one-condition-out predictions above a practical Aitchison-distance threshold. We therefore propose a lightweight QC gate: include one pooled-community sample on each run, predict its composition from the archived calibration, and reuse the calibration only if the compositional error is below the threshold. This provides an operational recipe for labs to treat PCR-bias calibration as a reusable asset while still detecting non-portable situations triggered by reagent changes.
