LAMPrey: a standardised method for analysing quantitative LAMP reactions using the inflection cycle threshold

Quantitative loop-mediated isothermal amplification (qLAMP) is a gene expression quantification method that has gained popularity in recent years, particularly in disease identification, including during the recent SARS-CoV-2 pandemic. Unlike conventional quantitative PCR (qPCR), qLAMP features reaction kinetics that may diverge from sigmoidal expectation, and may not include ROX dye in commercial kits. Determining cycle threshold (Ct or Cq) values through automatic thresholding may therefore produce inaccurate results, and the nature of these thresholds complicates comparability between studies and softwares. We introduce a new method for transforming sigmoidal amplification curves into inflection cycle threshold curves (iCt) to address issues with auto thresholds and analysis of qLAMP. This method is implemented as a set of R functions named LAMPrey, suitable for analysis of both qPCR and qLAMP reactions performed in the two most commonly used real-time thermocyclers. We simulate qLAMP amplification differences, demonstrate that iCt and Ct methods perform equivalently for conventional qPCR with an Illumina library quantitation kit, and show that iCt values outperform Ct and the sigmoid curve-fitting metric FDM for quantifying 2416 qLAMP reactions in of zebrafish embryos. All scripts developed for this article are available at https://github.com/dodged13/LAMPrey.