Population Genetics: Absolute and Relative Quantification of DNA Using Molecular Counting

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Although many different techniques are available to measure amounts of DNA, each has tradeoffswith respect to sensitivity, selectivity, dynamic range, convenience, robustness and cost.Digital methods (digital PCR [dPCR], NanoString, single-molecule sequencing) are graduallysuperseding analog alternatives (quantitative PCR [qPCR], microarrays) because of inherentstatistical advantages. Population Genetics (PG), and others, have recently described newdigital counting methods based on next-generation sequencing (NGS) termed‘CounterStamping’. DNA molecules in a sample of interest are labelled with a DNA countersequence from a large repertoire of counter sequences. Sequencing of the counter and itsattached labelled molecule can provide information on the relative abundance of differentmolecular species and estimate the absolute molecule number. The generated data therebyallow quantification of both known and previously unidentified genetic variation, unlikeexisting methods that can generally only quantify known variation.Despite significant benefits, use of counting methods is hampered because existing protocolsare complicated and inconvenient compared to qPCR/dPCR. This proposal is to develop proofof concept to streamline and simplify PCR-based counting for absolute and relativequantification. PG’s proposed method is quicker, easier and has reduced possibility ofcontamination compared to current approaches. PG also proposes to develop algorithms tocorrect errors in counter sequences to improve the accuracy of the method. Continuingreductions in NGS costs, and increased adoption of desktop sequencers, make counting PCRpotentially disruptive to existing qPCR and dPCR technologies. Further, the proposedtechnology will drive new applications in disease monitoring, companion diagnostics andmolecular diagnostics where greater multiplexing and precise measurements are required.