Abstract

Optimizing enzymatic reactions in next-generation sequencing (NGS) library preparation is critical for achieving high sensitivity and reproducibility, especially in low-input and single-cell workflows. However, conventional sequencing-based evaluation is expensive, time-consuming, and limit rapid iteration.

Here, we present Countable PCR, a quantitative single-molecule linkage analysis method that enables rapid and precise measurement of enzymatic efficiency without the need for sequencing. By counting and characterizing millions of individual DNA molecules in parallel, Countable PCR provides absolute quantification of molecular conversion outcomes. All measurements are performed within a single PCR, enabling fast, scalable assessment.
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We demonstrate the use of Countable PCR to evaluate reverse transcriptase (RT) performance across a panel of commercially available enzymes. This method directly measures cDNA generation, positional bias along transcripts, and template-switching rates—key factors governing library complexity and bias. Single-molecule linkage analysis revealed differences in processivity and template-switching efficiency across RTs, whereas qPCR-based evaluation was inconclusive due to target-dependent amplification bias.

Beyond reverse transcription, the same framework using Countable PCR can be extended to assess other enzymatic steps central to NGS library preparation, such as ligation. By eliminating the need for sequencing during optimization, the use of Countable PCR significantly reduces experimental cost and turnaround time, eliminating complicated bioinformatics analysis, thereby accelerating NGS assay development cycles.