Abstract
Here, we describe an electrophoresis free assay for characterizing Tn5 transposomes fragmentation efficiency in a tagmentation reaction, in which double-stranded DNA is fragmented and tagged with adapter sequences. The assay uses plasmid DNA as a reference tagmentation substrate. Fragmentation efficiency is analyzed by comparative qPCR which measures the difference (ΔCt) in amplification of a specific plasmid region before and after tagmentation: more efficient fragmentation is characterized by a larger number of cleavage events within the amplified region, a delayed increase in the amplification curve and as a result, a larger ΔCt. Tagmentation reactions characterized with the same ΔCt exhibit the same fragment size distribution on an agarose gel. The ΔCt values measured can be used to quantitatively determine the relative performance of Tn5 transposome assemblies in optimization experiments and to standardize between batch variations in transposomes for use in next-generation sequencing library preparation. Moreover, the use of a reference tagmentation template added during next-generation sequencing library preparation enabled monitoring of the input DNA fragmentation. The presented qPCR-based assay is quick, contamination-safe, high-throughput and cost-efficient.
Development of a robust DNA quality and quantity assessment qPCR assay for targeted next-generation sequencing library preparation
