QPCR-based characterization of DNA fragmentation efficiency of Tn5 transposomes.

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 (?Ct) 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 ?Ct. Tagmentation reactions characterized with the same ?Ct exhibit the same fragment size distribution on an agarose gel. The ?Ct 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.

SUBMITTER: Rykalina V 

PROVIDER: S-EPMC6994069 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature