Facilitated Large-Scale Sequence Validation Platform Using Tn5-Tagmented Cell Lysates.

A typical molecular cloning procedure requires Sanger sequencing for sequence validation, which is cost-prohibitive and labor-intensive for large-scale clone analysis in genotype-phenotype studies. Here we present the cost-effective clone analysis platform TnClone, which uses next-generation sequencing based on Tn5 tagmentation to rapidly analyze a large number of clones from cell lysates. This method bypasses the extensive plasmid purification step. We also developed a user-friendly graphical user interface and provided general guidelines for conducting validation experiments. We tested our program with 1023 plasmids (222 from cell lysates and 801 from purified clones) and achieved 92% and 99.3% sensitivity with cell lysates and purified DNA, respectively. Our platform provides rapid turnaround with minimal hands-on time for secondary evaluation, as next-generation sequencing technology continues to evolve.