Large-Scale Low-Cost NGS Library Preparation Using a Robust Tn5 Purification and Tagmentation Protocol

G3 (Bethesda). 2018 Jan 4;8(1):79-89. doi: 10.1534/g3.117.300257.


Efficient preparation of high-quality sequencing libraries that well represent the biological sample is a key step for using next-generation sequencing in research. Tn5 enables fast, robust, and highly efficient processing of limited input material while scaling to the parallel processing of hundreds of samples. Here, we present a robust Tn5 transposase purification strategy based on an N-terminal His6-Sumo3 tag. We demonstrate that libraries prepared with our in-house Tn5 are of the same quality as those processed with a commercially available kit (Nextera XT), while they dramatically reduce the cost of large-scale experiments. We introduce improved purification strategies for two versions of the Tn5 enzyme. The first version carries the previously reported point mutations E54K and L372P, and stably produces libraries of constant fragment size distribution, even if the Tn5-to-input molecule ratio varies. The second Tn5 construct carries an additional point mutation (R27S) in the DNA-binding domain. This construct allows for adjustment of the fragment size distribution based on enzyme concentration during tagmentation, a feature that opens new opportunities for use of Tn5 in customized experimental designs. We demonstrate the versatility of our Tn5 enzymes in different experimental settings, including a novel single-cell polyadenylation site mapping protocol as well as ultralow input DNA sequencing.

Keywords: Tn5; single cell; tagmentation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Cloning, Molecular / methods
  • DNA / genetics
  • DNA / metabolism
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Gene Expression
  • Gene Library*
  • Genetic Vectors / chemistry
  • Genetic Vectors / metabolism
  • HeLa Cells
  • High-Throughput Nucleotide Sequencing / economics
  • High-Throughput Nucleotide Sequencing / methods*
  • Humans
  • Point Mutation*
  • Polyadenylation
  • Protein Binding
  • Recombinant Fusion Proteins / genetics*
  • Recombinant Fusion Proteins / metabolism
  • Transposases / genetics*
  • Transposases / metabolism


  • Recombinant Fusion Proteins
  • Tn5 transposase
  • DNA
  • Transposases