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

Autor: Hennig BP; Genome Biology Unit, European Molecular Biology Laboratory, Heidelberg 69117, Germany., Velten L; Genome Biology Unit, European Molecular Biology Laboratory, Heidelberg 69117, Germany., Racke I; Protein Expression and Purification Core Facility, European Molecular Biology Laboratory, Heidelberg 69117, Germany., Tu CS; Genome Biology Unit, European Molecular Biology Laboratory, Heidelberg 69117, Germany., Thoms M; Biochemistry Center Heidelberg, Heidelberg University, 69120, Germany., Rybin V; Protein Expression and Purification Core Facility, European Molecular Biology Laboratory, Heidelberg 69117, Germany., Besir H; Protein Expression and Purification Core Facility, European Molecular Biology Laboratory, Heidelberg 69117, Germany., Remans K; Protein Expression and Purification Core Facility, European Molecular Biology Laboratory, Heidelberg 69117, Germany kim.remans@embl.de lars.steinmetz@embl.de., Steinmetz LM; Genome Biology Unit, European Molecular Biology Laboratory, Heidelberg 69117, Germany kim.remans@embl.de lars.steinmetz@embl.de.; Department of Genetics, Stanford University School of Medicine, California 94305.; Stanford Genome Technology Center, Stanford University School of Medicine, Palo Alto, California 94304.
Jazyk: angličtina
Zdroj: G3 (Bethesda, Md.) [G3 (Bethesda)] 2018 Jan 04; Vol. 8 (1), pp. 79-89. Date of Electronic Publication: 2018 Jan 04.
DOI: 10.1534/g3.117.300257
Abstrakt: 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 His 6 -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.
(Copyright © 2018 Hennig et al.)
Databáze: MEDLINE
Externí odkaz: