Tn5 transposase and tagmentation procedures for massively-scaled sequencing projects
- Simone Picelli1,
- Åsa K Björklund2,
- Björn Reinius2,
- Sven Sagasser1,
- Gösta Winberg2 and
- Rickard Sandberg2,3
- ↵* Corresponding author; email: rickard.sandberg{at}ki.se
Abstract
Massively parallel DNA sequencing of thousands of samples in a single machine-run is now possible, but the preparation of the individual sequencing libraries is expensive and time-consuming. Tagmentation-based library construction, using the Tn5 transposase, is an efficient way to generate sequencing libraries but currently relies on undisclosed reagents which severely limits novel application development and the execution of large scale projects. Here, we present simple and robust procedures for Tn5 transposase production and optimized reaction conditions for tagmentation-based sequencing library construction. We further show how molecular crowding agents both modulate library lengths and enable efficient tagmentation from sub-picogram amounts of cDNA. Comparison of single-cell RNA-sequencing libraries generated with produced and commercial Tn5 demonstrated equal performances in terms of gene detection and library characteristics. Finally, as naked Tn5 can be annealed to any oligonucleotide of choice, such as molecular barcodes in single-cell assays or methylated oligonucleotides for bisulfite sequencing, custom Tn5 production and tagmentation enables innovation in sequencing-based applications.
- Received May 4, 2014.
- Accepted July 24, 2014.
- Published by Cold Spring Harbor Laboratory Press
This manuscript is Open Access.
This article, published in Genome Research, is available under a Creative Commons License (Attribution-NonCommercial 4.0 International license), as described at http://creativecommons.org/licenses/by-nc/4.0/.
