Robust library preparation methods that ensure high-quality libraries from a wide range of samples are critical for research applications as next-generation sequencing progresses to the clinic. In translational genomics where NGS is used extensively with oncological samples, the emphasis is on compatibility with a wide range of input types, quantities, and quality. Also important is the ability to accommodate various levels of sample degradation, especially with FFPE tissue, and overall simplicity of the workflow. In this application note, we describe a library preparation workflow that combines the ThruPLEX DNA-seq Kit and the Covaris truSHEAR microTUBE-15 for a fully optimized low DNA input solution.
The Covaris truSHEAR mechanical shearing technology is recognized as the gold standard for high-quality, robust, and highly reproducible fragmentation of nucleic acids. It employs patented Adaptive Focused Acoustics (AFA) technology for the controlled and easily tunable shearing of nucleic acids. Compared to enzymatic-based fragmentation or tagmentation, AFA hydrodynamic shearing generates a tight and completely random fragmentation fundamental to obtaining uniform genome coverage (Van Nieuwerburgh et al. 2014; Lan et al. 2015) and high-complexity libraries (Samorodnitsky et al. 2015).
AFA-based hydrodynamic shearing is highly versatile, with the unique ability to deliver the same level of performance across a broad combination of DNA input sources, concentrations, and volumes. The AFA microTUBE-15 is optimized for a 15-μl sample volume and is ideal for the ThruPLEX DNA-seq input requirements. The microTUBE-15 incorporates AFA-Beads that enable fully controllable, easy-to-use, and highly reproducible DNA shearing in very low sample volumes with high recovery.
The ThruPLEX DNA-seq library preparation kit uses a patented stem-loop technology to repair DNA, reduce background, and generate high-complexity libraries. The ThruPLEX DNA-seq kit can generate DNA libraries from as little as 50 pg of DNA while providing up to 96 indexes for multiplexing. This kit can be used with fragmented DNA from any sample source—biofluids such as cell-free DNA, DNA from FFPE materials, and cDNA (Murtaza et al. 2013). The entire ThruPLEX DNA-seq kit workflow is performed in a single tube or well in about 2.5 hours and requires no intermediate purification steps or sample transfers. It can be used in a variety of applications, including DNA-seq, RNA-seq, and ChIP-seq, and offers robust target enrichment performance with all of the leading platforms.
The combination of AFA-based DNA mechanical shearing and library preparation with ThruPLEX DNA-seq enables a uniquely simple, fast, and robust workflow optimized not only for routine use, but also for precious and rare samples, since DNA input can be as low as 50 pg. The total processing time of the present workflow is less than 3 hours (Figure 1).