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Preparation of next-generation DNA sequencing libraries from ultra-low amounts of input DNA: Application to single-molecule, real-time (SMRT) sequencing on the Pacific Biosciences RS II.

By Castle Raley, David Munroe, Kristie Jones, Yu-Chih Tsai, Yan Guo, Bao Tran, Sujatha Gowda, Jennifer L. Troyer, Daniel R. Soppet, Claudia Stewart, Robert Stephens, Jack Chen, TF Skelly, Cheryl Heiner, Jonas Korlach, Dwight Nissley

Posted 25 Mar 2014
bioRxiv DOI: 10.1101/003566

We have developed and validated an amplification-free method for generating DNA sequencing libraries from very low amounts of input DNA (500 picograms - 20 nanograms) for single-molecule sequencing on the Pacific Biosciences (PacBio) RS II sequencer. The common challenge of high input requirements for single-molecule sequencing is overcome by using a carrier DNA in conjunction with optimized sequencing preparation conditions and re-use of the MagBead-bound complex. Here we describe how this method can be used to produce sequencing yields comparable to those generated from standard input amounts, but by using 1000-fold less starting material.

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