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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

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
doi: https://doi.org/10.1101/003566
Castle Raley
Sequencing Facility
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  • For correspondence: raleyjc@mail.nih.gov
David Munroe
Cancer Research Technology Program
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Kristie Jones
Cancer Genomics Research Laboratory
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Yu-Chih Tsai
Pacific Biosciences Inc., Menlo Park, CA, 94025
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Yan Guo
Pacific Biosciences Inc., Menlo Park, CA, 94025
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Bao Tran
Sequencing Facility
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Sujatha Gowda
Sequencing Facility
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Jennifer L. Troyer
Laboratory of Molecular Technology
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Daniel R Soppet
Laboratory of Molecular Technology
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Claudia Stewart
Laboratory of Molecular Technology
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Robert Stephens
Cancer Research Technology Program
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Jack Chen
Advanced Biological Computing Center, Leidos Biomedical Research Inc. (formerly SAIC-Frederick, Inc.), Frederick National Laboratory for Cancer Research, Frederick, MD, 21702.
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TF Skelly
Cancer Research Technology Program
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Cheryl Heiner
Pacific Biosciences Inc., Menlo Park, CA, 94025
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Jonas Korlach
Pacific Biosciences Inc., Menlo Park, CA, 94025
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Dwight Nissley
Cancer Research Technology Program
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Abstract

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 singlemolecule 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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Posted March 25, 2014.
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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
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
bioRxiv 003566; doi: https://doi.org/10.1101/003566
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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
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
bioRxiv 003566; doi: https://doi.org/10.1101/003566

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