Facilitating Genomic Library Preparation for Next-Generation
Sequencing Platforms
Aric Joneja, Xiaohua Huang
Bioengineering Department, University of California at San Diego, CA 92093-0412
Tel: 858-822-4702 E-mail: ajoneja@ucsd.edu
Summary: A device for the automatic, random shearing of genomic DNA is assembled. A DNA
amplification method entitled Nicking Endonuclease Mediated Strand Displacement
Amplification is optimized for the unbiased replication of long fragments of genomic DNA.
The fragmentation of chromosomal DNA and production of unbiased genomic DNA libraries is a critical and
sometimes rate-limiting step in the DNA sequencing pipeline, especially for many next-generation sequencing
platforms. We have developed an inexpensive device for automated fragmentation of genomic DNA using
hydrodynamic shearing. DNA molecules are fragmented by hydrodynamic shear stress when they are forced
through uniform pores with micron or sub-micron dimensions at high velocity. Genomic DNA can be sheared
randomly with a tight 2–3-fold size distribution, and the cloning efficiency of the sheared DNA fragments is very
high [1]. The length of the fragments is determined by the flow velocity of the solution and the diameter of the
pores, independent of the initial DNA size/concentration and salt concentration. Our device is user-friendly,
automated, and economical, making it an attractive alternative to current commercialized instruments.
Strand Displacement Amplification (SDA) [2] is a method that, until recently, was restricted to the replication of
short (<200 bp) DNA fragments. We have optimized this technique for the unbiased amplification of genomic DNA
for sequencing library preparation using nicking endonuclease-mediated SDA. We demonstrate that DNA templates
up to 5000 nucleotides can be linearly amplified using a nicking endonuclease with 7-bp recognition sequence and
Sequenase version 2.0 in the presence of single-stranded DNA binding proteins. We also show that a mixture of
three templates of 500, 1000, and 5000 nucleotides in length is linearly amplified with the original molar ratios of
the templates preserved. Moreover, we demonstrate that a complex library of hydrodynamically sheared genomic
DNA from bacteriophage lambda can be amplified linearly.
A.
B.
Fig. 1. a) Schematic representation of the syringe-pump based shearing device. A screen with uniform pores is set in a stainless steel housing.
b) Diagram of nicking endonuclease-mediated strand displacement amplification. The continuous combined actions of the nicking
endonuclease and DNA polymerase result in the linear amplification of one strand of the DNA.
[1] Oefner, P.J., S.P. Hunicke-Smith, L. Chiang, F. Dietrich, J. Mulligan, and R.W. Davis. “Efficient random subcloning of DNA sheared in a
recirculating point-sink flow system”, Nucleic Acids Res. 24:3879-3886. 1996.
[2] G.T. Walker, M.C. Little, J.G. Nadeau, D.D. Shank. “Isothermal in vitro amplification of DNA by a restriction enzyme/DNA polymerase
system”, Proc. Natl. Acad. Sci. USA 89 392–396. 1992