Digital PCR provides sensitive and absolute calibration for high throughput sequencing.pdf

Quake et al page 1 of 19 Digital PCR provides sensitive and absolute calibration for high throughput sequencing Richard A. White III#, Paul Blainey#, H. Christina Fan and Stephen R. Quake* Department of Bioengineering, Stanford University and Howard Hughes Medical Institute, Stanford, California 94305 Abstract Several of the next generation sequencers are limited in their sample preparation process by the need to make an absolute measurement of the number of template molecules in the library to be sequenced. As currently practiced, the practical effects of this requirement compromise sequencing performance, both by requiring large amounts of sample DNA and by requiring extra sequencing runs to be performed. We used digital PCR to quantitate sequencing libraries, and demonstrated its sensitivity and robustness by preparing and sequencing libraries from subnanogram amounts of bacterial and human DNA on the 454 and Solexa sequencing platforms. This assay allows absolute quantitation and eliminates uncertainties associated with the construction and application of standard curves. The digital PCR platform consumes subfemptogram amounts of the sequencing library and gives highly accurate results, allowing the optimal DNA concentration to be used in setting up sequencing runs without costly and time-consuming titration techniques. This approach also reduces the input sample requirement more than 1000-fold: from micrograms of DNA to less than a nanogram. keywords: next-generation sequencing, sequencing library quantitation, digital PCR, universal template PCR, absolute quantitation *To whom correspondence should be addressed. E-mail: quake@. Phone (650) 724-7890 Fax (650) 736-1961 # These authors contributed equally to this work . Quake et al page 2 of 19 Introduction A new generation of sequencing technologies based on “sequencing by synthesis” are revolutionizing biology, biotechnology, and medicine. A key advance facilitating