Digital PCR provides sensitive and absolute calibration for high throughput sequencing

Department of Bioengineering at Stanford University and Howard Hughes Medical Institute, Stanford, CA 94305, USA.
BMC Genomics (Impact Factor: 3.99). 04/2009; 10(1):116. DOI: 10.1186/1471-2164-10-116
Source: PubMed


Next-generation DNA sequencing on the 454, Solexa, and SOLiD platforms requires absolute calibration of the number of molecules to be sequenced. This requirement has two unfavorable consequences. First, large amounts of sample-typically micrograms-are needed for library preparation, thereby limiting the scope of samples which can be sequenced. For many applications, including metagenomics and the sequencing of ancient, forensic, and clinical samples, the quantity of input DNA can be critically limiting. Second, each library requires a titration sequencing run, thereby increasing the cost and lowering the throughput of sequencing.
We demonstrate the use of digital PCR to accurately quantify 454 and Solexa sequencing libraries, enabling the preparation of sequencing libraries from nanogram quantities of input material while eliminating costly and time-consuming titration runs of the sequencer. We successfully sequenced low-nanogram scale bacterial and mammalian DNA samples on the 454 FLX and Solexa DNA sequencing platforms. This study is the first to definitively demonstrate the successful sequencing of picogram quantities of input DNA on the 454 platform, reducing the sample requirement more than 1000-fold without pre-amplification and the associated bias and reduction in library depth.
The digital PCR assay allows absolute quantification of sequencing libraries, eliminates uncertainties associated with the construction and application of standard curves to PCR-based quantification, and with a coefficient of variation close to 10%, is sufficiently precise to enable direct sequencing without titration runs.

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Available from: Richard Allen White III, Oct 13, 2015
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    • "to the manufacturer ' s instructions , by qPCR using a microfluidic digital PCR quantified standard curve ( White III et al . , 2009 ) . The resulting libraries were pooled , and sequenced using both 250 and 100 bp paired - end sequencing on the MiSeq ( GenoSeq UCLA Los Angeles , CA ) and HiSeq ( McGill University / Génome Québec , Montreal , QC ) platforms , respectively . Analysis of Illumina Sequencing Data Raw Illumina data was screened for PhiX spike - in contam"
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    • "Several authors have already suggested that next-generation sequencing library preparation protocols can be started with a lower amount of DNA than that required by the standard protocol. They stated that the true limiting factor, in the case of 454 sequencing, is to reach the number of enriched beads required by the platform [23], [24], [27]–[29]. In this work, we demonstrate that it is possible to prepare a 454 shotgun library for direct sequencing, starting with the amount of DNA needed to reach the exact minimal number of molecules required to fill the target picotiterplate (PTP) region (e.g. "
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