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Updated: Jun 24, 2026

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella
07:11

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Published on: May 13, 2019

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

Richard A White1, Paul C Blainey, H Christina Fan

  • 1Department of Bioengineering at Stanford University and Howard Hughes Medical Institute, Stanford, CA 94305, USA. raw937@sbcglobal.net

BMC Genomics
|March 21, 2009
PubMed
Summary

Digital PCR accurately quantifies DNA sequencing libraries, enabling library preparation from nanograms of input material. This method reduces sample requirements over 1000-fold, eliminating costly titration runs for next-generation sequencing.

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Area of Science:

  • Molecular Biology
  • Genomics
  • Biotechnology

Background:

  • Next-generation sequencing (NGS) platforms (454, Solexa, SOLiD) require precise molecule quantification for library preparation.
  • Current methods necessitate large DNA input (micrograms) and costly titration runs, limiting applications and throughput.

Purpose of the Study:

  • To demonstrate digital PCR (dPCR) as a method for accurate absolute quantification of NGS libraries.
  • To enable library preparation from significantly reduced DNA input quantities (nanograms).
  • To eliminate the need for titration sequencing runs.

Main Methods:

  • Development and application of a digital PCR assay for absolute quantification of 454 and Solexa sequencing libraries.
  • Preparation and sequencing of libraries derived from nanogram quantities of bacterial and mammalian DNA.

Main Results:

  • Digital PCR enabled accurate quantification of sequencing libraries, allowing preparation from nanogram input DNA.
  • Successful sequencing of low-nanogram scale DNA samples on 454 FLX and Solexa platforms.
  • Demonstrated sequencing of picogram quantities of DNA on the 454 platform, a >1000-fold reduction in sample requirement without pre-amplification bias.

Conclusions:

  • Digital PCR provides precise (CV ~10%) absolute quantification of sequencing libraries, surpassing traditional PCR methods.
  • This approach eliminates uncertainties in quantification and the need for titration runs.
  • Enables efficient NGS library preparation from minute DNA samples, broadening application scope.