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

White RA, Blainey PC, Fan HC, Quake SR - BMC Genomics (2009)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

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

ABSTRACT

Background: 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.

Results: 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.

Conclusion: 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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A Schematic of the universal template (UT) PCR assay. The forward primer (as drawn) includes a short sequence complementary to the 8 bp dual-labeled locked nucleic acid probe on its 5' end, followed by the sequence of one of the adaptors ligated to the library molecules on its 3' end. The reverse primer (as drawn) is complementary to the sequence of the other adaptor. As the polymerase encounters the probe during strand extension, its 5' to 3' exonuclease activity cleaves the probe, releasing the fluorophore from its quencher, thus producing fluorescent signal by dequenching. B The assay is performed on a commercial microfluidic digital PCR chip. At the end of the PCR, compartments that contain amplifiable DNA molecules with sequencing adaptors properly appended give positive signal, while compartments that do not remain dark. The count of positive compartments corresponds to the number of productive library molecules in the volume loaded onto the microfluidic chip, thereby allowing measurement of the concentration of amplifiable library molecules.
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Figure 1: A Schematic of the universal template (UT) PCR assay. The forward primer (as drawn) includes a short sequence complementary to the 8 bp dual-labeled locked nucleic acid probe on its 5' end, followed by the sequence of one of the adaptors ligated to the library molecules on its 3' end. The reverse primer (as drawn) is complementary to the sequence of the other adaptor. As the polymerase encounters the probe during strand extension, its 5' to 3' exonuclease activity cleaves the probe, releasing the fluorophore from its quencher, thus producing fluorescent signal by dequenching. B The assay is performed on a commercial microfluidic digital PCR chip. At the end of the PCR, compartments that contain amplifiable DNA molecules with sequencing adaptors properly appended give positive signal, while compartments that do not remain dark. The count of positive compartments corresponds to the number of productive library molecules in the volume loaded onto the microfluidic chip, thereby allowing measurement of the concentration of amplifiable library molecules.

Mentions: TaqMan PCR has the advantage of yielding a fluorescence signal proportional to the number of molecules that have been amplified, rather than the total mass of dsDNA in the sample [9]. This method works by the addition of a double-labeled oligonucleotide probe in a PCR reaction powered by a polymerase with 5' to 3' exonuclease activity. The probe must be complementary to one of the two product strands such that the extending polymerase will encounter the probe and cleave it, separating the probe's two labels and activating the probe's fluorescence through its exonuclease activity. Conventional TaqMan detection chemistry requires that the probe is complementary to the region within the amplified portion of the template between the two amplification primers. This strategy is not applicable to the sequencing libraries, which have inserts of unknown or random sequence between short adaptor sequences. To overcome the challenge of probe design for templates of random sequence, we adapted the universal template (UT) approach where a probe-binding sequence is appended to one of the PCR primers [10] (Figure 1A). One amplification primer includes a short sequence complementary to the probe on its 5' end, followed by a sequence complementary to one of the sequencing adaptors ligated to the library molecules. The second amplification primer in the UT scheme is complementary to the sequence of the second sequencing adaptor. To decrease reaction times, we replaced the published 20 bp UT probe-binding region with an 8 bp sequence target for a probe containing a locked nucleic acid nucleotide as applied in Roche's UPL (Universal Probe Library) probes (see methods for details). The shorter amplicon-probe interaction length allows the reduction of PCR run times from 2.5 hours to less than 50 minutes. In practice, we often use the UT-quantitative PCR assay in the real-time mode (with a calibration standard) to range the library concentration so that an appropriate dilution can be made for absolute quantification by UT-digital PCR.


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

White RA, Blainey PC, Fan HC, Quake SR - BMC Genomics (2009)

A Schematic of the universal template (UT) PCR assay. The forward primer (as drawn) includes a short sequence complementary to the 8 bp dual-labeled locked nucleic acid probe on its 5' end, followed by the sequence of one of the adaptors ligated to the library molecules on its 3' end. The reverse primer (as drawn) is complementary to the sequence of the other adaptor. As the polymerase encounters the probe during strand extension, its 5' to 3' exonuclease activity cleaves the probe, releasing the fluorophore from its quencher, thus producing fluorescent signal by dequenching. B The assay is performed on a commercial microfluidic digital PCR chip. At the end of the PCR, compartments that contain amplifiable DNA molecules with sequencing adaptors properly appended give positive signal, while compartments that do not remain dark. The count of positive compartments corresponds to the number of productive library molecules in the volume loaded onto the microfluidic chip, thereby allowing measurement of the concentration of amplifiable library molecules.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2667538&req=5

Figure 1: A Schematic of the universal template (UT) PCR assay. The forward primer (as drawn) includes a short sequence complementary to the 8 bp dual-labeled locked nucleic acid probe on its 5' end, followed by the sequence of one of the adaptors ligated to the library molecules on its 3' end. The reverse primer (as drawn) is complementary to the sequence of the other adaptor. As the polymerase encounters the probe during strand extension, its 5' to 3' exonuclease activity cleaves the probe, releasing the fluorophore from its quencher, thus producing fluorescent signal by dequenching. B The assay is performed on a commercial microfluidic digital PCR chip. At the end of the PCR, compartments that contain amplifiable DNA molecules with sequencing adaptors properly appended give positive signal, while compartments that do not remain dark. The count of positive compartments corresponds to the number of productive library molecules in the volume loaded onto the microfluidic chip, thereby allowing measurement of the concentration of amplifiable library molecules.
Mentions: TaqMan PCR has the advantage of yielding a fluorescence signal proportional to the number of molecules that have been amplified, rather than the total mass of dsDNA in the sample [9]. This method works by the addition of a double-labeled oligonucleotide probe in a PCR reaction powered by a polymerase with 5' to 3' exonuclease activity. The probe must be complementary to one of the two product strands such that the extending polymerase will encounter the probe and cleave it, separating the probe's two labels and activating the probe's fluorescence through its exonuclease activity. Conventional TaqMan detection chemistry requires that the probe is complementary to the region within the amplified portion of the template between the two amplification primers. This strategy is not applicable to the sequencing libraries, which have inserts of unknown or random sequence between short adaptor sequences. To overcome the challenge of probe design for templates of random sequence, we adapted the universal template (UT) approach where a probe-binding sequence is appended to one of the PCR primers [10] (Figure 1A). One amplification primer includes a short sequence complementary to the probe on its 5' end, followed by a sequence complementary to one of the sequencing adaptors ligated to the library molecules. The second amplification primer in the UT scheme is complementary to the sequence of the second sequencing adaptor. To decrease reaction times, we replaced the published 20 bp UT probe-binding region with an 8 bp sequence target for a probe containing a locked nucleic acid nucleotide as applied in Roche's UPL (Universal Probe Library) probes (see methods for details). The shorter amplicon-probe interaction length allows the reduction of PCR run times from 2.5 hours to less than 50 minutes. In practice, we often use the UT-quantitative PCR assay in the real-time mode (with a calibration standard) to range the library concentration so that an appropriate dilution can be made for absolute quantification by UT-digital PCR.

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

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

ABSTRACT

Background: 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.

Results: 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.

Conclusion: 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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