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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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Library quantification results. A. Reproducibility of UT-PCR assays. Twelve 454 libraries were assayed with six to eight replicates by both UT-digital PCR and UT-quantitative PCR. UT-quantitative PCR was calibrated using a library quantified by digital PCR. The CV for dPCR is significantly lower than that for qPCR. B. Accurate digital PCR quantification of 454 libraries from trace quantities of input E. coli genomic or amplicon DNA. E. coli shotgun and amplicon DNA were first quantified by mass-based methods and the indicated amounts (0.5 to 35 ng) used for library preparation. The input quantity and yield are correlated with R2 = 0.88. The library yield was assessed based on replicate UT-digital PCR quantification. Useful numbers of library molecules were recovered in all cases.
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Figure 3: Library quantification results. A. Reproducibility of UT-PCR assays. Twelve 454 libraries were assayed with six to eight replicates by both UT-digital PCR and UT-quantitative PCR. UT-quantitative PCR was calibrated using a library quantified by digital PCR. The CV for dPCR is significantly lower than that for qPCR. B. Accurate digital PCR quantification of 454 libraries from trace quantities of input E. coli genomic or amplicon DNA. E. coli shotgun and amplicon DNA were first quantified by mass-based methods and the indicated amounts (0.5 to 35 ng) used for library preparation. The input quantity and yield are correlated with R2 = 0.88. The library yield was assessed based on replicate UT-digital PCR quantification. Useful numbers of library molecules were recovered in all cases.

Mentions: Digital PCR gives an absolute, calibration-free measurement of the concentration of amplifiable library molecules. To demonstrate the utility of digital PCR in preparing sequencing libraries from small amounts of starting material, twelve libraries were created from starting amounts of E. coli DNA ranging from 35 ng to as low as 500 pg. Six of the libraries were constructed with E. coli genomic DNA and six were prepared from the same quantities of an E. coli 16S rRNA amplification product (of 466 bp), all according to the 454 shotgun protocol using molecular barcodes, "MIDs" (or Multiplex IDentifiers, which allow multiple samples to be mixed and sequenced as a pool). The resulting DNA libraries, undetectable by UV spectrophotometry or capillary gel electrophoresis, were quantified by UT-digital PCR. Useful numbers of library molecules were recovered from all twelve library preparations. The quantity of input DNA and the library yield for each sample is listed in Table 2 and shown in Figure 3A.


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

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

Library quantification results. A. Reproducibility of UT-PCR assays. Twelve 454 libraries were assayed with six to eight replicates by both UT-digital PCR and UT-quantitative PCR. UT-quantitative PCR was calibrated using a library quantified by digital PCR. The CV for dPCR is significantly lower than that for qPCR. B. Accurate digital PCR quantification of 454 libraries from trace quantities of input E. coli genomic or amplicon DNA. E. coli shotgun and amplicon DNA were first quantified by mass-based methods and the indicated amounts (0.5 to 35 ng) used for library preparation. The input quantity and yield are correlated with R2 = 0.88. The library yield was assessed based on replicate UT-digital PCR quantification. Useful numbers of library molecules were recovered in all cases.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC2667538&req=5

Figure 3: Library quantification results. A. Reproducibility of UT-PCR assays. Twelve 454 libraries were assayed with six to eight replicates by both UT-digital PCR and UT-quantitative PCR. UT-quantitative PCR was calibrated using a library quantified by digital PCR. The CV for dPCR is significantly lower than that for qPCR. B. Accurate digital PCR quantification of 454 libraries from trace quantities of input E. coli genomic or amplicon DNA. E. coli shotgun and amplicon DNA were first quantified by mass-based methods and the indicated amounts (0.5 to 35 ng) used for library preparation. The input quantity and yield are correlated with R2 = 0.88. The library yield was assessed based on replicate UT-digital PCR quantification. Useful numbers of library molecules were recovered in all cases.
Mentions: Digital PCR gives an absolute, calibration-free measurement of the concentration of amplifiable library molecules. To demonstrate the utility of digital PCR in preparing sequencing libraries from small amounts of starting material, twelve libraries were created from starting amounts of E. coli DNA ranging from 35 ng to as low as 500 pg. Six of the libraries were constructed with E. coli genomic DNA and six were prepared from the same quantities of an E. coli 16S rRNA amplification product (of 466 bp), all according to the 454 shotgun protocol using molecular barcodes, "MIDs" (or Multiplex IDentifiers, which allow multiple samples to be mixed and sequenced as a pool). The resulting DNA libraries, undetectable by UV spectrophotometry or capillary gel electrophoresis, were quantified by UT-digital PCR. Useful numbers of library molecules were recovered from all twelve library preparations. The quantity of input DNA and the library yield for each sample is listed in Table 2 and shown in Figure 3A.

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.

Show MeSH