Digital PCR provides sensitive and absolute calibration for high throughput sequencing

Richard A White, Paul C Blainey, H Christina Fan and 1 more

BMC Genomics · 2009

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.

Full text

Its licence does not allow us to show the full text. Read it at PubMed Central:

References 13

1.
Holt RA, Jones SJM. The new paradigm of flow cell sequencing. Genome Research. 2008;18:839-846.
Not in our library10.1101/gr.073262.107
2.
Gupta PK. Single-molecule DNA sequencing technologies for future genomics research. Trends in Biotechnology. 2008;26:602-611.
Not in our library10.1016/j.tibtech.2008.07.003
3.
Bing DH, Boles C, Rehman FN, Audeh M, Belmarsh M, Kelley B, Adams CP. Bridge amplification: a solid phase PCR system for the amplification and detection of allelic differences in single copy genes. Genetic Identity Conference Proceedings, Seventh International Symposium on Human Identification. 1996.
Not in our library
4.
Margulies M, et al. Genome sequencing in microfabricated high-density picoliter reactors. Nature. 2005;437:376-380.
Not in our library10.1038/nature03959
5.
Mackelprang R, Rubin EM. Paleontology: New Tricks with Old Bones. Science. 2008;321:211-212.
Not in our library10.1126/science.1161890
6.
Assessment of whole genome amplification-induced bias through high-throughput, massively parallel whole genome sequencing · 2006
No notice
7.
Kalinina O, Lebedeva I, Brown J, Silver J. Nanoliter scale PCR with TaqMan detection. Nucleic Acids Research. 1997;25:1999-2004.
Not in our library10.1093/nar/25.10.1999
8.
Digital PCR · 1999
Not checked
9.
Heid CA, Stevens J, Livak KJ, Williams PM. Real-time quantitative PCR. Genome Research. 1996;6:986-994.
Not in our library10.1101/gr.6.10.986
10.
Zhang Y, Zhang D, Wenquan L, Chen J, Peng Y, Cao W. A novel real-time quantitative PCR method using attached universal template probe. Nucleic Acids Research. 2003;31:e123.
Not in our library10.1093/nar/gng123
11.
Meyer M, Briggs AW, Maricic T, Höber B, Höffner B, Krause J, Weihmann A, Pääbo S, Hofreiter M. From micrograms to picograms: quantitative PCR reduces the material demands of high-throughput sequencing. Nucleic Acids Research. 2008;36:e5.
Not in our library10.1093/nar/gkm1095
12.
Quail MA, Kozarewa I, Smith F, Scally A, Stephens PJ, Durbin R, Swerdlow H, Turner DJ. A large genome center's improvements to the Illumina sequencing system. Nature Methods. 2008;5:1005-1010.
Not in our library10.1038/nmeth.1270
13.
Simpson D, Feeney S, Boyle C, Stitt AW. Retinal VEGF mRNA measured by SYBR Green I fluorescence: A versatile approach to quantitative PCR. Molecular Vision. 2000;6:178-183.
Not in our library

Cited by papers we hold 10

ddPCR: a more accurate tool for SARS-CoV-2 detection in low viral load specimens · 2020
No notice
Estrogen receptor profiles across tissues from male and female Rattus norvegicus · 2019
No notice
Picoliter Well Array Chip-Based Digital Recombinase Polymerase Amplification for Absolute Quantification of Nucleic Acids. · 2016
Not checked
Comparison of Droplet Digital PCR and Seminested Real-Time PCR for Quantification of Cell-Associated HIV-1 RNA · 2014
No notice
Evaluation of Digital PCR for Absolute RNA Quantification · 2013
No notice
Highly Precise Measurement of HIV DNA by Droplet Digital PCR · 2013
No notice
Methods for Applying Accurate Digital PCR Analysis on Low Copy DNA Samples · 2013
No notice
Metagenomics - a guide from sampling to data analysis · 2012
No notice
Genome of a Low-Salinity Ammonia-Oxidizing Archaeon Determined by Single-Cell and Metagenomic Analysis · 2011
No notice
One Bacterial Cell, One Complete Genome · 2010
No notice

Reads

Read by people on this page

None yet.

Checked or read by AI through naulon

None yet.

A person counts once a day per paper. We keep no cookie and no address: a reader is a hash that changes every day. Any month under five reads shows no number.

Are you an author?

Is this your paper?

Claim it to add your note and be paid when agents cite it. Free.