The equation relating amplicon size and fluorescence amplitude can be applied either to the average (mean or median) fluorescence amplitude of a sample or to the fluorescence amplitude of individual droplets. Applying the equation to individual droplets allows for a more detailed analysis of the distribution of product sizes present in a sample. We applied the equation generated by the adapter-ligated size standards to the fluorescence amplitude of individual droplets containing library DNA to calculate the expected amplicon size within each droplet and compared the distribution of sizes to the distribution of read sizes measured by the MiSeq (Figure 4). The frequency distribution shows a high degree of overlap and a common center point for the estimation made using the QuantiSize assay and the observations from the MiSeq. As depicted in Figure 2A, there is an inherent variance in droplet amplitude that occurs even within a completely homogeneous sample of amplicon lengths. This variance likely accounts for the wider distribution of product sizes estimated by ddPCR than were observed in the MiSeq data. Size determination with QuantiSize provides a detailed calculation of the distribution of fragment sizes present in a sample, whereas gel or capillary electrophoresis provide only a range of sizes.
The QuantiSize assay demonstrates accuracy, reliability, and flexibility through the strength of the correlation between fluorescence amplitude and amplicon size in ddPCR experiments, and the ease with which the assay can be adjusted to fit specific experimental needs. Applying the QuantiSize assay to NGS library preparation avoids the limitations of other independent quantification and size determination methods and has the potential to increase the average yield of usable data generated from sequencing runs, thereby increasing the efficiency and throughput. The ability to determine the absolute quantity and the detailed size distribution of target DNA with a single experiment will be useful for a broad range of applications that require the quantification and sizing of target DNA.
We would like to thank Nolan Ericson and Mariola Kulawiec for helpful discussion and critical reading of our manuscript. This work was supported by an Ellison Medical Foundation New Scholar award (AG-NS-0577-09), an Outstanding New Environmental Scientist Award (ONES) (R01) from the National Institute of Environmental Health Sciences (R01ES019319), and a grant (W81XWH-10-1-0563) from the Congressionally Directed Medical Research Programs/U.S. Department of Defense. This paper is subject to the NIH Public Access Policy
The authors declare no competing interests.
Correspondence Address correspondence to Jason H. Bielas, Fred Hutchinson Cancer Research Center, Seattle, WA. E-mail: [email protected]">[email protected]References
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