Abstract
Standard units of measurement are required for the quantitative description of nature; however, few standard units have been established for genomics to date. Here, we have developed a synthetic DNA ladder that defines a quantitative standard unit that can measure DNA sequence abundance within a next-generation sequencing library. The ladder can be spiked into a DNA sample, and act as an internal scale that measures quantitative genetics features. Unlike previous spike-ins, the ladder is encoded within a single molecule, and can be equivalently and independently synthesized by different laboratories. We show how the ladder can measure diverse quantitative features, including human genetic variation and microbial abundance, and also estimate uncertainty due to technical variation and improve normalization between libraries. This ladder provides an independent quantitative unit that can be used with any organism, application or technology, thereby providing a common metric by which genomes can be measured.
Highlights
Standard units of measurement are required for the quantitative description of nature; few standard units have been established for genomics to date
In a hypothetical “perfect” library that is unaffected by experimental variables, a comparison of the read counts to cn will assemble a linear and graduated quantitative ladder structure, that can be used as an internal scale for the quantification of DNA sequences in the NGS library (Fig. 1c)
We demonstrated the performance of the synthetic DNA ladder in an experimental NGS library that is subject to technical variation
Summary
Standard units of measurement are required for the quantitative description of nature; few standard units have been established for genomics to date. The use of a common ladder allows the size of DNA fragments to be compared between experiments, and under different migration conditions Despite these advantages, no similar quantitative DNA ladder is available against which to measure abundance in nextgeneration sequencing. No similar quantitative DNA ladder is available against which to measure abundance in nextgeneration sequencing Natural genetic materials, such as reference human genomes or small phage genomes, do not provide a graduated scale[5,6,7]. These irregular errors result in differences between batches of spike-in controls To address these limitations, we have designed a quantitative DNA ladder that is encoded within a single synthetic DNA sequence. These sub-sequences form an exact scale across a dynamic quantitative range within an NGS library
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