Standard units of measurement are required for the empirical and quantitative description of natural phenomena. However, despite their importance, few standard units have been developed for genome analysis. We have developed a set of synthetic DNA ladders that define a reference unit for measuring abundance of DNA sequences with next generation sequencing. Each ladder is comprised of elements present at known copy-numbers. Prior to library preparation the synthetic ladders are added to a DNA sample and undergo concurrent sequencing. In the resulting library, reads derived from the ladder can be easily partitioned due to their unique sequence. The number of reads derived from each element is proportional to its copy-number and in constant ratio to the other elements within the same ladder. The reference unit defined by the ladder is sensitive to technical errors and biases and can be used to assess the accuracy of different library preparation methods or sequencing technologies. The ladder provides uncertainty estimates associated with quantification and acts as reference scale, which accurately measures genomic features from an accompanying human DNA sample. Due to its reproducibility, the ladder can also be used as a common scale to compare quantitative features of different sequencing libraries independent to a reference genome. We show, for example, how using the ladder improves the detection of genetic variants between a family trio of genomes in a reference-free comparison. Furthermore, this reference unit can be applied to any organism due to the lack of homology to natural nucleotide sequences. We show how even highly-divergent metagenomic samples can be calibrated by the ladder to achieve best-in-class normalization. Here, we present the first standardized unit purpose-built for genomics to be used for quantification, measuring technical variation and normalizing samples independently to any reference assembly.