The identification of genetic variation with next-generation sequencing is confounded by the complexity of the human genome sequence and by biases that arise during library preparation, sequencing and analysis. We have developed a set of synthetic DNA standards, termed ‘sequins’, that emulate instances of human genetic variation and constitute qualitative
 and quantitative spike-in controls for genome sequencing¹. To design sequins, we sample naturally occurring genetic variants, along with 1.5-10kb of surrounding genome sequence, and synthesize these fragments in reverse-orientation (ie 3’-5’). Sequencing reads derived from sequin DNA therefore align exclusively to a reverse-orientation copy of the human reference genome (hg38-rev), rather than hg38 itself. Thus, sequins provide mirror-image representations of bona fide variants, in which all relevant properties are preserved, but that can be unambiguously distinguished from natural human DNA, based on alignment. Here we use this approach to represent common, challenging and clinically relevant genetic variation, ranging from single nucleotide variants to large structural rearrangements and copy-number variation. We provide sequins as a standardized, quantitative resource against which human genetic variation can be measured and diagnostic performance assessed.

¹ Deveson IW, Chen WY, Wong T, Hardwick SA, Andersen SB, Nielsen LK, Mattick JS & Mercer TR. Representing genetic variation with synthetic DNA standards. Nature Methods. 9, 784-91 (2016).