Representing genetic variation with synthetic DNA standards

Abstract

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 human genetic features and constitute qualitative and quantitative spike-in controls for genome sequencing. Sequencing reads derived from sequins align exclusively to an artificial in silico reference chromosome, rather than the human reference genome, which allows them them to be partitioned for parallel analysis. Here we use this approach to represent common and clinically relevant genetic variation, ranging from single nucleotide variants to large structural rearrangements and copy-number variation. We validate the design and performance of sequin standards by comparison to examples in the NA12878 reference genome, and we demonstrate their utility during the detection and quantification of variants. We provide sequins as a standardized, quantitative resource against which human genetic variation can be measured and diagnostic performance assessed.

Featherweight long read alignment using partitioned reference indexes

“…Sensitivity and error rate calculations for different window sizes (Minimap2 parameter w ) were performed using DNA sequins-chiral, synthetic human genome spike-in standards. The particular Sequins we employed (Deveson et al, under review) were unpublished at the time this manuscript was written, but are conceptually similar to what is reported in [18]. The Sequins were sequenced on ONT MinION sequencers with R9.4.1 flow cells, using LSK108 library preparation kits and the results were base called with ONT's Albacore sequencing pipeline software version 1.2.6.…”

Quality control materials for pharmacogenomic testing in the clinic

“…Sequins (http://www.sequin.xyz/), a set of novel synthetic DNA standards that simulate the features of a real human chromosome, may serve as qualitative and quantitative spike-in controls for NGS [49]. This control system is based on an artificial in silico reference chromosome, which constitutes sequins and "background" derived from reference genome sequences.…”

A multiplex ligation‑dependent probe amplification‑based next‑generation sequencing approach for the detection of copy number variations in the human genome

“…Compared with conventional assays, including Southern blotting, fluorescence in situ hybridization and Sanger sequencing, MLPA is a good alternative to array-based techniques and has high accuracy, specificity and efficiency. In addition, MLPA is cost-effective and has less technical complexity relative to array comparative genomic hybridization, which often requires further validation A multiplex ligation-dependent probe amplification-based next-generation sequencing approach for the detection of copy number variations in the human genome using other methods, such as quantitative polymerase chain reaction (qPCR) (13)(14)(15)(16)(17)(18)(19)(20)(21)(22). More importantly, MLPA may overcome the limitations of CMA and SNP array to some extent.…”