Reference human genome samples and synthetic mirrored representation of human genome features.

Next-generation sequencing (NGS) can identify mutations in the human genome that cause disease, and has been widely adopted in clinical diagnosis. However, the human genome contains many polymorphic, low complexity, and repetitive regions that are difficult to sequence and analyse. Despite their difficulty, these regions include many clinically-important features, and their accurate diagnosis can inform the treatment of a range of human diseases. To evaluate the accuracy by which these difficult regions are analysed using sequencing, we built an in silico chromosome, and corresponding synthetic DNA reference standards that encode difficult and clinically important sequences of the human genome, including repeats, microsatellites, HLA genes and immune-receptors. Unlike natural genome materials, that can be difficult to unambiguously characterise at these difficult regions, the synthetic DNA standards provide a known ground-truth to evaluate the performance of a diverse sequencing technologies, reagents, and bioinformatic tools. Here we provide a comprehensive and detailed evaluation of short- and long-read sequencing instruments, PCR-based and -free library reagents, and a range of leading bioinformatic tools. This evaluation provides analytical validation for using sequence to diagnose a range of clinical important features of the human genome, and highlight the challenges in resolving these difficult regions using genome sequencing.