Biography:  I am currently Professor of Medicine and Pathology, and George Isaac Chair for Cancer Research with more than 25 years of continuous funding from the National Institutes of Health in translational research.  We develop methods to optimize to quality control in next generation sequencing analysis of germline and somatic variants in clinical specimens, including circulating tumor DNA.  Since 2005, my laboratory has participated in United States Food and Drug Administration (FDA) MAQC collaborative projects to improve quality control in genetic testing.  Through these FDA consortia, as of 2021 I have co-authored twelve articles reporting results from these collaborative projects, including nine in Nature Biotechnology articles, three Genome Biology, and one in Cell Reports Methods.

Abstract: The primary objective of the FDA-led Sequencing and Quality Control Phase 2 (SEQC2) project is to develop standard analysis protocols and quality control metrics for use in DNA testing to enhance scientific research and precision medicine. This study reports a targeted next generation sequencing (NGS) method that enables more accurate detection of actionable mutations in circulating tumor DNA (ctDNA) clinical specimens. This advancement was enabled by designing a synthetic internal standard spike-in for each actionable mutation target, suitable for use in NGS following hybrid-capture enrichment and unique molecular index (UMI) or non-UMI library preparation. When mixed with contrived ctDNA reference samples, internal standards enabled calculation of technical error rate, limit of blank, and limit of detection for each variant at each nucleotide position, in each sample.  True positive mutations with variant allele fraction too low for detection by current practice were detected with this method, thereby increasing sensitivity.

Biography:  Timothy Mercer is a Group Leader at the Australian Institute for Bioengineering and Nanotechnology (AIBN) at The University of Queensland where he is the Scientific Director of the BASE nucleic-acid synthesis facility. He also leads a diverse laboratory and bioinformatic research group into the expression and splicing of synthetic genes.  Prior to this, he was Group Leader at the Garvan Institute for Medical Research, Sydney, where he pioneered the use of synthetic RNA and DNA controls to improve the accuracy of clinical genome sequencing. He also developed targeted RNA sequencing approaches for the diagnosis of fusion genes in cancer. Together, this reflects his ongoing interest in the development of genome biotechnologies.   Before joining the Garvan, Tim Mercer received his PhD in Genomics from UQ and completed postdoctoral studies in transcriptomics, long-noncoding RNAs and splicing at Broad Institute, United States, Centre for Gene Regulation, Spain, and Max Plank Institute for Cell Biology, Germany.

Abstract: 

Resolving the complexity of the human genome using synthetic chromosomal controls: 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 sequences that can inform the treatment of human diseases and improve the diagnostic yield of NGS. To evaluate the accuracy by which these difficult regions are analysed with NGS, we built an in silico decoy chromosome, along with corresponding synthetic DNA reference controls, that encode difficult and clinically-important human genome regions, including repeats, microsatellites, HLA genes and immune-receptors. These synthetic chromosome controls provide a known ground-truth reference against which to measure the performance of diverse sequencing technologies, reagents, and bioinformatic tools. Using this approach, we provide a comprehensive evaluation of short- and long-read sequencing instruments, library preparation methods, and software tools, and identify the errors and systematic bias that confound our resolution of these remaining difficult regions. This study provides an analytical validation of diagnosis using NGS in difficult regions of the human genome and highlights the challenges that remain to resolve these difficult regions.

Building a synthetic genome that encodes DNA, mRNA and protein controls. PhiX-174 was the first genome to be sequenced in 1974, and has become the most commonly used standard in sequencing, molecular and synthetic biology. However, given the advent of affordable DNA synthesis and de novo gene design, we considered whether we could build a new genome, termed SynX, that is optimized for use as a molecularstandard. The SynX genome encodes synthetic genes that are organised into paralogous gene families and provide qualitative and quantitative evaluation of next-generation sequencing performance. The synthetic genes can be in vitrotranscribed to form matched synthetic mRNA controls to evaluate RNA sequencing performance. Finally, the synthetic mRNA controls can be in vitro translated to form a matched protein controls for high throughput proteomics methods, such as mass spectrophotometry. The SynX genome can be independently and sustainably prepared, modified and shared by recipient laboratories using common molecular biology techniques, and be widely used as a universal molecular standard.