The central theme of this year's AGBT was the application of sequencing technologies to clinical needs. Recurring topics of discussion included the need for speed in processing pipelines (from samples to interpretation), validation of results and clinical reporting, particularly for incidental findings.
How can results from NGS be validated?
Zivana Tezak (Food and Drug Administration, USA) gave an overview of regulatory considerations and questioned whether NGS technologies have the required quality for clinical use, if Sanger sequencing - the currently established standard - is still required for validation. Tezak discussed decoupling instrument validation from clinical test validation, and the need to define the minimal set of markers tested and percentage of genome covered for an instrument to be deemed reliable for clinical use. However, the question remains: to what extent can an instrument be deemed 'validated' given that failure modes are strongly sequence-specific?
How should NGS results be reported?
Elizabeth Worthey (Medical College of Wisconsin, USA) and several others cited the reporting recommendations of the American College of Medical Genetics. Worthey discussed the difficulty of establishing causality for each patient ('this variant/in this gene/causes this disease/in this case') and described WGS analysis yielding 100 to 120 variants flagged for in-depth review, only approximately six of which were considered clinically reportable. Worthey also highlighted the danger of 'wrong annotation creep' in databases and conference reports. Jonathan Berg (University of North Carolina at Chapel Hill, USA) described 'binning' the genome into three or four actionability levels and a reportability score combining disease severity, likelihood of causality, effectiveness and acceptability of interventions, and available knowledge. The exact cutoff used for reporting findings would then be left to patients' personal preferences.
An additional recurring topic was the need to end the 'diagnostic odyssey': the grueling, painful, expensive and sometimes decades-long journey from negative test to negative test, failing to diagnose a rare disease. Christine Eng (Baylor College of Medicine, USA) referred to the Undiagnosed Diseases Program at the National Institutes of Health (NIH) - soon to offer support for extramural research - and described an exome sequencing pipeline currently processing 140 samples a month, largely pediatric and neurologic in nature, with a conservative diagnosis rate of 25%. When asked how frequently diagnosis affected healthcare, Eng stressed that the focus was on reaching a diagnosis and ending the odyssey. Stephen Kingsmore (Children's Mercy Hospital, USA) described STAT-Seq, a WGS program to deliver (within 50 hours) a provisional report to the ordering neonatologist, including indications for pharmacogenomic dose adjustment. Kingsmore stressed the psychosocial benefits of rapid and definitive diagnosis, even in the absence of a cure.
Several advances were described using NGS to tackle cancer. Rebecca Leary (Johns Hopkins Kimmel Cancer Center, USA) described Personalized Analysis of Rearrangement Ends (PARE), a method for tracking cancer progression in a patient. Most promising, she described the ability to perform 'digital karyotyping' by sequencing circulating DNA in a patient's plasma - a sensitive method for early detection. Olivier Elemento (Weill Cornell Medical College, USA) performed deep sequencing of VDJ junctions and phylogenetic analysis to elucidate the personalized history of lymphoma - from primary tumor to relapse. Ira Hall (University of Virginia, USA) reanalyzed data from The Cancer Genome Atlas to identify chromosomal rearrangements, mapping thousands of somatic breakpoints to base resolution. This revealed non-random breakpoint clustering: Hall described three main modes of chromothripsis ('chromosome shattering'): focused, diffuse and multifocal, and reported significant enrichment of chromothripsis in glioblastoma.
Sequencing technologies are now being applied 'at all stages of life', not just for research and clinical diagnosis. Kevin Hrusovsky (Perkin Elmer, USA) described 'consumer genomics' uses of sequencing for dating, parental testing, neonatal diagnosis and 'no phenotype' personalized genomics. Dagan Wells (University of Oxford, UK) reported improved in vitro fertilization results by using multiplexed, low-coverage WGS on an Ion Torrent PGM to detect aneuploidies in single cells - a feat performed in just 12.5 hours, for a mere $70 per sample. Sunney Xie (Harvard University, USA) described a highly detailed preimplantation genetic diagnosis procedure that involves sequencing the first and second meiotic polar bodies, to deduce from them the genome of the female pronucleus.