Ambry Genetics (“Ambry”) today publishes data in The Journal of Molecular Diagnostics from the largest study to date1, 20,000 cases submitted for hereditary cancer panel testing, supporting its practice of Sanger sequencing next generation sequencing (NGS) variant calls. This step, integrated into Ambry’s workflow, allows for optimal results.
Study findings show that if Sanger confirmation is not utilized in addition to NGS, 1.3% of mutations identified would be false positives. A laboratory could control its bioinformatics pipeline to eliminate these false positives, but that would result in missing 2% of real mutations. The Ambry publication identified conservative thresholds that a laboratory can use to determine when Sanger confirmation is necessary. When those thresholds are not met, Sanger sequencing is necessary to ensure quality results.
NGS has rapidly replaced Sanger sequencing as the method of choice for diagnostic gene panel testing. For hereditary cancer testing, the technical sensitivity and specificity of the assays are paramount, as clinicians use results to make important clinical management and treatment decisions. There is significant debate within the diagnostic community regarding the necessity of confirming NGS variant calls by Sanger sequencing. Numerous laboratories report having 100% specificity from the NGS data alone; however, the current study conducted by Ambry shows that will sacrifice the mutation detection rate and false positive rate.
Ambry’s results come from 20,000 hereditary cancer NGS panels spanning 47 genes, where all 7,845 non-polymorphic variants were Sanger sequenced. Of these, 98.7% were concordant between NGS and Sanger sequencing and 1.3% were identified as NGS false positives, located mainly in complex genomic regions. Simulating a zero false positive rate by adjusting the variant calling Q score thresholds decreased the sensitivity of the assay from 100% to 97.8%. This resulted in the missed detection of 176 Sanger confirmed variants, the majority in complex genomic regions (n=114) and mosaic mutations (n=7).
The data illustrate the importance of setting quality thresholds for panel testing only after thousands of samples have been processed, and the necessity of Sanger confirmation of NGS variants to maintain the highest possible sensitivity.
Many laboratories are resistant to Sanger sequencing because it requires many additional steps, all that increase both the cost and turnaround time of testing. Guidance on NGS laboratory standards from the College of American Pathologists (CAP) leaves it up to the laboratory performing the assay to determine if confirmation testing is appropriate.2 In contrast, the American College of Medical Genetics and Genomics (ACMG) recommends secondary confirmation for all NSG reported variants.3
Ambry Chief Executive Officer and study author Aaron Elliott, PhD, said, “There’s a very dangerous trend in the diagnostic industry to drive pricing down by eliminating quality control steps such as NGS confirmation. Ambry will never sacrifice quality of testing to save money. The true false positive rate of an assay cannot be determined from running a few hundred samples during validation. Our data clearly show that if you do not perform Sanger confirmation you will either report out false positives or will miss more than 2% of real mutations.”
Ambry is a pioneer in NGS, having adopted the technology in 2007 and integrated it into operations since 2010. Ambry has been the first to market for a wide variety of NGS test offerings, including clinical exome sequencing and commercial multi-gene hereditary cancer panels. Having processed more than 1 million tests by NGS, Ambry has learned about the limitations in the technology. By maintaining the most sensitive bioinformatics pipeline available along with Sanger sequencing NGS variants, Ambry ensures the removal of false positives while keeping accuracy as high as possible.
To learn more about Ambry’s quality and approach to Sanger sequencing confirmation, please visit here.
Ambry engages in ongoing research, both internally and with collaborators. For a listing of peer-reviewed research publications, please visit here.
ABOUT AMBRY GENETICS®
Ambry Genetics is a privately-held healthcare company with the most comprehensive suite of genetic testing solutions for inherited diseases. Since 1999, Ambry has tested approximately nearly one million patient samples benefiting >94% of all U.S. patients covered by public and private insurers. Ambry is dedicated to scientific collaboration by offering its rapidly growing database of anonymized genomic data (variant frequencies) free to the global medical research community to fulfill the promise of the human genome to cure or manage all human disease. Ambry is dedicated to the belief that human health should not be patented or owned, and genomic data should be freely shared so we can try to understand all human disease. For more information about Ambry Genetics, visit ambrygen.com.
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Last updated on: 06/10/2016
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