Software-assisted manual review of clinical next-generation sequencing data An alternative to routine Sanger sequencing confirmation with equivalent results in >15,000 germline DNA screens

Publication date: Available online 4 December 2018Source: The Journal of Molecular DiagnosticsAuthor(s): Dale Muzzey, Shera Kash, Jillian I. Johnson, Laura M. Melroy, Piotr Kaleta, Kelly A. Pierce, Kaylene Ready, Hyunseok P. Kang, Kevin R. HaasAbstractClinical genomic tests increasingly utilize a next-generation sequencing (NGS) platform due in part to the high fidelity of variant calls, yet rare errors are still possible. In germline DNA screening, failure to correct such errors could have serious consequences for patients, who may follow an unwarranted screening or surgical-management path. It has been suggested that routine orthogonal confirmation via Sanger sequencing is required to verify NGS results, especially low-confidence positives with depressed allele fraction (<30% of alternate allele). We evaluated whether an alternative method of confirmation—software-assisted manual call review—performed comparably to Sanger confirmation in>15,000 samples. Licensed reviewers manually inspected both raw and processed data at the batch-, sample-, and variant-level, including raw NGS read pileups. Of ambiguous variant calls with <30% allele fraction (1,707 total calls at 38 unique sites), manual call review classified>99% (1,701) as true positives (enriched for long insertions or deletions (“indels”) and homopolymers) or true negatives (often conspicuous NGS artifacts), with the remaining <1% (six) being mosaic. Critically, results from software-assisted manua...

Source: The Journal of Molecular Diagnostics - December 5, 2018 Category: Pathology Source Type: research