ABSTRACT
Systematic performance comparing the results of exome-sequencing as a single test replacing Sanger-sequencing of targeted gene(s) is still lacking. In this study we compared Sanger-sequencing results of 258 genes to those obtained from next generation sequencing (NGS) using two exome-sequencing enrichment kits: Agilent-SureSelectQXT and Illumina-Nextera. Sequencing was performed on leukocytes and buccal-derived DNA from a single individual, and all 258 genes were sequenced a total of 11 times (using different sequencing methods and DNA sources). Sanger-sequencing was completed for all exons, including flanking ±8bp regions. For the 258 genes, NGS mean coverage was >20x for >98% and >91% of the regions targeted by SureSelect and Nextera, respectively. Overall, 449 variants were identified in at least one experiment, and 407/449 (90.6%) were detected by all. Of the 42 discordant variants, 23 were determined as true calls, summing-up to a truth set of 430 variants. Sensitivity of true-variant detection was 99% for Sanger-sequencing and 97%-100% for the NGS experiments. Mean false-positive rates were 3.7E-6 for Sanger-sequencing, 2.5E-6 for SureSelect-NGS and 5.2E-6 for Nextera-NGS. Our findings suggest a high overall concordance between Sanger-sequencing and NGS. Both methods demonstrated false positive and false negative calls and similar performances. Consequently, high clinical suspicion for a specific diagnosis should override negative results of either Sanger-sequencing or NGS.
Competing Interest Statement
The lab work presented in this study was conducted in Gene by Gene Ltd, in which Doron M. Behar, Concetta Bormans, and Arjan Bormans declare stock ownership, and Daniel Au, Brent Manning, and Luisa Fernanda Sanchez are employees. The analytic pipeline used in this study was conducted in Genoox Ltd, in which Moshe Einhorn and Yuval Porat declare stock ownership. The other authors claim no competing financial interests associated with this paper.