Validation of a comprehensive cancer genomic profiling assay based on massively parallel DNA sequencing.

e13138 Background: Validation is very important for diagnostic assays, here we describe a validation process of a comprehensive cancer genomic profiling assay based on massively parallel DNA sequencing. This assay can detect base substitutions, short insertions and deletions, copy number alterations, tumor mutation burden (TMB) and microsatellite instability (MSI) across 543 cancer-related genes from tumor specimens or plasma. Methods: Analytical validation was conducted with 20 cell line pools whose mutation were verified by digital droplet PCR (ddPCR). Clinical validation was conducted with Proficiency Test samples and clinical samples whose mutation were verified by ddPCR. Sensitivity, positive predictive value (PPV) and precision of tumor specimens and plasma were assessed across the reportable range of the assay. For measurement of TMB， NGS libraries of a cohort of tumor samples were tested by whole exosome panel (WES) and this assay respectively. And clinical tumor tissue samples whose MSI status were identified by fluorescent PCR-capillary electrophoresis were also tested by this assay. Results: The SNV/Indel LOD95 was 0.6% for hot-spot variants, and 1.3% for panel-wised variants of tumor tissue samples with high specificity (PPV >99%). The SNV/Indel LOD95 was 0.4% for hot-spot variants, and 1.1% for panel-wised variants of plasma samples (cfDNA≥15ng), with PPV >99%. LOD95 of CNV was 2.2-2.3, with PPV>99%. Precision of CNV was higher than 95%, precision of SNV/Indel was higher than 97%. Concordance between TMB results tested by WES and this assay was 0.94. MSI results identified by this assay was the same as fluorescent PCR-capillary electrophoresis method. Conclusions: In summary, we present the analytical and clinical validation of a comprehensive NGS-based diagnostic assay for comprehensive tumor genomic profiling.

Identification of Pathogenic Genes of Nonsyndromic Hearing Loss in Uyghur Families Using Massively Parallel DNA Sequencing Technique

We aim to identify the mutations of deafness genes using massively parallel DNA sequencing in the 12 Uyghur families. SNPscan method was used to screen against the 124 sites in the common deafness genes in probands. Subjects with SNPscan negativity were subject to massively parallel DNA sequencing for the sequencing of 97 genes known to be responsible for hearing loss. Eight families (66.7%) showed biallelic mutations in probands, including MYO15A mutation (6892C>T in J02 family, 9514C>T/7894G>T in J07 family, and 9514C>T in J16 family), MYO7A mutation (1258A>T in J03 family), TMC1 mutation (773G>A in J09 family and 1247T>G/1312G>A in J11 family), and PCDH15 mutation (4658delT in J08 and J13 families). Six novel types of mutation were identified including 6892C>T, 9514C>T/7894G>T, and 9514C>T in MYO15A gene, 1258A>T in MYO7A, 773G>A in TMC1, and 4658delT in PCDH15. The ratio of nonsense mutation and frameshift mutation was comparatively high. All these indicated that the mutation types reported in this study were rare. In conclusion, rare deafness genes were identified in the Uyghur families using massively parallel DNA sequencing, part of which were suggested to be related to the pathogenesis of the disease.