Bioinformatic Challanges in clinical diagnostic application of targeted next generation sequencing. Experience from Pheochromocytoma.

Background: Recent studies have demonstrated equal quality of targeted next generation sequencing (NGS) compared to Sanger Sequencing. Whereas these novel sequencing processes have a validated robust performance, genomic enrichment and bioinformatic software needs to be further investigated in a diagnostic setting. Methods: DNA from 21 patients with genetic variants in SDHB, VHL, EPAS1, RET, (n=17) or clinical criteria of NF1 syndrome (n=4) were included. Targeted NGS was performed using Truseq custom amplicon enrichment sequenced on an Illumina MiSEQ instrument. Results were analysed in parallel using three bioinformatics pipelines; (1) MiSEQ Reporter, fully automatized and integrated software, (2) CLC Genomics Workbench, graphical interface based software, and ICP (3) an in-house scripted custom pipeline. Results: A tenfold read coverage was achieved in between 95-98% of targeted bases. All workflows had alignment of reads to SDHA and NF1 pseudogenes. Compared to Sanger sequencing, variant calling revealed a sensitivity ranging from 83 to 100% and a specificity of 99.9-100%. Only MiSEQ reporter identified all pathogenic variants in both sequencing runs. Conclusions: We conclude that targeted next generation sequencing have equal quality compared to Sanger sequencing. Enrichment specificity and the bioinformatic performance need to be carefully assessed in a diagnostic setting. As acceptable accuracy was noted for a fully automated bioinformatic workflow, we suggest that processing of NGS data could be performed without expert bioinformatics skills.