title: Bioinformatic Challenges in Clinical Diagnostic Application of Targeted Next Generation Sequencing: Experience from Pheochromocytoma.
abstract: 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, choice of enrichment method and different available bioinformatic software as reliable analysis tool 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 different bioinformatics pipelines; (1) Commercially available MiSEQ Reporter, fully automatized and integrated software, (2) CLC Genomics Workbench, graphical interface based software, also commercially available, and ICP (3) an in-house scripted custom bioinformatic tool.
  
  
  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 utilizing already existing commercially available bioinformatics tools.
authors.affiliation: Department of Surgical Sciences, Uppsala University, SE-75185, Uppsala, Sweden.
authors.forename: Joakim
authors.initials: J
authors.lastname: Crona
authors.affiliation: Department of Immunology, Genetics and Pathology, Uppsala University, SE-75185, Uppsala, Sweden.
authors.forename: Viktor
authors.initials: V
authors.lastname: Ljungström
authors.affiliation: Departments of Medical Sciences, Uppsala University, SE-75185, Uppsala, Sweden.
authors.forename: Staffan
authors.initials: S
authors.lastname: Welin
authors.affiliation: Department for Surgery and Centre of Minimal Invasive Surgery, Kliniken Essen-Mitte, Academic Teaching Hospital of the University of Duisburg-Essen, DE-45136 Essen, Germany.
authors.forename: Martin K
authors.initials: MK
authors.lastname: Walz
authors.affiliation: Department of Surgical Sciences, Uppsala University, SE-75185, Uppsala, Sweden.
authors.forename: Per
authors.initials: P
authors.lastname: Hellman
authors.affiliation: Department of Surgical Sciences, Uppsala University, SE-75185, Uppsala, Sweden.
authors.forename: Peyman
authors.initials: P
authors.lastname: Björklund
mesh_terms: D000293:Adolescent; D000310:Adrenal Gland Neoplasms / Q000175:diagnosis* / Q000235:genetics; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D019295:Computational Biology; D005260:Female; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008297:Male; D008875:Middle Aged; D010673:Pheochromocytoma / Q000175:diagnosis* / Q000235:genetics; D012189:Retrospective Studies; D055815:Young Adult