Bioinformatic Challenges in Clinical Diagnostic Application of Targeted Next Generation Sequencing: Experience from Pheochromocytoma.

Crona J, Ljungström V, Welin S, Walz MK, Hellman P, Björklund P

PLoS ONE 10 (7) e0133210 [2015-07-31; online 2015-07-31]

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. 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. 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. 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.

NGI Uppsala (SNP&SEQ Technology Platform)

National Genomics Infrastructure

PubMed 26230854

DOI 10.1371/journal.pone.0133210

Crossref 10.1371/journal.pone.0133210

pii: PONE-D-14-27974
pmc: PMC4521794