uu.seUppsala University Publications
Change search
ReferencesLink to record
Permanent link

Direct link
Bioinformatic Challenges in Clinical Diagnostic Application of Targeted Next Generation Sequencing: Experience from Pheochromocytoma
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Endocrine Surgery.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Experimental and Clinical Oncology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Endocrine Oncology.
Show others and affiliations
2015 (English)In: PLoS ONE, ISSN 1932-6203, Vol. 10, no 7, e0133210Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2015. Vol. 10, no 7, e0133210
National Category
Endocrinology and Diabetes Cancer and Oncology
URN: urn:nbn:se:uu:diva-261294DOI: 10.1371/journal.pone.0133210ISI: 000358838400029PubMedID: 26230854OAI: oai:DiVA.org:uu-261294DiVA: diva2:851176
Swedish Cancer Society
Available from: 2015-09-03 Created: 2015-09-01 Last updated: 2015-09-03Bibliographically approved

Open Access in DiVA

fulltext(2593 kB)67 downloads
File information
File name FULLTEXT01.pdfFile size 2593 kBChecksum SHA-512
Type fulltextMimetype application/pdf

Other links

Publisher's full textPubMed

Search in DiVA

By author/editor
Crona, JoakimLjungström, ViktorWelin, StaffanHellman, PerBjörklund, Peyman
By organisation
Endocrine SurgeryExperimental and Clinical OncologyEndocrine Oncology
In the same journal
Endocrinology and DiabetesCancer and Oncology

Search outside of DiVA

GoogleGoogle Scholar
Total: 67 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

Altmetric score

Total: 193 hits
ReferencesLink to record
Permanent link

Direct link