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Parallel Sequencing Used in Detection of Mosaic Mutations: Comparison With Four Diagnostic DNA Screening Techniques
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Neurology.
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2009 (English)In: Human Mutation, ISSN 1059-7794, E-ISSN 1098-1004, Vol. 30, no 6, 1012-1020 p.Article in journal (Refereed) Published
Abstract [en]

We have made an evaluation of mutation detection techniques for their abilities to detect mosaic mutations. In this study, Sanger sequencing, single-strand conformation polymorphism (SSCP)/heteroduplex analysis (HD), protein truncation test (PTT), and denaturating high-performance liquid chromatography (DHPLC) were compared with parallel sequencing. In total DNA samples from nine patients were included in this study. Mosaic mutations were artificially constructed from seven of these samples, which were from heterozygote mutation carriers with the mutant allele present at 50%. The mutations analyzed were as follows: c.646C>T, c.2626C>T, c.2828C>A, c.1817_1818insA, c.2788dupA, c.416_419delAAGA, and c.607delC in the APC gene. The lowest degree of mutant alleles detected with SSCP/HD and DHPLC varied between 5% and 25%, and between 15% and 50% for Sanger sequencing. Three of the mutations were analyzed with PTT with considerable variations in detection levels (from 10 to 100%). Using parallel sequencing a detection frequency down to 1% was reached, but to achieve this high sensitivity sufficient coverage was required. Two patients with natural mosaic mutations were also included in this study. These two mutations had previously been identified with Sanger sequencing (NF2 c.1026_1027delGA) and SSCP/HD (APC c.2700_2701delTC). In conclusion, all the evaluated methods are applicable for mosaic mutation screening even though combinations of the conventional methods should be used to reach an adequate sensitivity. Sanger sequencing alone is not sensitive enough to detect low mosaic levels. Parallel sequencing seems to be the ultimate choice but the possibilities to use this technique is today limited by its complexity, economics, and availability of instruments. Hum Mutat 30, 1012-1020, 2009.

Place, publisher, year, edition, pages
2009. Vol. 30, no 6, 1012-1020 p.
Keyword [en]
mosaicism, massively parallel sequencing, FAP, familial adenomatous polyposis, APC, NF2, neurofibromatosis type 2, mutation screening
National Category
Medical and Health Sciences
URN: urn:nbn:se:uu:diva-152613DOI: 10.1002/humu.20980ISI: 000267635100020OAI: oai:DiVA.org:uu-152613DiVA: diva2:413556
Available from: 2011-04-28 Created: 2011-04-28 Last updated: 2011-04-28Bibliographically approved

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