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Assessment of HaloPlex Amplification for Sequence Capture and Massively Parallel Sequencing of Arrhythmogenic Right Ventricular Cardiomyopathy-Associated Genes
KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Linkoping University, Sweden.
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2015 (English)In: Journal of Molecular Diagnostics, ISSN 1525-1578, E-ISSN 1943-7811, Vol. 17, no 1, 31-42 p.Article in journal (Refereed) Published
Abstract [en]

The genetic basis of arrhythmogenic right ventricular cardiomyopathy (ARVC) is complex. Mutations in genes encoding components of the cardiac desmosomes have been implicated as being causally related to ARVC. Next-generation sequencing allows parallel sequencing and duplication/deletion analysis of many genes simultaneously, which is appropriate for screening of mutations in disorders with heterogeneous genetic backgrounds. We designed and validated a next-generation sequencing test panel for ARVC using HaloPlex. We used SureDesign to prepare a HaloPlex enrichment system for sequencing of DES, DSC2, DSG2, DSP, JUP, PKP2, RYR2, TGFB3, TMEM43, and TIN from patients with ARVC using a MiSeq instrument. Performance characteristics were determined by comparison with Sanger, as the gold standard, and TruSeq Custom Amplicon sequencing of DSC2, DSG2, DSP, JUP, and PKP2. All the samples were successfully sequenced after HaloPlex capture, with >99% of targeted nucleotides covered by >20x. The sequences were of high quality, although one problematic area due to a presumptive context-specific sequencing error causing motif Located in exon 1 of the DSP gene was detected. The mutations found by Sanger sequencing were also found using the HaloPlex technique. Depending on the bioinformatics pipeline, sensitivity varied from 99.3% to 100%, and specificity varied from 99.90/0 to 100%. Three variant positions found by Sanger and HaloPlex sequencing were missed by TruSeq Custom Amplicon owing to Loss of coverage.

Place, publisher, year, edition, pages
2015. Vol. 17, no 1, 31-42 p.
Keyword [en]
Task-Force Criteria, Generation, Genetics, Identification, Mutations, Dysplasia, Diagnosis, Families
National Category
Biological Sciences
URN: urn:nbn:se:kth:diva-159361DOI: 10.1016/j.jmoldx.2014.09.006ISI: 000347143100006ScopusID: 2-s2.0-84964307877OAI: diva2:784726
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Available from: 2015-01-30 Created: 2015-01-29 Last updated: 2015-01-30Bibliographically approved

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Green, Henrik
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