Title: Microfluidic Amplification as a Tool for Massive Parallel Sequencing of Familial Hypercholesterolemia Genes
Authors: Hollants, Silke
Redeker, Egbert J W
Matthijs, Gert # ×
Issue Date: Apr-2012
Publisher: P.B. Hoeber
Series Title: Clinical Chemistry vol:58 issue:4 pages:717-724
Abstract: BACKGROUND:Familial hypercholesterolemia (FH) is an autosomal dominant disorder that affects cholesterol metabolism and is an important risk factor for heart disease. Three different genes were causally linked to this disorder: LDLR (low density lipoprotein receptor), APOB [apolipoprotein B (including Ag(x) antigen)], and PCSK9 (proprotein convertase subtilisin/kexin type 9). We evaluated a new amplicon preparation tool for resequencing these genes on next generation sequencing (NGS) platforms.METHODS:For the 3 genes, 38 primer pairs were designed and loaded on the Fluidigm Access Array, a microfluidic array in which a PCR was performed. We amplified 144 DNA samples (73 positive controls and 71 patient samples) and performed 3 sequencing runs on a GS FLX Titanium system from Roche 454, using pyrosequencing. Data were analyzed with the SeqNext module of the Sequence Pilot software.RESULT:From the 38 amplicons, 37 were amplified successfully, without any further optimization. Sequencing resulted in a mean coverage of the individual amplicons of 71-fold, 74-fold, and 117-fold for the 3 runs, respectively. In the positive controls, all known mutations were identified. In 29% of the patient samples, a pathogenic point mutation or small deletion/insertion was found. Large rearrangements were not detectable with NGS, but were picked up by multiplex ligation-dependent probe amplification.CONCLUSIONS:Combining a microfluidic amplification system with massive parallel sequencing is an effective method for mutation scanning in FH patients, which can be implemented in diagnostics. For data analysis, we propose a minimum variant frequency threshold of 20% and a minimum coverage of 25-fold.
ISSN: 0009-9147
Publication status: published
KU Leuven publication type: IT
Appears in Collections:Department of Human Genetics - miscellaneous
Laboratory for Molecular Diagnosis
× corresponding author
# (joint) last author

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