TY - JOUR
T1 - Microfluidic Amplification as a Tool for Massive Parallel Sequencing of the Familial Hypercholesterolemia Genes
AU - Hollants, Silke
AU - Redeker, Egbert J. W.
AU - Matthijs, Gert
PY - 2012
Y1 - 2012
N2 - 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. (C) 2011 American Association for Clinical Chemistry
AB - 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. (C) 2011 American Association for Clinical Chemistry
U2 - https://doi.org/10.1373/clinchem.2011.173963
DO - https://doi.org/10.1373/clinchem.2011.173963
M3 - Article
C2 - 22294733
SN - 0009-9147
VL - 58
SP - 717
EP - 724
JO - Clinical Chemistry
JF - Clinical Chemistry
IS - 4
ER -