ena-STUDY-University of Cyprus-08-10-2014-09:55:52:978-33
Frequency of COL4A3/COL4A4 mutations among families segregating glomerular microscopic hematuria and evidence for activation of the unfolded protein response
In this study, we used a custom-design Ampliseq panel that included the coding sequence of CFHR5, FN1, COL4A3, COL4A4 and COL4A5 genes to screen patients diagnosed with Thin Basement Membrane Nephropathy (TBMN) for mutations. This panel was comprised of 260 primer pairs that amplify exonic sequences with ±10bp padding (59.23 kb per patient in total). Amplicon sizes ranged from 125 – 175bp. Library was built by amplifying 10ng of genomic DNA, using the Ion Ampliseq Library Kit v2.0. Each sample was barcoded for multiplexing. Ampliseq library quantification and enrichment validation was performed using the Taqman™ Ion Library Quantitation Kit (LifeTechnologies) on a ViiA-7 Real Time PCR machine (Applied Biosystems, Foster City, CA, USA).  Amplified libraries were pooled and loaded onto the Ion OneTouch 2 system for emulsion PCR and enrichment of Ion Sphere Particles (ISPs) followed, using the OneTouch ES system. Enriched ISPs were then loaded on an Ion 316 chip and sequenced on the Ion Torrent PGM sequencer.
In this study, we used a custom-design Ampliseq panel that included the coding sequence of CFHR5, FN1, COL4A3, COL4A4 and COL4A5 genes to screen patients diagnosed with Thin Basement Membrane Nephropathy (TBMN) for mutations. This panel was comprised of 260 primer pairs that amplify exonic sequences with ±10bp padding (59.23 kb per patient in total). Amplicon sizes ranged from 125 – 175bp. Library was built by amplifying 10ng of genomic DNA, using the Ion Ampliseq Library Kit v2.0. Each sample was barcoded for multiplexing. Ampliseq library quantification and enrichment validation was performed using the Taqman™ Ion Library Quantitation Kit (LifeTechnologies) on a ViiA-7 Real Time PCR machine (Applied Biosystems, Foster City, CA, USA).  Amplified libraries were pooled and loaded onto the Ion OneTouch 2 system for emulsion PCR and enrichment of Ion Sphere Particles (ISPs) followed, using the OneTouch ES system. Enriched ISPs were then loaded on an Ion 316 chip and sequenced on the Ion Torrent PGM sequencer.