Albertine Donker

IRIDA: a Heterogeneous Disease 167 4 SUPPLEMENTAL FILES Supplemental Table 1. Primers used for Sanger and Ion Torrent sequencing Exon Forward primer (5’ → 3’) Reverse primer (5’ → 3’) 1-2 tgtaaaacgacggccagtTGAGACCTCCGTCTGTCCTC caggaaacagctatgaccGCTACAGTCACCCCAAGTCC 3 tgtaaaacgacggccagtACAGGAGACTTTCCCACCTG caggaaacagctatgaccCATGTGATCAGACCCCACC 4 tgtaaaacgacggccagtGTAGGAAGTGGGCCTGTCTG caggaaacagctatgaccCCCCTCCCATTCTTTGAATC 5 tgtaaaacgacggccagtAGCTGGGGCCAGACCTC caggaaacagctatgaccCCCTTGGAGCCAGCCTTGTC 6 tgtaaaacgacggccagtAAGGCTGGCTCCAAGGG caggaaacagctatgaccTGCTTGGGACACATCGCTGA 7 tgtaaaacgacggccagtGTCCCCTCCTTCTGGCTC caggaaacagctatgaccTGACTTTCAACTCCCCCATC 8 tgtaaaacgacggccagtCCACTCCCCTCCCAGAC caggaaacagctatgaccAAAGGTGAGCAGTGAGCCC 9 tgtaaaacgacggccagtGTGGGGTTACAAGCTGCG caggaaacagctatgaccACCAGGGACCTGTAGTGTGC 10 tgtaaaacgacggccagtTTTGTTGTTAGGGAGGTGGG caggaaacagctatgaccCTGAGATTGGGGACTTGGG 11 tgtaaaacgacggccagtCACGGGACCCAGGAGAC caggaaacagctatgaccTTGGTGGTTCCAGGGATG 12 tgtaaaacgacggccagtGCTACGCATGGCCTAATGG caggaaacagctatgaccCTCAGCTCAGAGCAGGAAAG 13 tgtaaaacgacggccagtCGTGCAATACAGCACACCTT caggaaacagctatgaccCTTTTGCTGAAGCATGTAGCAG 14 tgtaaaacgacggccagtCTTTGGAAGGTTTCCTTGGG caggaaacagctatgaccAGCTTCCTGCTGTGGGC 15 tgtaaaacgacggccagtTCTGTCTGCTTCTCCCCTTC caggaaacagctatgaccGACACAGTGCACCTCCCAC 16 tgtaaaacgacggccagtCAGCTTCCTCCCACCTCAC caggaaacagctatgaccTTCTCCAGGCCAGGTGTTAC 17 tgtaaaacgacggccagtTAGAGAACAGGGGCTCCAGG caggaaacagctatgaccATGTGAGCAAAGGGCCAG 18 tgtaaaacgacggccagtTCCCTATGGCTCTTCACCTG caggaaacagctatgaccTTAGGCAGCAGTGGAGGAAG Lower case letters indicate the universal primersequences used for Sanger Sequencing, capital letters indicate the specific parts of the TMPRSS6 exons Multiplex Ligation-dependent Probe Amplification (MLPA) All reagents for the MLPA reaction and subsequent PCR amplification were purchased from MRC- Holland (Amsterdam, The Netherlands), with exception of the TMRPSS6 and control probes. The two- color MLPA was performed as described previously. In short, in a one-tube format, combinations of two adjacently annealing oligonucleotide probes were hybridized and ligated. After ligation, the common ends of the probes served as a template for PCR amplification with one primer pair and due to the fluorescent labeling of the primer the resulting products could be separated according to size using capillary electrophoresis on the ABI3130XL (Applied Biosystems). Fragment data were analyzed in GeneScan (Applied Biosystems). Peak heights of patient’s samples were compared with control probes and ratios were calculated for all fragments (originating from TMPRSS6 exons) using an Excel spreadsheet. Thresholds for deletions and duplications were set at 0.8 and 1.2 respectively, and all samples were tested at least twice (Table 2).