Mj tetrad

All 145 tissue samples have been previously analyzed by whole-exome sequencing, and the somatic mutation data have been published elsewhere^{8 (link)}. Sequence variants from three different genes (SPOP, FOXA1 and IDH1) were nominated from the exome sequencing analysis and here validated on genomic DNA with PCR and Sanger sequencing (using the same set up, kit, and cycling conditions as the RT-PCR described previously). The Primer3 software was used for primer design (Supplementary Table S1). Sanger-sequencing was performed on a MJ tetrad (BIO-RAD, Ca, USA) and an AB3730 DNA analyzer (Applied Biosystems) according to the manufacturers’ protocols.

QIAGEN Fast Cycling PCR Kit (Qiagen, UK) was used for both the GYPB DEL1 and DEL2 PCR reactions as detailed in Table 3a and 3b, with primers purchased from IDT (Leuven, Belgium). Restriction enzymes AciI (Catalog number: R0551L, NEB, UK) and BsrBI (Catalog number: R0102L, NEB, UK) were purchased (Table 4a). Reactions were prepared in 96-well plates (#AB-800, ThermoFisher Scientific, UK) and cycled on an MJ Tetrad (BioRad, UK) as described in Table 3a and 3b. The PCR products were digested with the relevant restriction enzymes (NEB, UK) for 2 hours at 37°C and then the digestion fragments were separated on 1% agarose gel electrophoresis containing ethidium bromide (3 ng/uL) for 2–2½ hours. Products were visualized under UV light and photographed to allow genotype assignment. All plates contained control samples obtained from the NHGRI repository (Table 2).