sec three cycles of 95 u00b0 c for 30 sec 60 u00b0 c for 30 seconds 72 u00b0 c for 60 seconds 37 cycles of 95 u00b0 c for 30 seconds 58 u00b0 c for 30 seconds 72 u00b0 c for 60 seconds and one final cycle at 70 u00b0 c for 5 minutes the resulting dna sequencing templates were purified using agencourt ampure beckman coulter inc www beckmangenomics com the purified pcr reaction products were split in two and sequenced bidirectionally with m13 forward and reverse primers and bigdye terminator v3 1 cycle sequencing kit applied biosystems dye terminators were removed using the agencourt cleanseq kit beckman coulter inc and sequence reactions were run on an abi prism 3730xl sequencing apparatus applied biosystems mutations were detected using an automated detection pipeline at the mskcc bioinformatics core bi directional sequencing reads and mapping tables to link read names to sample identifiers gene names read direction and amplicon were subjected to a filter that excludes reads that have an average phred score of lt 10 for bases 100 200 passing reads were assembled against the reference sequences for each gene containing all coding and untranslated exons including 5 kb upstream and downstream of the gene using command line consed 16 0 gordon abajian and green 1998 assemblies were passed on to polyphred 6 02b nickerson tobe and taylor.