Hiseq2500 reagent kit

Central tumor DNA sequencing for HER2 mutation was performed at the GPS laboratory. Sanger technology was initially used to sequence exons 8 and 18 to 24 of HER2, which applied to approximately 20% of samples by October 2014. Subsequently, a PCR based next-generation sequencing (NGS) assay was used to sequence all HER2 coding exons. PCR was performed using the 48.48 high-throughput access array system (Fluidigm Corp.). Cluster generation and sequencing were performed using Illumina’s HiSeq2500 Reagent Kit (200 cycles), and 2 × 101 paired-end sequence reads were generated. Each patient’s DNA was processed in three independent technical replicates to generate and sequence three independent amplicon libraries. Variant call was performed on all BAM files together by a combination of commercially available and custom-developed scripts to generate a multi-sample variant call file. Variants were called if at least three of the four BAM files had evidence for the variant, and the average variant frequency was ≥10%.

PIK3CA (ref seq# NM_006218) sequencing of exons 2, 5, 8, 10 and 21, and exon-intron splice junctions was performed on tumor DNA from the baseline biopsy at the CLIA certified Washington University Genomic and Pathology Service initially by Sanger (n=18), subsequently by next-generation sequencing (NGS) (n=32). The targeted exons were PCR amplified using the (Fluidigm Corp., South San Francisco, USA) 48.48 high-throughput access array system. Cluster generation and sequencing was performed using Illumina's HiSeq2500 Reagent Kit (200 cycles) and 2x101 paired-end sequence reads were generated. Each patient's tumor DNA was processed and sequenced in three independent technical replicates. The three fastq files were each aligned independently to the human reference genome hg19 NCBI build 37.2 to generate three BAM files, then merged to a single BAM file. Sequence analysis was performed on all four BAM files using a combination of commercially available and custom developed scripts to generate a multisample VCF file. Alignment to the human reference genome was performed using Novoalign 2.08.02, sorting and indexing was performed by SAMtools 0.1.18-1, with coverage calculation by BedTools 2.13.3 and variants calling by Freebayes 0.9.7. Variants were called if present in at least three of the four BAM files and the average variant allele frequency (VAF) ≥10%.