Figure 1

Experimental approach. (A) Each tumor was divided into three to five regions to assay intratumoral heterogeneity. Each individual region was subdivided into four pieces for use in next generation sequencing (NGS), histology, cell culture and xenotransplantation. (B) Molecular inversion probe method. Oligonucleotide probes were previously designed against 33 cancer genes [6]. MIPs have a common backbone sequence, molecular tag sequence as well as targeting arms homologous to regions flanking targets of interest. After polymerase extension and ligation, targeted sequence is captured within a circular molecule. Captured sequences are amplified in a barcoding-PCR reaction and multiple samples are pooled and sequenced on the same lane. After tag-correction (not shown), reads corresponding to each tumor region are mapped to the human reference sequence to be used to identify copy number amplifications and point mutations specific to one region or another. Additional details are provided in Figure S1 in Additional file 1. (C) Example of comparisons: MIP captures of regions C and D can detect both TP53 point mutation heterogeneity and EGFR amplification heterogeneity within a tumor. Tumors with mutational heterogeneity were required to share either a point mutation or copy number alteration (in this case mutation of PTEN) across all regions to ensure that differences in observed mutation were not due to varying levels of tumor cellularity.