Palindromic amplification of the ERBB2 oncogene in human primary breast tumors. A common pattern of copy number transition of chromosome 17 in HER2-

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1 Supplemental Figures Table of Contents Palindromic amplification of the ERBB2 oncogene in human primary breast tumors Michael Marotta 1,5, Taku Onodera 3,5, Jeffrey Johnson 4, Thomas Budd 2, Takaaki Watanabe 1,4, Xiaojiang Cui 4, Armando Giuliano 4, Atsushi Niida 3 and Hisashi Tanaka 1,4 Figure 1 A common pattern of copy number transition of chromosome 17 in HER2- positive breast tumors Figure 2 Figure 3 Figure 4 Figure 5 Procedure of GAPF-seq Local genomic environment of the region surrounding ERBB2 gene Copy number alterations of chromosome 17 in HER2-positive breast cancer Mechanisms for generating a hairpin-capped chromosome 17 (model)

2 Supplementary Figure 1 - Marotta et al, Chr17 (hg19) 17q p12 p11.2 q q12 17q q25.3 ERBB2 A common pattern of copy number transition of chromosome 17 in HER2-positive breast tumors A heat map showing the copy number alterations in 117 HER2-positive breast tumors. Blue indicates genomic regions with copy-number gains and red indicates regions with copy-number loss.

3 Supplementary Figure 2 - Marotta et al, Tumor DNA palindromic DNA Normal DNA de-naturation re-naturation S1 nuclease digestion sequencing Procedure for enriching palindromic DNA from tumor genomic DNA. Palindromic DNA is shown by the pair of a blue and a red arrow that are complementary to each other. After denaturation and quick renaturation, palindromic DNA become double-stranded (dsdna), while non-palindromic DNA remains single-stranded (ssdna). Single-stranded nucleic acid specific nuclease S1 digests ssdna, but not dsdna. Thus the treatment by S1 results in the enrichment of DNA that derives from palindromic DNA.

4 Supplementary Figure 3 - Marotta et al, Scale chr17: Chromosome Band Hg19 Diff Common SNPs(144) Segmental Dups SYNRG DDX52 MIR378J RP11-697E22.2 HNF1B AC Mb hg38 38,000,000 38,500,000 39,000,000 39,500,000 40,000,000 40,500,000 41,000,000 41,500,000 17q12 Chromosome Bands Localized by FISH Mapping Clones 17q q21.2 Contigs New to GRCh38/(hg38), Not Carried Forward from GRCh37/(hg19) U6 TBC1D3K TBC1D3D 5S_rRNA TBC1D3L TBC1D3C TBC1D3D TBC1D3E Y_RNA Y_RNA ARHGAP23 MIR4734 RPL23 PLXDC1 TBC1D3E MRPL45 SRCIN1 PSMB3 FBXO47 ARL5C TBC1D3 SOCS7 C17orf96 U6 RP1-56K13.5 CACNB1 5S_rRNA MRPL45 CTB-58E17.2 CWC25 AC RPL19 GPR179 CISD3 LASP1 SOCS7 CTB-58E17.1 PLXDC1 ARHGAP23 CTB-58E17.3 AC AC MLLT6 RP5-906A24.1 CTB-58E17.9 MIR4726 MIR6779 RP5-906A24.2 AC CTD-2206N4.2 5S_rRNA PCGF2 CTB-58E17.5 PIP4K2B MIR4727 C17orf98 SNORA21 RP1-56K13.2 RP1-56K13.3 LINC00672 STAC2 FBXL20 AC Simple Nucleotide Polymorphisms (dbsnp 144) Found in >= 1% of Samples Duplications of >1000 Bases of Non-RepeatMasked Sequence CTB-131K11.1 NEUROD2 GRB7 MED1 PPP1R1B IKZF3 CDK12 TCAP RNU6-981P STARD3 RNU6-233P PNMT RP11-390P24.1 AC GRB7 PGAP3 MIR4728 MIEN1 ERBB2 GSDMB THRA CDC6 TNS4 KRT222 KRT28 KRT39 KRTAP4-5 KRT33A KRT36 KRT17 JUP CSF3 MIR6867 RARA CCR7 KRT24 KRT12 KRTAP2-1 KRTAP9-3 KRT34 KRT15 EIF1 P3H4 ZPBP2 MED24 WIPF2 IGFBP4 SMARCE1 KRT10 KRT40 KRTAP4-3 KRT33B KRT13 GAST ORMDL3 THRA RARA-AS1 KRT25 KRT23 KRTAP4-6 KRT31 KRT9 HAP1 RP11-387H17.4 NR1D1 CTD-2267D19.1 RP5-1028K7.1 KRT26 KRTAP4-4 AC KRT14 JUP LRRC3C MSL1 CTD-2267D19.3 RP11-458J1.1 KRT27 KRTAP4-2 KRT37 KRT16 GSDMA CASC3 GJD3 AC TMEM99 KRTAP1-5 KRTAP9-2 KRT38 RNA5SP442 PSMD3 RNA5SP441 KRT20 KRTAP1-4 KRTAP9-9 KRT32 HAP1 TOP2A KRTAP2-4 KRTAP9-4 RP11-387H17.6 RN7SL399P AC KRTAP4-1 AC MIR6884 KRTAP3-3 KRTAP9-2 RNU2-32P SNORD124 KRTAP3-2 KRTAP9-1 KRT35 RP11-749I16.3 KRTAP3-1 KRTAP9-8 KRT36 AC RP11-605F20.1 KRTAP1-3 KRTAP9-9 KRT13 RAPGEFL1 RP5-1110E20.1 KRTAP1-1 KRTAP9-6 Y_RNA KRTAP2-1 KRTAP9-7 AC RP11-58O9.2 KRTAP2-2 KRTAP29-1 KRT15 RP5-1028K7.2 KRTAP2-3 KRTAP16-1 MIR6510 KRTAP4-7 KRTAP17-1 KRT19 KRTAP4-8 LINC00974 RP13-415G19.2 KRTAP4-16P KRTAP4-9 KRTAP4-11 KRTAP4-12 FKBP10 NT5C3B KLHL10 RP11-156E6.1 KLHL11 Local genomic environment of the surrounding ERBB2 gene. Coordinates of chromosome 17, chromosome bands, contigs discordant from the previous genome assembly, dbsnps, segmental duplications, and genes are shown (modified from a figure taken from UCSC genome browser). Note that the centromeric side is flanked by the large discordant contigs and a large block of segmental duplications, whereas the telomeric side is flanked by the mumber of KRTAP and KRT gene families. As a result, these regions lack definitive SNP markers.

5 Supplementary Figure 4 - Marotta et al, 5T BT5 11T 40T BT4 ERBB2 Copy number alterations of chromosome 17 in 5 HER2-positive breast tumors. The location of ERBB2 amplicon is shown on the chromatograph. The regions on the 17p that retain copy numbers are indicated by black lines.

6 Supplementary Figure 5 - Marotta et al, fork stalling and reversal resolution resolution hairpin resolution end resection intra-strand annealing telomere centromere inverted repeats Inverted repeats-guided formation of a hairpin-capped chromosome (model). Replication forks stall at the inverted repeat (left). Fork reversal creates a four-way junction, the resolution of which results in a one-ended DSB. Processing of the end and intra-strand annealing lead to the formation of a hairpin-capped chromosome. Alternatively, a cruciform structure formed by the intra-strand annealing is resolved to form a hairpincapped chromosome (right).