a b 2250 1.0

2000

1750 0.8

1500 0.6 1250

1000 0.4

depth read gDNA 750 cfDNA allele frequency allele cfDNA 500 0.2

250

0 0 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 gDNA allele frequency gDNA allele frequency

Supplementary Figure 1. Detection of germline BRCA2, ATM, and CDK12 variants in patient-matched gDNA and cfDNA. a. Scatterplot showing the variant allele frequencies (VAFs) of deleterious germline BRCA2, ATM, and CDK12 mutations versus sequencing read depth. b. Scatterplot showing the VAFs of deleterious germline BRCA2, ATM, and CDK12 mutations as detected in both leukocyte (germline DNA; gDNA) and cell-free DNA (cfDNA) samples. VAF distribution is highly suggestive of a germline origin for all variants (i.e. inconsistent with hematopoietic etiology) and shows features consistent with somatic loss-of- heterozygosity.

1 3 P37-2 2

ctDNA fraction = 75% 1

0

1 Coverage logratio Coverage

2 BRCA2 3

AR APC CLU ATR RB1 ERG MET ATM MYC IDH2 IDH1 TP53 AKT2 AKT1 AKT3 PTEN RYBP CUL1 SPOP BRAF KRAS CDK6 CDK4 MLH1 GNAS CHD1 MSH2 MSH6 MDM2 MDM4 PALB2 ASXL1 FANCF FOXP1 RNF43 ZFHX3 FANCE ERCC2 ERCC1 ERCC5 ERCC4 ERCC3 FOXA1 FANCA CDK12 BRCA1 BRCA2 FANCC KMT2C FANCG RUNX1 PIK3R1 MED12 KMT2D CCND1 NCOA2 FBXW7 PIK3CA PIK3CB KDM6A NKX3-1 ARID1A NFE2L2 ZBTB16 RAD51B FANCD2 HSD3B1 RAD51C CTNNB1 CDKN2A CDKN1B TMPRSS2 SMARCA1 100

90

80

70 SNP allele fraction allele SNP 60

50 AR APC CLU ATR RB1 ERG MET ATM MYC IDH2 IDH1 TP53 AKT2 AKT1 AKT3 PTEN RYBP CUL1 SPOP BRAF KRAS CDK6 CDK4 MLH1 GNAS CHD1 MSH2 MSH6 MDM2 MDM4 PALB2 ASXL1 FANCF FOXP1 RNF43 ZFHX3 FANCE ERCC2 ERCC1 ERCC5 ERCC4 ERCC3 FOXA1 FANCA CDK12 BRCA1 BRCA2 FANCC KMT2C FANCG RUNX1 PIK3R1 MED12 KMT2D CCND1 NCOA2 FBXW7 PIK3CA PIK3CB KDM6A NKX3-1 ARID1A NFE2L2 ZBTB16 RAD51B FANCD2 HSD3B1 RAD51C CTNNB1 CDKN2A CDKN1B TMPRSS2 SMARCA1

3 P24-4 2 1 ctDNA fraction = 60% 0

1

logratio Coverage 2 3 BRCA2 AR APC CLU ATR RB1 ERG MET ATM MYC IDH2 IDH1 TP53 AKT2 AKT1 AKT3 PTEN RYBP CUL1 SPOP BRAF

KRAS CDK6 CDK4 MLH1 GNAS CHD1 MSH2 MSH6 MDM2 MDM4 PALB2 ASXL1 FANCF FOXP1 RNF43 ZFHX3 FANCE ERCC2 ERCC1 ERCC5 ERCC4 ERCC3 FOXA1 FANCA CDK12 BRCA1 BRCA2 FANCC KMT2C FANCG RUNX1 PIK3R1 MED12 KMT2D CCND1 NCOA2 FBXW7 PIK3CA PIK3CB KDM6A NKX3-1 ARID1A NFE2L2 ZBTB16 RAD51B FANCD2 HSD3B1 RAD51C CTNNB1 CDKN2A CDKN1B TMPRSS2 SMARCA1 100

90

80

70 SNP allele fraction allele SNP 60

50

AR APC CLU ATR RB1 ERG MET ATM MYC IDH2 IDH1 TP53 AKT2 AKT1 AKT3 PTEN RYBP CUL1 SPOP BRAF KRAS CDK6 CDK4 MLH1 GNAS CHD1 MSH2 MSH6 MDM2 MDM4 PALB2 ASXL1 FANCF FOXP1 RNF43 ZFHX3 FANCE ERCC2 ERCC1 ERCC5 ERCC4 ERCC3 FOXA1 FANCA CDK12 BRCA1 BRCA2 FANCC KMT2C FANCG RUNX1 PIK3R1 MED12 KMT2D CCND1 NCOA2 FBXW7 PIK3CA PIK3CB KDM6A NKX3-1 ARID1A NFE2L2 ZBTB16 RAD51B FANCD2 HSD3B1 RAD51C CTNNB1 CDKN2A CDKN1B TMPRSS2 SMARCA1

3 P12-6 2

ctDNA fraction = 39% 1 0

1 logratio Coverage BRCA2 2 3 AR APC CLU ATR RB1 ERG MET ATM MYC IDH1 IDH2 TP53 AKT2 AKT3 AKT1 PTEN RYBP CUL1 SPOP BRAF KRAS CDK6 CDK4 MLH1 GNAS CHD1 MSH2 MSH6 MDM4 MDM2 PALB2 ASXL1 FANCF FOXP1 RNF43 ZFHX3 FANCE ERCC2 ERCC1 ERCC3 ERCC5 ERCC4 FOXA1 FANCA CDK12 BRCA1 BRCA2 FANCC KMT2C FANCG RUNX1 PIK3R1 MED12 KMT2D CCND1 NCOA2 FBXW7 PIK3CA PIK3CB KDM6A NKX3-1

ARID1A NFE2L2 ZBTB16 RAD51B FANCD2 HSD3B1 RAD51C CTNNB1 CDKN2A CDKN1B TMPRSS2 SMARCA1 100 90 80

70

fraction allele SNP 60 50 AR APC CLU ATR RB1 ERG MET ATM MYC IDH2 IDH1 TP53 AKT2 AKT1 AKT3 PTEN RYBP CUL1 SPOP BRAF KRAS CDK4 CDK6 MLH1 GNAS CHD1

MSH2 MSH6 MDM2 MDM4 PALB2 ASXL1 FANCF FOXP1 RNF43 ZFHX3 FANCE ERCC2 ERCC1 ERCC5 ERCC4 ERCC3 FOXA1 FANCA CDK12 BRCA1 BRCA2 FANCC KMT2C FANCG RUNX1 PIK3R1 MED12 KMT2D CCND1 NCOA2 FBXW7 PIK3CA PIK3CB KDM6A NKX3-1 ARID1A NFE2L2 ZBTB16 RAD51B FANCD2 HSD3B1 RAD51C CTNNB1 CDKN2A CDKN1B TMPRSS2 SMARCA1

Supplementary Figure 2. Homozygous BRCA2 deletions. Evidence for BRCA2 homozygous deletion from initial targeted cfDNA sequencing in three representative cases. Copy number violin plots are plotted on top and swarmplots showing intragenic heterozygous single nucleotide polymorphism (SNP) variant allele frequencies are shown below.

2 3 3

2 2

1 1 P25 0 P47 0 1 48% TC 1

26% TC logratio Coverage logratio Coverage Monoallelic 2 2nd mutation 2 3 3 AR AR APC APC CLU CLU ATR ATR RB1 RB1 ERG ERG MET MET ATM ATM MYC MYC IDH1 IDH2 IDH1 IDH2 TP53 TP53 AKT2 AKT2 AKT3 AKT1 AKT3 AKT1 PTEN PTEN RYBP RYBP CUL1 CUL1 SPOP SPOP BRAF BRAF KRAS KRAS CDK6 CDK6 CDK4 CDK4 MLH1 MLH1 GNAS GNAS CHD1 CHD1 MSH2 MSH6 MSH2 MSH6 MDM4 MDM2 MDM4 MDM2 PALB2 PALB2 ASXL1 ASXL1 FANCF FANCF FOXP1 FOXP1 RNF43 RNF43 ZFHX3 ZFHX3 FANCE FANCE ERCC2 ERCC1 ERCC2 ERCC1 ERCC3 ERCC5 ERCC4 ERCC3 ERCC5 ERCC4 FOXA1 FOXA1 FANCA FANCA CDK12 CDK12 BRCA1 BRCA1 BRCA2 BRCA2 FANCC FANCC KMT2C KMT2C FANCG FANCG RUNX1 RUNX1 PIK3R1 PIK3R1 MED12 MED12 KMT2D KMT2D CCND1 CCND1 NCOA2 NCOA2 FBXW7 FBXW7 PIK3CA PIK3CA PIK3CB PIK3CB KDM6A KDM6A NKX3-1 NKX3-1 ARID1A ARID1A NFE2L2 NFE2L2 ZBTB16 ZBTB16 RAD51B RAD51B FANCD2 FANCD2 HSD3B1 HSD3B1 RAD51C RAD51C CTNNB1 CTNNB1 CDKN2A CDKN2A CDKN1B CDKN1B TMPRSS2 TMPRSS2 SMARCA1 SMARCA1 100 100

3 3 90 90

2 2 80 80 1 1

70 70 P30 0 P60 0 SNP allele fraction allele SNP fraction allele SNP

60 60 1 48% TC 1 32% TC logratio Coverage Coverage logratio Coverage

2 2nd mutation 50 Monoallelic 250 AR AR APC APC CLU CLU ATR ATR RB1 RB1 ERG ERG MET MET ATM ATM MYC MYC IDH1 IDH2 IDH1 IDH2 TP53 TP53 AKT2 AKT2 AKT3 AKT1 AKT3 AKT1 PTEN PTEN RYBP RYBP CUL1 CUL1 SPOP SPOP BRAF BRAF KRAS KRAS CDK6 CDK6 CDK4 CDK4 MLH1 MLH1 GNAS GNAS CHD1 CHD1 3 MSH2 MSH6 MSH2 MSH6 MDM4 MDM2 MDM4 MDM2 PALB2 PALB2 ASXL1 ASXL1 FANCF FANCF FOXP1 FOXP1 RNF43 RNF43 ZFHX3 ZFHX3 FANCE FANCE ERCC2 ERCC1 ERCC2 ERCC1 ERCC3 ERCC5 ERCC4 ERCC3 ERCC5 ERCC4 FOXA1 FOXA1 FANCA FANCA CDK12 CDK12 BRCA1 BRCA1 BRCA2 BRCA2

FANCC 3 FANCC KMT2C KMT2C FANCG FANCG RUNX1 RUNX1 PIK3R1 PIK3R1 MED12 MED12 KMT2D KMT2D CCND1 CCND1 NCOA2 NCOA2 FBXW7 FBXW7 PIK3CA PIK3CA PIK3CB PIK3CB KDM6A KDM6A NKX3-1 NKX3-1 ARID1A ARID1A NFE2L2 NFE2L2 ZBTB16 ZBTB16 RAD51B RAD51B FANCD2 FANCD2 HSD3B1 HSD3B1 RAD51C RAD51C CTNNB1 CTNNB1 CDKN2A CDKN2A CDKN1B CDKN1B AR TMPRSS2 TMPRSS2 AR SMARCA1 SMARCA1 APC CLU ATR RB1 ERG MET ATM MYC APC CLU ATR RB1 IDH1 IDH2 ERG MET TP53 ATM MYC AKT2 AKT3 AKT1 PTEN RYBP CUL1 SPOP BRAF KRAS CDK6 CDK4 MLH1 IDH1 IDH2 GNAS CHD1 TP53 MSH2 MSH6 AKT2 AKT3 AKT1 PTEN RYBP CUL1 SPOP BRAF KRAS MDM4 MDM2 CDK6 CDK4 PALB2 MLH1 ASXL1 GNAS CHD1 MSH2 MSH6 FANCF FOXP1 RNF43 ZFHX3 FANCE ERCC2 ERCC1 ERCC3 ERCC5 ERCC4 FOXA1 FANCA CDK12 BRCA1 BRCA2 FANCC KMT2C FANCG RUNX1 MDM4 MDM2 PIK3R1 MED12 KMT2D PALB2 CCND1 NCOA2 FBXW7 PIK3CA PIK3CB ASXL1 KDM6A FANCF FOXP1 RNF43 ZFHX3 FANCE NKX3-1 ERCC2 ERCC1 ERCC3 ERCC5 ERCC4 FOXA1 ARID1A FANCA CDK12 BRCA1 BRCA2 FANCC NFE2L2 KMT2C FANCG RUNX1 PIK3R1 MED12 KMT2D CCND1 NCOA2 ZBTB16 FBXW7 PIK3CA PIK3CB KDM6A NKX3-1 RAD51B FANCD2 HSD3B1 RAD51C CTNNB1 ARID1A NFE2L2 CDKN2A CDKN1B ZBTB16 RAD51B FANCD2 HSD3B1 RAD51C CTNNB1 CDKN2A CDKN1B TMPRSS2 SMARCA1 TMPRSS2 SMARCA1 100 100

3 3 90 90 2 2 80 80 1 1 70 70 P78 0 P82 0 SNP allele fraction allele SNP SNP allele fraction allele SNP 60 1 52% TC 601 Coverage logratio Coverage

39% TC logratio Coverage

2 Monoallelic 50 2nd mutation 502 AR AR APC CLU ATR RB1 ERG MET ATM MYC APC CLU ATR RB1 IDH1 IDH2 ERG MET TP53 ATM MYC AKT2 AKT3 AKT1 PTEN RYBP CUL1 SPOP BRAF KRAS CDK6 CDK4 MLH1 IDH1 IDH2 GNAS CHD1 TP53 MSH2 MSH6 AKT2 AKT3 AKT1 PTEN RYBP CUL1 SPOP BRAF KRAS MDM4 MDM2 CDK6 CDK4 PALB2 MLH1 ASXL1 GNAS CHD1

3 MSH2 MSH6 FANCF FOXP1 RNF43 ZFHX3 FANCE ERCC2 ERCC1 ERCC3 ERCC5 ERCC4 FOXA1 FANCA CDK12 BRCA1 BRCA2 FANCC KMT2C FANCG RUNX1 MDM4 MDM2 PIK3R1 MED12 KMT2D 3 PALB2 CCND1 NCOA2 FBXW7 PIK3CA PIK3CB ASXL1 KDM6A FANCF FOXP1 RNF43 ZFHX3 FANCE NKX3-1 ERCC2 ERCC1 ERCC3 ERCC5 ERCC4 FOXA1 ARID1A FANCA CDK12 BRCA1 BRCA2 FANCC NFE2L2 KMT2C FANCG RUNX1 PIK3R1 MED12 KMT2D CCND1 NCOA2 ZBTB16 FBXW7 PIK3CA PIK3CB KDM6A NKX3-1 RAD51B FANCD2 HSD3B1 RAD51C CTNNB1 ARID1A NFE2L2 CDKN2A CDKN1B ZBTB16 RAD51B FANCD2 HSD3B1 RAD51C CTNNB1 CDKN2A CDKN1B AR TMPRSS2 AR SMARCA1 TMPRSS2 APC CLU ATR RB1 SMARCA1 ERG MET ATM MYC APC CLU ATR RB1 ERG MET ATM MYC IDH1 IDH2 TP53 AKT2 AKT3 AKT1 PTEN RYBP CUL1 SPOP BRAF KRAS IDH1 IDH2 TP53 CDK6 CDK4 MLH1 GNAS CHD1 AKT2 AKT3 AKT1 PTEN RYBP MSH2 MSH6 CUL1 SPOP BRAF KRAS CDK6 CDK4 MLH1 GNAS CHD1 MDM4 MDM2 MSH2 MSH6 PALB2 ASXL1 FANCF FOXP1 RNF43 ZFHX3 FANCE ERCC2 ERCC1 ERCC3 ERCC5 ERCC4 FOXA1 FANCA CDK12 BRCA1 BRCA2 FANCC MDM4 MDM2 PALB2 KMT2C ASXL1 FANCG RUNX1 PIK3R1 MED12 KMT2D CCND1 NCOA2 FANCF FOXP1 RNF43 FBXW7 PIK3CA PIK3CB KDM6A ZFHX3 FANCE ERCC2 ERCC1 ERCC3 ERCC5 ERCC4 FOXA1 FANCA CDK12 BRCA1 BRCA2 FANCC NKX3-1 KMT2C FANCG ARID1A RUNX1 NFE2L2 PIK3R1 MED12 KMT2D CCND1 NCOA2 FBXW7 PIK3CA PIK3CB KDM6A ZBTB16 NKX3-1 ARID1A NFE2L2 RAD51B FANCD2 HSD3B1 RAD51C CTNNB1 CDKN2A CDKN1B ZBTB16 RAD51B FANCD2 HSD3B1 RAD51C CTNNB1 CDKN2A CDKN1B TMPRSS2 SMARCA1 TMPRSS2 SMARCA1 100 100

3 3 90 90 2 2 80 80 1 1 70 70 P54 0 P58 0 SNP allele fraction allele SNP SNP allele fraction allele SNP 60 41% TC 1 56% TC 601 Coverage logratio Coverage logratio Coverage

Monoallelic 250 Monoallelic 502 AR AR APC CLU ATR RB1 ERG MET ATM MYC APC CLU ATR RB1 ERG MET ATM MYC IDH2 IDH1 TP53 AKT2 AKT1 AKT3 PTEN RYBP CUL1 SPOP BRAF KRAS IDH2 IDH1 TP53 CDK6 CDK4 MLH1 GNAS CHD1 AKT2 AKT1 AKT3 PTEN RYBP MSH2 MSH6 CUL1 SPOP BRAF KRAS CDK6 CDK4 MLH1 GNAS CHD1 MDM2 MDM4 MSH2 MSH6 PALB2 ASXL1 FANCF FOXP1 RNF43 ZFHX3 FANCE ERCC2 ERCC1 ERCC5 ERCC4 ERCC3 FOXA1 FANCA CDK12 BRCA1 BRCA2 FANCC MDM2 3 MDM4 PALB2 KMT2C ASXL1 FANCG

3 RUNX1 PIK3R1 MED12 KMT2D CCND1 NCOA2 FANCF FOXP1 RNF43 FBXW7 PIK3CA PIK3CB KDM6A ZFHX3 FANCE ERCC2 ERCC1 ERCC5 ERCC4 ERCC3 FOXA1 FANCA CDK12 BRCA1 BRCA2 FANCC NKX3-1 KMT2C FANCG ARID1A RUNX1 NFE2L2 PIK3R1 MED12 KMT2D CCND1 NCOA2 FBXW7 PIK3CA PIK3CB KDM6A ZBTB16 NKX3-1 ARID1A NFE2L2 RAD51B FANCD2 HSD3B1 RAD51C CTNNB1 CDKN2A CDKN1B

ZBTB16 RAD51B FANCD2 HSD3B1 RAD51C CTNNB1 CDKN2A CDKN1B TMPRSS2 AR AR SMARCA1 TMPRSS2 SMARCA1 APC CLU ATR RB1 APC CLU ATR RB1 ERG MET ATM ERG MYC MET ATM MYC IDH2 IDH1 TP53 IDH2 IDH1 TP53 AKT2 AKT1 AKT3 PTEN RYBP CUL1 SPOP BRAF AKT2 AKT1 AKT3 PTEN RYBP KRAS CUL1 SPOP BRAF KRAS CDK6 CDK4 MLH1 CDK6 CDK4 GNAS CHD1 MLH1 MSH2 MSH6 GNAS CHD1 MSH2 MSH6 MDM2 MDM4 PALB2 ASXL1 MDM2 MDM4 PALB2 ASXL1 FANCF FOXP1 RNF43 ZFHX3 FANCE FANCF FOXP1 RNF43 ERCC2 ERCC1 ERCC5 ERCC4 ERCC3 FOXA1 FANCA CDK12 BRCA1 BRCA2 ZFHX3 FANCC FANCE ERCC2 ERCC1 ERCC5 ERCC4 ERCC3 FOXA1 FANCA KMT2C CDK12 BRCA1 BRCA2 FANCC FANCG RUNX1 PIK3R1 KMT2C MED12 KMT2D FANCG CCND1 NCOA2 RUNX1 FBXW7 PIK3CA PIK3CB PIK3R1 MED12 KMT2D KDM6A CCND1 NCOA2 FBXW7 PIK3CA PIK3CB KDM6A NKX3-1 ARID1A NFE2L2 NKX3-1 ARID1A NFE2L2 ZBTB16 ZBTB16 RAD51B FANCD2 HSD3B1 RAD51C CTNNB1 RAD51B FANCD2 HSD3B1 RAD51C CDKN2A CDKN1B CTNNB1 CDKN2A CDKN1B TMPRSS2 TMPRSS2 SMARCA1 SMARCA1 100 100

3 3 90 90

2 2 80 80 1 1

70 70 P36 0 P85 0 SNP allele fraction allele SNP SNP allele fraction allele SNP

43% TC 601 601

60% TC logratio Coverage logratio Coverage 2 CNLOH 502 2nd mutation 50 AR AR APC CLU ATR RB1 APC CLU ATR RB1 ERG MET ATM ERG MYC MET ATM MYC IDH2 IDH1 TP53 IDH2 IDH1 TP53 AKT2 AKT1 AKT3 PTEN RYBP CUL1 SPOP BRAF AKT2 AKT1 AKT3 PTEN RYBP KRAS CUL1 SPOP BRAF KRAS CDK6 CDK4 MLH1 CDK6 CDK4 GNAS CHD1 MLH1 MSH2 MSH6 GNAS CHD1 MSH2 MSH6 MDM2 3 MDM4 PALB2 ASXL1 MDM2 MDM4 PALB2 ASXL1 FANCF FOXP1 RNF43

3 ZFHX3 FANCE FANCF FOXP1 RNF43 ERCC2 ERCC1 ERCC5 ERCC4 ERCC3 FOXA1 FANCA CDK12 BRCA1 BRCA2 ZFHX3 FANCC FANCE ERCC2 ERCC1 ERCC5 ERCC4 ERCC3 FOXA1 FANCA KMT2C CDK12 BRCA1 BRCA2 FANCC FANCG RUNX1 PIK3R1 KMT2C MED12 KMT2D FANCG CCND1 NCOA2 RUNX1 FBXW7 PIK3CA PIK3CB PIK3R1 MED12 KMT2D KDM6A CCND1 NCOA2 FBXW7 PIK3CA PIK3CB KDM6A NKX3-1 ARID1A NFE2L2 NKX3-1 ARID1A NFE2L2 ZBTB16 ZBTB16 RAD51B FANCD2 HSD3B1 RAD51C CTNNB1 RAD51B FANCD2 HSD3B1 RAD51C CDKN2A CDKN1B CTNNB1 CDKN2A CDKN1B AR TMPRSS2 AR TMPRSS2 SMARCA1 SMARCA1 APC CLU ATR RB1 ERG MET ATM APC CLU ATR MYC RB1 ERG MET ATM MYC IDH2 IDH1 TP53 AKT2 AKT1 AKT3 PTEN RYBP IDH2 CUL1 IDH1 SPOP BRAF TP53 KRAS AKT2 AKT1 AKT3 CDK6 CDK4 PTEN RYBP CUL1 SPOP MLH1 BRAF GNAS KRAS CHD1 MSH2 MSH6 CDK6 CDK4 MLH1 GNAS CHD1 MSH2 MSH6 MDM2 MDM4 PALB2 ASXL1 FANCF FOXP1 RNF43 MDM2 MDM4 ZFHX3 FANCE PALB2 ERCC2 ERCC1 ERCC5 ERCC4 ERCC3 FOXA1 ASXL1 FANCA CDK12 BRCA1 BRCA2 FANCC FANCF FOXP1 RNF43 KMT2C FANCG RUNX1 ZFHX3 FANCE PIK3R1 ERCC2 ERCC1 ERCC5 ERCC4 ERCC3 MED12 KMT2D FOXA1 CCND1 NCOA2 FANCA CDK12 BRCA1 BRCA2 FBXW7 PIK3CA PIK3CB FANCC KDM6A KMT2C FANCG RUNX1 PIK3R1 MED12 KMT2D CCND1 NKX3-1 NCOA2 FBXW7 PIK3CA PIK3CB ARID1A KDM6A NFE2L2 NKX3-1 ARID1A ZBTB16 NFE2L2 RAD51B ZBTB16 FANCD2 HSD3B1 RAD51C CTNNB1 CDKN2A CDKN1B RAD51B FANCD2 HSD3B1 RAD51C CTNNB1 CDKN2A CDKN1B TMPRSS2 TMPRSS2 SMARCA1 SMARCA1 100 100

3 3 90 90

2 2 80 80

1 1

70 70 P67 0 P10 0 SNP allele fraction allele SNP SNP allele fraction allele SNP

60 60 44% TC 1 1

Coverage logratio Coverage 72% TC logratio Coverage 2nd mutation 502 LOH 502 AR AR APC CLU ATR RB1 ERG MET ATM APC CLU ATR MYC RB1 ERG MET ATM MYC IDH2 IDH1 TP53 AKT2 AKT1 AKT3 PTEN RYBP IDH2 CUL1 IDH1 SPOP BRAF TP53 KRAS AKT2 AKT1 AKT3 CDK6 CDK4 PTEN RYBP CUL1 SPOP MLH1 BRAF GNAS KRAS CHD1 MSH2 MSH6 CDK6 CDK4 MLH1 GNAS CHD1 MSH2 MSH6 MDM2 MDM4 PALB2 ASXL1 FANCF FOXP1 RNF43 MDM2 MDM4 ZFHX3 FANCE PALB2 ERCC2 ERCC1 ERCC5 ERCC4 ERCC3 FOXA1 ASXL1 FANCA CDK12 BRCA1 BRCA2 3 FANCC FANCF FOXP1 RNF43 KMT2C FANCG RUNX1 ZFHX3 FANCE PIK3R1 ERCC2 ERCC1 ERCC5 ERCC4 ERCC3 MED12 KMT2D FOXA1 CCND1 NCOA2 FANCA CDK12 BRCA1 BRCA2 FBXW7 PIK3CA PIK3CB FANCC KDM6A

KMT2C 3 FANCG RUNX1 PIK3R1 MED12 KMT2D CCND1 NKX3-1 NCOA2 FBXW7 PIK3CA PIK3CB ARID1A KDM6A NFE2L2 NKX3-1 ARID1A ZBTB16 NFE2L2 RAD51B ZBTB16 FANCD2 HSD3B1 RAD51C CTNNB1 CDKN2A CDKN1B RAD51B FANCD2 HSD3B1 RAD51C CTNNB1 CDKN2A CDKN1B TMPRSS2 AR TMPRSS2 SMARCA1 AR SMARCA1 APC CLU ATR RB1 ERG MET ATM MYC APC CLU ATR RB1 ERG MET ATM MYC IDH2 IDH1 TP53 AKT2 AKT1 AKT3 PTEN RYBP CUL1 SPOP BRAF IDH2 IDH1 KRAS TP53 CDK6 CDK4 AKT2 AKT1 AKT3 PTEN RYBP MLH1 CUL1 SPOP GNAS BRAF CHD1 KRAS MSH2 MSH6 CDK6 CDK4 MLH1 GNAS CHD1 MSH2 MSH6 MDM2 MDM4 PALB2 ASXL1 FANCF FOXP1 RNF43 ZFHX3 MDM2 MDM4 FANCE ERCC2 ERCC1 ERCC5 ERCC4 ERCC3 FOXA1 PALB2 FANCA CDK12 BRCA1 BRCA2 ASXL1 FANCC KMT2C FANCG FANCF FOXP1 RNF43 RUNX1 PIK3R1 MED12 KMT2D ZFHX3 FANCE CCND1 NCOA2 ERCC2 ERCC1 ERCC5 ERCC4 ERCC3 FBXW7 FOXA1 PIK3CA PIK3CB FANCA CDK12 BRCA1 BRCA2 KDM6A FANCC KMT2C FANCG RUNX1 NKX3-1 PIK3R1 MED12 KMT2D CCND1 NCOA2 ARID1A FBXW7 PIK3CA PIK3CB NFE2L2 KDM6A NKX3-1 ZBTB16 ARID1A NFE2L2 RAD51B FANCD2 HSD3B1 RAD51C CTNNB1 ZBTB16 CDKN2A CDKN1B RAD51B FANCD2 HSD3B1 RAD51C CTNNB1 CDKN2A CDKN1B TMPRSS2 TMPRSS2 SMARCA1 SMARCA1 100 100 3 3 90 90 2 2 80 80 1 1

70 70 P88 0 P28 0

fraction allele SNP SNP allele fraction allele SNP

601 601 Coverage logratio Coverage 45% TC 83% TC logratio Coverage

2 2 2nd mutation 50 CNLOH 50 AR AR APC CLU ATR RB1 ERG MET ATM MYC APC CLU ATR RB1 ERG MET ATM MYC IDH2 IDH1 TP53 AKT2 AKT1 AKT3 PTEN RYBP CUL1 SPOP BRAF IDH2 IDH1 KRAS TP53 CDK6 CDK4 AKT2 AKT1 AKT3 PTEN RYBP MLH1 CUL1 SPOP GNAS BRAF CHD1 KRAS MSH2 MSH6 CDK6 CDK4 MLH1 GNAS CHD1 MSH2 MSH6 MDM2 MDM4 PALB2 3 ASXL1 FANCF FOXP1 RNF43 ZFHX3 MDM2 MDM4 FANCE ERCC2 ERCC1 ERCC5 ERCC4 ERCC3

FOXA1 3 PALB2 FANCA CDK12 BRCA1 BRCA2 ASXL1 FANCC KMT2C FANCG FANCF FOXP1 RNF43 RUNX1 PIK3R1 MED12 KMT2D ZFHX3 FANCE CCND1 NCOA2 ERCC2 ERCC1 ERCC5 ERCC4 ERCC3 FBXW7 FOXA1 PIK3CA PIK3CB FANCA CDK12 BRCA1 BRCA2 KDM6A FANCC KMT2C FANCG RUNX1 NKX3-1 PIK3R1 MED12 KMT2D CCND1 NCOA2 ARID1A FBXW7 PIK3CA PIK3CB NFE2L2 KDM6A NKX3-1 ZBTB16 ARID1A NFE2L2 RAD51B FANCD2 HSD3B1 RAD51C CTNNB1 ZBTB16 CDKN2A CDKN1B RAD51B FANCD2 HSD3B1 RAD51C CTNNB1 CDKN2A CDKN1B AR TMPRSS2 AR TMPRSS2 SMARCA1 SMARCA1 APC CLU ATR RB1 APC CLU ATR ERG MET RB1 ATM MYC ERG MET ATM MYC IDH2 IDH1 TP53 IDH2 IDH1 AKT2 AKT1 TP53 AKT3 PTEN RYBP CUL1 SPOP BRAF KRAS AKT2 AKT1 AKT3 PTEN RYBP CUL1 SPOP BRAF CDK6 CDK4 KRAS MLH1 GNAS CHD1 CDK6 CDK4 MSH2 MSH6 MLH1 GNAS CHD1 MSH2 MSH6 MDM2 MDM4 PALB2 ASXL1 MDM2 MDM4 PALB2 FANCF FOXP1 RNF43 ASXL1 ZFHX3 FANCE ERCC2 ERCC1 ERCC5 ERCC4 ERCC3 FOXA1 FANCF FOXP1 FANCA RNF43 CDK12 BRCA1 BRCA2 FANCC ZFHX3 FANCE ERCC2 ERCC1 ERCC5 ERCC4 ERCC3 KMT2C FOXA1 FANCG FANCA RUNX1 CDK12 BRCA1 BRCA2 FANCC PIK3R1 MED12 KMT2D CCND1 NCOA2 KMT2C FBXW7 PIK3CA PIK3CB FANCG RUNX1 KDM6A PIK3R1 MED12 KMT2D CCND1 NCOA2 FBXW7 PIK3CA PIK3CB KDM6A NKX3-1 ARID1A NFE2L2 NKX3-1 ARID1A NFE2L2 ZBTB16 ZBTB16 RAD51B FANCD2 HSD3B1 RAD51C CTNNB1 CDKN2A CDKN1B RAD51B FANCD2 HSD3B1 RAD51C CTNNB1 CDKN2A CDKN1B TMPRSS2 TMPRSS2 SMARCA1 SMARCA1 100 100 3 4 90 90

2 3 80 80 2 1

70 701 P45 0 P9 SNP allele fraction allele SNP fraction allele SNP 0 60 60 47% TC 1 90% TC Coverage logratio Coverage Coverage logratio Coverage 1

2nd mutation 502 50 CNLOH 2 AR AR APC CLU ATR RB1 APC CLU ATR ERG MET RB1 ATM MYC ERG MET ATM MYC IDH2 IDH1 TP53 IDH2 IDH1 AKT2 AKT1 TP53 AKT3 PTEN RYBP CUL1 SPOP BRAF KRAS AKT2 AKT1 AKT3 PTEN RYBP CUL1 SPOP BRAF CDK4 CDK6 KRAS MLH1 GNAS CHD1 CDK4 CDK6 MSH2 MSH6 MLH1 GNAS CHD1 MSH2 MSH6 MDM2 MDM4 PALB2 ASXL1 MDM2 MDM4 PALB2 FANCF FOXP1 RNF43 ASXL1 ZFHX3 FANCE ERCC2 ERCC1 ERCC5 ERCC4 ERCC3 FOXA1 FANCF FOXP1 FANCA RNF43 CDK12 BRCA1 BRCA2 FANCC ZFHX3 FANCE ERCC2 ERCC1 ERCC5 ERCC4 ERCC3 KMT2C FOXA1 FANCG FANCA RUNX1 CDK12 BRCA1 BRCA2

3 FANCC PIK3R1 MED12 KMT2D CCND1 NCOA2 KMT2C FBXW7 PIK3CA PIK3CB FANCG RUNX1 KDM6A PIK3R1 MED12 KMT2D CCND1 NCOA2 FBXW7 PIK3CA PIK3CB KDM6A NKX3-1 3 ARID1A NFE2L2 NKX3-1 ARID1A NFE2L2 ZBTB16 ZBTB16 RAD51B FANCD2 HSD3B1 RAD51C CTNNB1 CDKN2A CDKN1B RAD51B FANCD2 HSD3B1 RAD51C CTNNB1 CDKN2A CDKN1B TMPRSS2 TMPRSS2 AR SMARCA1 SMARCA1 AR APC CLU ATR RB1 ERG MET ATM MYC APC CLU ATR RB1 ERG MET ATM MYC IDH2 IDH1 TP53 AKT2 AKT1 AKT3 PTEN RYBP CUL1 SPOP BRAF KRAS IDH1 IDH2 TP53 CDK4 CDK6 MLH1 AKT3 AKT1 AKT2 PTEN RYBP GNAS CHD1 CUL1 SPOP BRAF MSH2 MSH6 KRAS CDK4 CDK6 MLH1 GNAS CHD1 MSH2 MSH6 MDM2 MDM4 PALB2 ASXL1 FANCF FOXP1 RNF43 ZFHX3 FANCE ERCC2 ERCC1 ERCC5 ERCC4 ERCC3 FOXA1 MDM4 MDM2 FANCA CDK12 BRCA1 BRCA2 FANCC PALB2 ASXL1 KMT2C FANCG RUNX1 FANCF PIK3R1 FOXP1 RNF43 KMT2D MED12 CCND1 NCOA2 FBXW7 PIK3CA PIK3CB ZFHX3 FANCE ERCC3 ERCC5 ERCC4 ERCC2 ERCC1 FOXA1 KDM6A FANCA CDK12 BRCA1 BRCA2 FANCC KMT2C FANCG NKX3-1 RUNX1 PIK3R1 ARID1A MED12 KMT2D NFE2L2 CCND1 NCOA2 FBXW7 PIK3CA PIK3CB KDM6A ZBTB16 NKX3-1 ARID1A NFE2L2 RAD51B FANCD2 HSD3B1 RAD51C CTNNB1 ZBTB16 CDKN2A CDKN1B RAD51B FANCD2 HSD3B1 RAD51C CTNNB1 CDKN2A CDKN1B TMPRSS2 SMARCA1 TMPRSS2 SMARCA1 100 100 90 90

80 80

70 70 SNP allele fraction allele SNP fraction allele SNP 60 60

50 50

Supplementary Figure 3. CharacteristicAR copy number profiles associated with CDK12 AR APC CLU ATR RB1 ERG MET ATM MYC APC CLU ATR RB1 ERG MET ATM MYC IDH2 IDH1 TP53 AKT2 AKT1 AKT3 PTEN RYBP CUL1 SPOP BRAF KRAS IDH2 IDH1 TP53 CDK6 CDK4 MLH1 AKT2 AKT1 AKT3 PTEN RYBP GNAS CHD1 CUL1 SPOP BRAF MSH2 MSH6 KRAS CDK6 CDK4 MLH1 GNAS CHD1 MSH2 MSH6 MDM2 MDM4 PALB2 ASXL1 FANCF FOXP1 RNF43 ZFHX3 FANCE ERCC2 ERCC1 ERCC5 ERCC4 ERCC3 FOXA1 MDM2 MDM4 FANCA CDK12 BRCA1 BRCA2 FANCC PALB2 ASXL1 KMT2C FANCG RUNX1 FANCF PIK3R1 FOXP1 RNF43 MED12 KMT2D CCND1 NCOA2 FBXW7 ZFHX3 PIK3CA PIK3CB FANCE ERCC2 ERCC1 ERCC5 ERCC4 ERCC3 KDM6A FOXA1 FANCA CDK12 BRCA1 BRCA2 FANCC KMT2C FANCG NKX3-1 RUNX1 PIK3R1 ARID1A MED12 KMT2D NFE2L2 CCND1 NCOA2 FBXW7 PIK3CA PIK3CB KDM6A ZBTB16 NKX3-1 ARID1A NFE2L2 RAD51B FANCD2 HSD3B1 RAD51C CTNNB1 ZBTB16 CDKN2A CDKN1B RAD51B FANCD2 HSD3B1 RAD51C CTNNB1 CDKN2A CDKN1B TMPRSS2 SMARCA1 TMPRSS2 inactivation. Representative copy number violin plots illustrating panel-wide copy-number gainsSMARCA1 from initial targeted cfDNA sequencing.

3 p = 0.009 p = 0.07 a p = 0.0002 b p = 0.0007 c p = 0.14 p = 0.05 Minimum 5% ctDNA threshold Control cohort: r = 0.06 , p = 6.33e-01 30 30 30 Study cohort: r = -0.22 , p = 9.23er-02 Overall: r = -0.09 , p = 3.30e-01

20 20 20

10 10 10 AR gene copies (#) copies gene AR (#) copies gene AR AR gene copies (#) copies gene AR

0 0 0 0.0 0.2 0.4 0.6 0.8 1.0 ATM ATM BRCA2 CDK12 Control BRCA2 CDK12 Control ctDNA fraction

Minimum 5% ctDNA threshold Minimum 20% ctDNA threshold

Supplementary Figure 4. Absolute AR copy number in samples with DNA damage repair defects. Comparison of absolute AR copy number restricted to the subset of study and control samples with ≥5% (a) and ≥20% (b) ctDNA fraction. c. Scatterplot showing absolute AR copy number versus ctDNA fraction suggesting that the relationship shown in A and B is not related to the ctDNA fraction-normalization approach. Linear least-squares regression was used to generate p- and r-values.

4 -3 p=p=0.024 5.6 x 10 0.8 a Fraction of patients Fraction of patients b with TP53 deletion with TP53 mutation p=0.003 0.5 0.25 0 0 0.2 0.4 0.6 0.6 p = 5.0 x 10-3 p=0.003 29 DDR-WT 60 0.4 3 ATM 1 BRCA2 14 11 p = 1.2 x 10-2 2 CDK12 2 0.2 copy-loss with Fraction 0.0 5 5 10 21 2 29 29 RB1 RB1 RB1 RB1 PTEN PTEN PTEN PTEN ATMATM BRCA2BRCA2 CDK12CDK12 DDR-WTDDR-WT

p = 0.029 c d e p = 0.0016 p = 1.0 x 10-4 p = 0.019 p = 2.67 x 10-3 0.6 1.0 30 0.5 0.8 -3 0.4 p = 1.3 x 10 20 0.6 0.3 0.4 0.2 10

0.2 Fraction with copy gain copy with Fraction 0.1 Fraction with AR copy gain copy AR with Fraction 1 copies gene AR of Number 0.0 1 1 3 2 7 3 4 5 15 1 0.0 7 14 16 60 0

ATM ATM MDM2 MDM2 MDM2 MDM2 BRCA2 CDK12 Control CCND1CDK4/6 CCND1CDK4/6 CCND1CDK4/6 CCND1CDK4/6 BRCA2 CDK12 DDR-WT ATM BRCA2 CDK12 DDR-WT Minimum 5% ctDNA threshold

Supplementary Figure 5. Genomic features of mPCa patients with evidence for biallelic disruption to BRCA2, ATM, or CDK12. Recapitulation of genomic alteration frequency comparisons between DDR-gene groups and the comparator cohort, restricted to the study cohort subset with confirmed biallelic functional loss from Figure 2 (n=57 of 101 patients with at least one ctDNA-positive cfDNA collection). Barplot descriptions (a through d) are identical to those provided in Figure 3 (a = Figure 3e; b = Figure 3c; c = Figure 3b; d = Figure 3d). e. Comparison of absolute AR copy number restricted to samples with ≥5% ctDNA fraction.

5 a ATM BRCA2 CDK12 SVSVSV * * * * Intragenic deletion CNVCNVCNV Truncating rearrangement SNVSNVSNV Events Events Events Tandem duplication P6 P8 P0 P4 P5 P7 P9 P6 P8 P6 P8 P0 P4 P0 P5 P4 P7 P5 P7 P9 P9 P39 P42 P48 P49 P52 P61 P62 P70 P74 P37 P81 P83 P84 P90 P92 P97 P73 P33 P22 P15 P20 P23 P32 P35 P40 P43 P46 P50 P31 P77 P98 P99 P16 P21 P86 P85 P88 P10 P47 P18 P82 P13 P13 P17 P13 P95 P17 P89 P95 P17 P89 P95 P89 P39 P42 P39 P48 P42 P49 P48 P52 P49 P61 P52 P62 P61 P70 P62 P74 P70 P37 P74 P81 P37 P83 P81 P84 P83 P90 P84 P92 P90 P97 P92 P97 P73 P73 P33 P22 P33 P15 P22 P20 P15 P23 P20 P32 P23 P35 P32 P40 P35 P43 P40 P46 P43 P50 P46 P31 P50 P77 P31 P77 P98 P99 P98 P99 P16 P21 P16 P86 P21 P85 P86 P88 P85 P10 P88 P47 P10 P18 P47 P18 P82 P82 Presence of defect P100 P102 P104 P107 P101 P101 P101 P100 P102 P100 P102 P104 P104 P107 P107 PatientPatientPatient No defect identified *Harbors two independent events

b RB1: chr13: 48303726 - chr13: 48599436 (hg38)

5’ 3’

Large rearrangements extending beyond gene boundaries

Identified in P40

Supplementary Figure 6. RB1 structural rearrangements in patients with deleterious DDR-gene defects. a. Heatmap summarizing RB1 gene copy number variants (CNV), deleterious single nucleotide variants (SNVs), and structural variants (SVs) with intragenic breakpoints in 57 DDR patients with targeted sequencing data across both introns and exons. Note that the CNV and SNV rows reflect the presence/absence of heterozygous gene-body loss and deleterious mutations as detected from initial exon-based targeted sequencing. b. Pileup of intragenic RB1 structural variants (relative to introns/exons) from 14 patients with BRCA2- defective tumors. Colour annotation of SVs is identical to a.

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