next - generation whole - genome and exome - sequenced cancer samples were previously sequenced by members of the international cancer genome consortium , the cancer genome atlas and other centres .
high - confidence somatic substitutions were obtained from these consortia or other peer - reviewed publications not related to these consortia , filtered further for potential false positive calls using dbsnp , 1000 genomes , the nhlbi go exome sequencing project , the 69 complete genomics panel as well as a bespoke panel of bam files of unmatched normal tissues containing more than 120 normal genomes and 500 exomes .
these data were then parsed through an algorithm previously developed to extract mutational signatures in human cancers called non - negative matrix factorisation ( nnmf ) .
six main substitution classes ( c > a : g > t , c > g : g > c , c > t : g > a , t > a : a > t , t > c : a > g and t > g : a > c ) were subdivided according to the 5 prime and 3 prime flanking sequence context .
since there are six classes of base substitution and 16 possible sequence contexts for each mutated base ( a , c , g or t at the 5 base and a , c , g or t at the 3 base ) , there are 96 possible mutated trinucleotides for each cancer .
herewith , the convention for describing a mutated trinucleotide will be xpcpx , where x can be any base and the mutated base is underlined .
the commonest signatures were signature 1a and signature 1b , both characterized by c > t mutations at a xpcpg trinucleotide ( supplementary figure 1a ) , and signature 2 and signature 13 , characterized by dominant c > t transitions at a tpcpx sequence context in signature 2 and c > g transversions at a tpcpx sequence context in signature 13 ( supplementary figure 1a - b ) .
signatures 1a/1b are likely to be caused by deamination at methylated cpgs whereas signatures 2/13 are thought to be due to the apobec family of cytidine deaminases . therefore , for the purposes of this analysis , signatures 2 and 13 are considered together .
nnmf is able to estimate the number of mutations associated with extracted mutation signatures for individual cancers in a given set of samples ( summarized in supplementary table 1b , figure 1c-1o ) . in this analysis ,
a total of 2,719 samples were previously characterized by nnmf and also had bam files available for inspection ( supplementary table 1a - b ) .
bam files were downloaded from cghub ( https://cghub.ucsc.edu/ ) between 9 may 2013 and 26 june 2013 . for the ease of tracking samples through this analysis ,
we have kept the naming convention attached to the cancer sample for tables and figures , even if the germline deletion polymorphism was sourced from a matched normal because the signatures of somatic mutagenesis will have been identified in the tumor samples in the first place .
this is also for the purpose of continuity between publications . for samples originating from the sanger institute
d  denoting tumor samples and suffix  b  for the matched normal sample . in order to detect this deletion polymorphism from next - generation sequencing data , multiple loci within and flanking the coordinates of the deletion were sampled ( supplementary figure 2a , supplementary table 2a ) from bam files ( overall workflow and directions to processing data in supplementary figure 2b ) .
raw short read data had been aligned back to the reference genome ( ncbi build 37 ) with duplicates and unmapped reads removed .
externally sourced bam files were sourced from the tcga data hub https://browser.cghub.ucsc.edu/. matched normal bam files were sought for calling this deletion allele .
however , a tumor bam file was used if a normal bam was not available ( supplementary table 2b ) .
samples that did not have bam files available for examination were excluded from analysis . in total , the apobec3a/3b polymorphism detection was sourced from 561 tumors ( 99 blca , 117 brca , 1 cesc , 19 hnsc , 2 kirp , 303 luad , 12 stad , 2 thca and 6 ucec ) and 2158 normals . for samples with whole genome data , the expected sequencing depth in the absence of the deletion polymorphism , i.e. wild - type copy number ( cn ) of 2 , d2 ,
the expected depth in the presence of a heterozygous deletion allele , d1 , is then given by d1 = d2 / 2 and the expected depth in the presence of a homozygous deletion allele , d0 , was set to the expected number of misreads , estimated as d2/20 .
a maximum likelihood test was performed to identify the most likely cn from the set , with corresponding expected depths represented as poisson distributions { pois(d0 ) , pois(d1 ) , pois(d2 ) } , given the observed sequencing depths within the region of the deletion polymorphism ( supplementary dataset 1 ) . for exome - sequenced samples , an expectation - maximisation algorithm was utilized , with the copy number ( cn ) of each sample and the ratio of sequencing depth within and outside the deletion polymorphism used as latent variables .
the cn of each sample was initialized as 2 and the depth ratio of loci within the deletion polymorphism region to those outside , r , was modelled non - parametrically by bootstrap resampling ( n=1000 ) .
for samples with cn of 2 , 1 and 0 , respectively , the expected depths within the deletion polymorphism region are then given by 
 din,2=rdoutdin,1=rdout2din,0=rdout20 at each maximisation step , the copy number of each sample was assigned as that whose distribution showed most overlap with din for that sample , after bootstrap resampling .
at each expectation step , r was recalculated using bootstrap resampling of loci within just those samples classified as cn=2 in the previous maximisation step .
the em algorithm was continued until no samples were reclassified from one iteration to the next , or for a maximum of 100 iterations ( supplementary figure 2b , supplementary dataset 2 ) .
the results of the calling of the polymorphism status in all the samples is provided in supplementary table 2b .
the reproducibility of the calling method was sought by examining the concordance between calling on the tumor and normal bam files from the same patient ( supplementary note , supplementary table 2c , supplementary figure 2c ) as well as concordance between genome- and exome - sequenced samples in the same patient .
the dataset comprised genome - sequenced ( 123 ) as well as exome - sequenced ( 800 ) cancers . in order to perform the analyses
, the rate of mutation was calculated for each cancer ( rate of signature 2/13 per mb ) , correcting for whether the samples had been genome- or exome - sequenced . because the rates of signatures 2/13 were not normally distributed ( supplementary 3a - d )
, a one - sided wilcoxon rank - sum test was performed to see whether carrying one copy of the deletion allele had an overall effect on the mutation rate of the signatures .
we sought to include more cancer samples in order to increase the power of the analyses .
there were no further available breast cancer samples with bam files ready for download , hence we sought inclusion of other cancer types that had previously been analyzed .
however , it was noted that the distribution of rates of signatures 2/13 varied considerably between cancer - types ( supplementary figure 3b - d ) and clear outliers were present in all the cancer types skewing the distribution of mutation rates ( supplementary figure 3a , d - e ) .
some cancers were observed to have a strikingly high proportion of total mutations associated with signatures 2/13 and/or have higher rates of mutagenesis associated with this signature ( supplementary figure 1c-1o , 5b ) . using the rate of signatures 2/13 mutagenesis ,
outliers were identified as patients with cancers that had a mutation rate exceeding 1.5 times the length of the interquartile range from the 75 percentile for each type of cancer .
hypermutators  although we do not suggest that there is an on - going biological process attached to this name .
given the considerable variation of the mutation rates for different cancer tissue - types ( supplementary figure 1a,1b ) , each cancer type was analyzed separately .
a summary of the hypermutators versus non - hypermutators is provided in the supplementary table 3a . in theory
, neighboring mutations could arise on either of two strands of a double - helix ( supplementary figure 4a ) particularly if they had arisen as independent events during different cycles of cell division .
if more mutations are observed to occur on the same strand than expected by chance ( supplementary figure 4b ) , this would imply one of two scenarios : either those neighboring mutations arose over different rounds of cell division with preferential targeting of one strand over another or they arose during a single round of cell division and potentially occurred in the same instance .
we therefore sought to formally document that neighboring mutations are occurring on the same strand more often than expected or  strand - coordinated mutagenesis  . in order to demonstrate genome - wide strand coordination ,
analysis was carried out on all whole - genome sequence data for which bam files were available ( supplementary table 4a ) .
given a set of mutations , each occurring at a base of type ( a , c , g , t ) on the + strand , we identify all pairs of mutations and classify them as  same if both mutations are of the same originating base and 
diff if not ( i.e. first and second mutations of each pair respectively : a > x and a > x ; g > x and g > x ; c > x and c > x ; and t > x and t > x , with no prior selection for mutations at a tpc context and where x can be any base ) . the distance between successive pairs or intermutation distance
is also calculated ( supplementary figure 4c ) . the proportion of pairs of mutations that are expected to occur on the same base assuming randomly ordered mutations is given by pa2+pc2+pg2+pt2 , where px is the fraction of mutations that occur at nucleotide x. to depict the deviation of the observed pairs of mutations found on the same strand from that of the expected pairs of mutations on the same strand , a standard forest plot was constructed ( data of expected and observed same strand mutations for all 124 samples are provided in supplementary table 4a , columns b - e ) . for the reason of space , only a subset of samples were presented in figure 2a .
because same - strand mutations were ascertained in an unbiased way from any mutation type ( not restricted to just cytosine mutations at tpcs ) , to see whether strand - coordinated mutations were a particular feature of signatures 2/13 , we sought a relationship between the degree of  strand - coordination  , given by the or of strand - coordination , and the fractional burden of signatures 2/13 in each cancer ( supplementary figure 4d ) .
we sought additional characteristics of the mutations in the whole - genome sequenced breast cancers that support the suggestion that mutations associated with signatures 2/13 have arisen due to the apobec family of enzymes ( supplementary note , supplementary figure 4d , supplementary table 4 ) .
rna - seq derived expression data was obtained from the https://browser.cghub.ucsc.edu/ for relevant samples . in total , there were 1691 patients for whom comparable data were obtainable .
expression levels for each apobec family member were standardized relative to the levels of tbp ( tata - binding protein ) and the relationship between the apobec3b expression levels and germline deletion allele status in these cancers ( supplementary figure 5a - b , supplementary table 5a ) .
the germline apobec3a/3b deletion polymorphism highlighted in this analysis was reported to display a strikingly differentiated worldwide distribution of allele frequencies5 .
the fst value ( measuring population differentiation ) was re - examined using the reported deletion allele frequencies and additional snp genotype data from the ceph - hgdp panel published after the cnp study .
this value will depend on the way the populations are grouped , and needs to be compared with other variants of similar frequency to measure how unusual it is .
we used two published grouping schemes , into five continental geographical / genetic groups ( 1 .
oceanians ) or into 32 population groups , and matched snp frequencies measured as minor allele frequency to  0.1% .
for the five continental groups , fst was 0.330 ( 97.4th percentile compared with 2,716 frequency - matched snps ) , and for the 32 population groups it was 0.285 ( 96.6th percentile compared to 2,059 frequency - matched snps ) .
the level of population differentiation was thus higher than expected by chance , which can result from positive selection , and we therefore examined other statistics sensitive to positive selection .
cross - population extended haplotype homozygosity ( xp - ehh ) and integrated haplotype score ( ihs ) values were obtained from the hgdp selection browser ( http://hgdp.uchicago.edu/ ) .
these haplotype - based tests for positive selection utilise information 500 kb upstream and downstream of the deletion , and thus the two sides can be examined separately .
neither side showed any significantly high xp - ehh ( > 2.5 ) or ihs value ( ihs>2.0 ) in any continental group or individual population .
finally , we looked at allele frequency spectrum - based tests ( tajima s d , fay & wu s h and nielsen et al.s composite likelihood ratio test ) using the 1000 genomes phase 1 re - sequenced data in the east asian populations ( chb , chs and jpt ) ( 1000 g phase 1 ) in the regions surrounding the deletion , as described ( 1000 g pilot ) .
there was no evidence for positive selection in these populations , although in this case , the power of these tests is limited because the frequency of the deletion in these populations is not high enough .
however , there remains a lack of other evidence for positive selection and so we can not convincingly conclude that this deletion has been positively selected in human populations .
next - generation whole - genome and exome - sequenced cancer samples were previously sequenced by members of the international cancer genome consortium , the cancer genome atlas and other centres .
high - confidence somatic substitutions were obtained from these consortia or other peer - reviewed publications not related to these consortia , filtered further for potential false positive calls using dbsnp , 1000 genomes , the nhlbi go exome sequencing project , the 69 complete genomics panel as well as a bespoke panel of bam files of unmatched normal tissues containing more than 120 normal genomes and 500 exomes .
these data were then parsed through an algorithm previously developed to extract mutational signatures in human cancers called non - negative matrix factorisation ( nnmf ) .
six main substitution classes ( c > a : g > t , c > g : g > c , c > t : g > a , t > a : a > t , t > c : a > g and t > g : a > c ) were subdivided according to the 5 prime and 3 prime flanking sequence context .
since there are six classes of base substitution and 16 possible sequence contexts for each mutated base ( a , c , g or t at the 5 base and a , c , g or t at the 3 base ) , there are 96 possible mutated trinucleotides for each cancer .
herewith , the convention for describing a mutated trinucleotide will be xpcpx , where x can be any base and the mutated base is underlined .
the commonest signatures were signature 1a and signature 1b , both characterized by c > t mutations at a xpcpg trinucleotide ( supplementary figure 1a ) , and signature 2 and signature 13 , characterized by dominant c > t transitions at a tpcpx sequence context in signature 2 and c > g transversions at a tpcpx sequence context in signature 13 ( supplementary figure 1a - b ) .
signatures 1a/1b are likely to be caused by deamination at methylated cpgs whereas signatures 2/13 are thought to be due to the apobec family of cytidine deaminases . therefore , for the purposes of this analysis , signatures 2 and 13 are considered together .
nnmf is able to estimate the number of mutations associated with extracted mutation signatures for individual cancers in a given set of samples ( summarized in supplementary table 1b , figure 1c-1o ) . in this analysis ,
a total of 2,719 samples were previously characterized by nnmf and also had bam files available for inspection ( supplementary table 1a - b ) .
bam files were downloaded from cghub ( https://cghub.ucsc.edu/ ) between 9 may 2013 and 26 june 2013 . for the ease of tracking samples through this analysis ,
we have kept the naming convention attached to the cancer sample for tables and figures , even if the germline deletion polymorphism was sourced from a matched normal because the signatures of somatic mutagenesis will have been identified in the tumor samples in the first place .
this is also for the purpose of continuity between publications . for samples originating from the sanger institute
d  denoting tumor samples and suffix  b  for the matched normal sample .
in order to detect this deletion polymorphism from next - generation sequencing data , multiple loci within and flanking the coordinates of the deletion were sampled ( supplementary figure 2a , supplementary table 2a ) from bam files ( overall workflow and directions to processing data in supplementary figure 2b ) .
raw short read data had been aligned back to the reference genome ( ncbi build 37 ) with duplicates and unmapped reads removed .
externally sourced bam files were sourced from the tcga data hub https://browser.cghub.ucsc.edu/. matched normal bam files were sought for calling this deletion allele .
however , a tumor bam file was used if a normal bam was not available ( supplementary table 2b ) .
samples that did not have bam files available for examination were excluded from analysis . in total , the apobec3a/3b polymorphism detection was sourced from 561 tumors ( 99 blca , 117 brca , 1 cesc , 19 hnsc , 2 kirp , 303 luad , 12 stad , 2 thca and 6 ucec ) and 2158 normals . for samples with whole genome data , the expected sequencing depth in the absence of the deletion polymorphism , i.e. wild - type copy number ( cn ) of 2 , d2 ,
the expected depth in the presence of a heterozygous deletion allele , d1 , is then given by d1 = d2 / 2 and the expected depth in the presence of a homozygous deletion allele , d0 , was set to the expected number of misreads , estimated as d2/20 .
a maximum likelihood test was performed to identify the most likely cn from the set , with corresponding expected depths represented as poisson distributions { pois(d0 ) , pois(d1 ) , pois(d2 ) } , given the observed sequencing depths within the region of the deletion polymorphism ( supplementary dataset 1 ) . for exome - sequenced samples ,
an expectation - maximisation algorithm was utilized , with the copy number ( cn ) of each sample and the ratio of sequencing depth within and outside the deletion polymorphism used as latent variables .
the cn of each sample was initialized as 2 and the depth ratio of loci within the deletion polymorphism region to those outside , r , was modelled non - parametrically by bootstrap resampling ( n=1000 ) . for samples with cn of 2 , 1 and 0 , respectively , the expected depths within the deletion polymorphism region are then given by 
 din,2=rdoutdin,1=rdout2din,0=rdout20 at each maximisation step , the copy number of each sample was assigned as that whose distribution showed most overlap with din for that sample , after bootstrap resampling .
at each expectation step , r was recalculated using bootstrap resampling of loci within just those samples classified as cn=2 in the previous maximisation step .
the em algorithm was continued until no samples were reclassified from one iteration to the next , or for a maximum of 100 iterations ( supplementary figure 2b , supplementary dataset 2 ) .
the results of the calling of the polymorphism status in all the samples is provided in supplementary table 2b .
the reproducibility of the calling method was sought by examining the concordance between calling on the tumor and normal bam files from the same patient ( supplementary note , supplementary table 2c , supplementary figure 2c ) as well as concordance between genome- and exome - sequenced samples in the same patient .
the dataset comprised genome - sequenced ( 123 ) as well as exome - sequenced ( 800 ) cancers . in order to perform the analyses ,
the rate of mutation was calculated for each cancer ( rate of signature 2/13 per mb ) , correcting for whether the samples had been genome- or exome - sequenced . because the rates of signatures 2/13 were not normally distributed ( supplementary 3a - d )
, a one - sided wilcoxon rank - sum test was performed to see whether carrying one copy of the deletion allele had an overall effect on the mutation rate of the signatures .
we sought to include more cancer samples in order to increase the power of the analyses .
there were no further available breast cancer samples with bam files ready for download , hence we sought inclusion of other cancer types that had previously been analyzed .
however , it was noted that the distribution of rates of signatures 2/13 varied considerably between cancer - types ( supplementary figure 3b - d ) and clear outliers were present in all the cancer types skewing the distribution of mutation rates ( supplementary figure 3a , d - e ) . some cancers were observed to have a strikingly high proportion of total mutations associated with signatures 2/13 and/or have higher rates of mutagenesis associated with this signature ( supplementary figure 1c-1o , 5b ) . using the rate of signatures 2/13 mutagenesis ,
outliers were identified as patients with cancers that had a mutation rate exceeding 1.5 times the length of the interquartile range from the 75 percentile for each type of cancer .
hypermutators  although we do not suggest that there is an on - going biological process attached to this name .
given the considerable variation of the mutation rates for different cancer tissue - types ( supplementary figure 1a,1b ) , each cancer type was analyzed separately .
a summary of the hypermutators versus non - hypermutators is provided in the supplementary table 3a .
in theory , neighboring mutations could arise on either of two strands of a double - helix ( supplementary figure 4a ) particularly if they had arisen as independent events during different cycles of cell division .
if more mutations are observed to occur on the same strand than expected by chance ( supplementary figure 4b ) , this would imply one of two scenarios : either those neighboring mutations arose over different rounds of cell division with preferential targeting of one strand over another or they arose during a single round of cell division and potentially occurred in the same instance .
we therefore sought to formally document that neighboring mutations are occurring on the same strand more often than expected or  strand - coordinated mutagenesis  . in order to demonstrate genome - wide strand coordination ,
analysis was carried out on all whole - genome sequence data for which bam files were available ( supplementary table 4a ) .
given a set of mutations , each occurring at a base of type ( a , c , g , t ) on the + strand , we identify all pairs of mutations and classify them as  same if both mutations are of the same originating base and 
diff if not ( i.e. first and second mutations of each pair respectively : a > x and a > x ; g > x and g > x ; c > x and c > x ; and t > x and t > x , with no prior selection for mutations at a tpc context and where x can be any base ) . the distance between successive pairs or intermutation distance
the proportion of pairs of mutations that are expected to occur on the same base assuming randomly ordered mutations is given by pa2+pc2+pg2+pt2 , where px is the fraction of mutations that occur at nucleotide x. to depict the deviation of the observed pairs of mutations found on the same strand from that of the expected pairs of mutations on the same strand , a standard forest plot was constructed ( data of expected and observed same strand mutations for all 124 samples are provided in supplementary table 4a , columns b - e ) . for the reason of space , only a subset of samples were presented in figure 2a . because same - strand mutations were ascertained in an unbiased way from any mutation type (
not restricted to just cytosine mutations at tpcs ) , to see whether strand - coordinated mutations were a particular feature of signatures 2/13 , we sought a relationship between the degree of  strand - coordination  , given by the or of strand - coordination , and the fractional burden of signatures 2/13 in each cancer ( supplementary figure 4d ) .
we sought additional characteristics of the mutations in the whole - genome sequenced breast cancers that support the suggestion that mutations associated with signatures 2/13 have arisen due to the apobec family of enzymes ( supplementary note , supplementary figure 4d , supplementary table 4 ) .
rna - seq derived expression data was obtained from the https://browser.cghub.ucsc.edu/ for relevant samples . in total , there were 1691 patients for whom comparable data were obtainable .
expression levels for each apobec family member were standardized relative to the levels of tbp ( tata - binding protein ) and the relationship between the apobec3b expression levels and germline deletion allele status in these cancers ( supplementary figure 5a - b , supplementary table 5a ) .
the germline apobec3a/3b deletion polymorphism highlighted in this analysis was reported to display a strikingly differentiated worldwide distribution of allele frequencies5 .
the fst value ( measuring population differentiation ) was re - examined using the reported deletion allele frequencies and additional snp genotype data from the ceph - hgdp panel published after the cnp study .
this value will depend on the way the populations are grouped , and needs to be compared with other variants of similar frequency to measure how unusual it is .
we used two published grouping schemes , into five continental geographical / genetic groups ( 1 .
oceanians ) or into 32 population groups , and matched snp frequencies measured as minor allele frequency to  0.1% .
for the five continental groups , fst was 0.330 ( 97.4th percentile compared with 2,716 frequency - matched snps ) , and for the 32 population groups it was 0.285 ( 96.6th percentile compared to 2,059 frequency - matched snps ) .
the level of population differentiation was thus higher than expected by chance , which can result from positive selection , and we therefore examined other statistics sensitive to positive selection .
cross - population extended haplotype homozygosity ( xp - ehh ) and integrated haplotype score ( ihs ) values were obtained from the hgdp selection browser ( http://hgdp.uchicago.edu/ ) . these haplotype - based tests for positive selection utilise information 500 kb upstream and downstream of the deletion , and
neither side showed any significantly high xp - ehh ( > 2.5 ) or ihs value ( ihs>2.0 ) in any continental group or individual population .
finally , we looked at allele frequency spectrum - based tests ( tajima s d , fay & wu s h and nielsen et al.s composite likelihood ratio test ) using the 1000 genomes phase 1 re - sequenced data in the east asian populations ( chb , chs and jpt ) ( 1000 g phase 1 ) in the regions surrounding the deletion , as described ( 1000 g pilot ) .
there was no evidence for positive selection in these populations , although in this case , the power of these tests is limited because the frequency of the deletion in these populations is not high enough .
however , there remains a lack of other evidence for positive selection and so we can not convincingly conclude that this deletion has been positively selected in human populations .
 introductionthe somatic mutations in a cancer genome are the aggregate outcome of one or more mutational processes operative through the life of the cancer patient1 - 3 . 
 each mutational process leaves a characteristic mutational signature determined by the mechanisms of dna damage and repair that constitute it . 
 a role was recently proposed for the apobec family of cytidine deaminases in generating particular genome - wide mutational signatures1,4 and a signature of localized hypermutation called kataegis1,4 . 
 a germline copy number polymorphism involving apobec3a and apobec3b , which effectively deletes apobec3b5 , has been associated with a modest increased risk of breast cancer6 - 8 . here 
 , we show that breast cancers in carriers of the deletion show more mutations of the putative apobec - dependent genome - wide signatures than cancers in non - carriers . 
 the results suggest that the apobec3a/3b germline deletion allele confers cancer susceptibility through increased activity of apobec - dependent mutational processes , although the mechanism by which this occurs remains unknown . 
Online methods
1. Background information
2. Detection of germline 
3. The relationship between the 
4: Strand-coordinated mutagenesis
5: Relationship between expression of APOBEC family members and rates of mutation of Signatures 2/13
6. Selection for the APOBEC3A/3B deletion
Supplementary Material