number of ( a ) insertion / deletions , ( b ) high - confidence substitutions and ( c ) chromosomal rearrangements are plotted across all the samples from the 10 patients that were whole - genome sequenced .
the primary means of validation was a deep sequencing validation experiment that included selected substitutions and indels from each sample , as described in extended data table 2 and supplementary information , section 2b .
in addition , indels and rearrangements identified in wgs represent datasets orthogonal to the substitution data from which the subclones were identified .
the subsets of samples in which validated substitutions , indels and rearrangements are found correlate strongly with the subclonal clusters identified from the clustering of substitutions from wgs , providing support for the existence of these subclones .
for each patient , hierarchical clustering of the variant allele fraction ( vaf ) was performed separately for substitutions ( a ) and indels ( b ) .
vafs are represented as a heatmap with deeper shades of red indicating a higher proportion of reads reporting the mutant allele . above each heatmap
, mutations are colour - coded according to the subclone they were assigned to by dirichlet process clustering of wgs data in the case of substitutions or by vaf for indels .
for a22 , additional samples not subject to wgs were included in the validation experiment .
for these patients the phylogenetic tree from figure 2 was modified to incorporate these additional samples ( c ) .
number of substitutions assigned to each subclone ( d ) and numbers of indels ( e ) and rearrangements ( f ) present in different subsets of samples are plotted as bar charts .
vafs from wgs and validation data , plotted as scatter plots ( g ) , are very highly correlated .
validation strategy as described in extended data figure 2 . for a31 and a32 , hierarchical clustering of the vaf was performed separately for substitutions ( a ) and ( j ) and indels ( b ) and ( k ) .
additional samples for a31 and a32 are incorporated into the phylogenetic trees ( c ) and ( l ) .
subclones for a31 cd and a32 ce are annotated in the corresponding 2d - dp plots ( d ) and ( m ) .
numbers of substitutions in wgs data assigned to each subclone are plotted in ( e ) and ( n ) .
vafs from wgs and validation data , plotted as scatter plots ( f ) and ( o ) , are very highly correlated .
number of indels ( g ) and ( p ) and rearrangements ( h ) and ( q ) present in different subsets of samples are plotted as bar charts .
subclone colour keys for a31 and a32 ( i and r ) respectively . validation strategy as described in extended data figure 2 .
for a24 and a34 , hierarchical clustering of the vaf was performed separately for substitutions ( a ) and ( i ) and indels ( b ) and ( j ) .
indels that could not be assigned to any cluster ( if any ) are annotated with black .
additional samples for a24 and a34 are incorporated into the phylogenetic tree ( c ) and ( k ) .
the additional cluster in a24 , supported by rearrangements only , is indicated by a light green branch in the tree .
numbers of substitutions in wgs data assigned to each subclone are plotted in ( d ) and ( l ) .
vafs from wgs and validation data , plotted as scatter plots ( e ) and ( m ) , are very highly correlated .
number of indels ( f ) and ( n ) and rearrangements ( g ) and ( o ) present in different subsets of samples are plotted as bar charts .
subclone colour keys for a24 and a34 ( h and p ) respectively . validation strategy as described in extended data figure 2 .
for a10 and a29 , hierarchical clustering of the vaf was performed separately for substitutions ( a ) and ( h ) and indels ( b ) and ( i ) .
indels that could not be assigned to any cluster ( if any ) are annotated with black .
the additional sample ( d ) for a29 is incorporated into the phylogenetic tree ( j ) .
validation experiment for a10-e , the prostate sample , gave very low coverage ( d ) .
subclones for a29-a and a29-c are annotated in the 2d - dp plot ( k ) .
numbers of substitutions in wgs data assigned to each subclone are plotted in ( c ) and ( l ) .
vafs from wgs and validation data , plotted as scatter plots ( d ) and ( m ) , are very highly correlated .
number of indels ( e ) and ( n ) and rearrangements ( f ) and ( o ) present in different subsets of samples are plotted as bar charts .
subclone colour keys for a10 and a29 ( g and p ) respectively . validation strategy as described in extended data figure 2 . for a17 and a12 , hierarchical clustering of the vaf was performed separately for substitutions ( a ) and ( i ) and indels ( b ) and ( j ) .
for a12 , the c - specific cluster that is not present in substitutions is shown in very light green .
subclones for a17 ad are annotated in the 2d - dp plot ( c ) .
numbers of substitutions in wgs data assigned to each subclone are plotted in ( d ) and ( l ) .
vafs from wgs and validation data , plotted as scatter plots ( e ) and ( m ) , are very highly correlated .
number of indels ( f ) and ( n ) and rearrangements ( g ) and ( o ) present in different subsets of samples are plotted as bar charts .
subclone colour keys for a17 and a12 ( h and p ) respectively . validation strategy as described in extended data figure 2 .
hierarchical clustering of the vaf was performed separately for substitutions ( a ) and indels ( b ) .
numbers of substitutions in wgs data assigned to each subclone are plotted in ( d ) .
number of indels ( e ) and rearrangements ( f ) present in different subsets of samples are plotted as bar charts .
vafs from wgs and validation data , plotted as scatter plots ( g ) , are very highly correlated .
rearrangements and copy number segments in the vicinity of the ar locus are shown for a31 , a21 , a29 and a10 . (
is a tandem duplication whose existence is supported by tumour reads in adef but not c. however pcr - gel validation confirms its existence in the prostate sample c - the faintness of the band suggesting that this rearrangement is present subclonally in a31-c - as well as the prostate sample i , which was not subject to wgs .
one tandem duplication is common to both prostate samples ( shown in green ) while the other is specific to sample c ( dark pink ) .
( b ) in a21 , there are 4 different sets of complex rearrangements , one shared by acdegh and the remainder specific to f , i and j. ( c ) rearrangements in the vicinity of the ar locus and inter - mutation distances for a29 plotted on a log10 scale for lesions specific to the metastasis ( left ) and specific to the prostate ( middle ) .
each sample has a different set of complex rearrangements , which are associated with distinct kataegis events .
( d ) in a10 , one tandem duplication is shared by cd while four others are each specific to a single sample . to determine validation rate for mutation calling ,
a custom capture sureselect design was used to sequence selected coding / non - coding loci to an average depth of 360 - 2000x . for loci with sufficient depth (
> = 20x ) , the validation rate ( the proportion of somatic variants ) was calculated as described in extended data table 2 and supplementary information , section 3c . on average 95% and 86% of the substitutions and indels , respectively , were somatic .
validation for rearrangement calls was performed by pcr - gel electrophoresis , as described in supplementary information , section 3d .
pcr - gel experiments yielded a high validation rate for three of the four patients included in the validation .
for this sample , we therefore assessed the veracity of the breakpoints by visual inspection of the associated copy number segments and confirmed that 82% were high - confidence events resulting in visible copy number changes . to identify potentially oncogenic events within regions of copy number changes , we intersected the affected genomic segments with genes previously shown to be recurrently amplified / deleted .
source column indicates the literature source of the gene as follows : pan_cancer = the cancer genome atlas ( tcga ) pan - cancer data set ( zach , 2013 ) , prostate = reports of genes specifically amplified / deleted in prostate cancer ( taylor , 2010 and barbieri , 2012 ) , cancer_gene_census = cancer gene census ( futreal , 2004 ) , literature = widely reported in cancer literature .
 cancers emerge from an on - going darwinian evolutionary process , often leading to multiple competing subclones within a single primary tumour1 - 4 . 
 this evolutionary process culminates in the formation of metastases , which is the cause of 90% of cancer - related deaths5 . 
 however , despite its clinical importance , little is known about the principles governing the dissemination of cancer cells to distant organs . 
 although the hypothesis that each metastasis originates from a single tumour cell is generally supported6 - 8 , recent studies using mouse models of cancer demonstrated the existence of polyclonal seeding from and inter - clonal cooperation between multiple subclones9,10 . in this study , we sought definitive evidence for the existence of polyclonal seeding in human malignancy and to establish the clonal relationship among different metastases in the context of androgen - deprived metastatic prostate cancer . using whole genome sequencing , we characterised multiple metastases arising from prostate tumours in ten patients . 
 integrated analyses of subclonal architecture revealed the patterns of metastatic spread in unprecedented detail . 
 metastasis - to - metastasis spread was found to be common , either through de novo monoclonal seeding of daughter metastases or , in five cases , through the transfer of multiple tumour clones between metastatic sites . 
 lesions affecting tumour suppressor genes usually occur as single events , whereas mutations in genes involved in androgen receptor signalling commonly involve multiple , convergent events in different metastases . 
 our results elucidate in detail the complex patterns of metastatic spread and further our understanding of the development of resistance to androgen deprivation therapy in prostate cancer . 