Frequent somatic transfer of mitochondrial DNA into the nuclear genome of human cancer cells

F equen soma ic ans e o mi ochond ial DNA in o he nuclea genome o human cance cells Young Seok Ju, 1 Jose M.C. Tubio, 1,45 William Mi sud, 1,45 Beiyuan Fu, 2 Helen R. Da ies, 1 Manasa Ramak ishna, 1 Yilong Li, 1 Lucy Ya es, 1 Gunes Gundem, 1 Pa ick S. Ta pey, 1 Sam Behja i, 1 Elli Papaemmanuil, 1 Sancha Ma in, 1 An hony Fullam, 1 Mo i z Ge s ung, 1 ICGC P os a e Cance Wo king G oup, 46 ICGC Bone Cance Wo king G oup, 46 ICGC B eas Cance Wo king G oup, 46 Jyo i Nangalia, 1,3,4 An hony R. G een, 3,4 Ca los Caldas, 3,5 Åke Bo g, 6,7,8 And ew Tu , 9 Ming Ta Michael Lee, 10,11 Lau a J. an’ Vee , 12,13 Beni a K.T. Tan, 14 Samuel Apa icio, 15 Paul N. Span, 16 John W.M. Ma ens, 17 S ian Knappskog, 18,19 Anne Vincen -Salomon, 20 Anne-Lise Bø esen-Dale, 21,22 Jó unn E la Ey jö d, 23 Ola Myklebos , 24 Ad ienne M. Flanagan, 25,26 Ch is ophe Fos e , 27 Da id E. Neal, 28,29 Colin Coope , 30,31 Rosalind Eeles, 32,33 G. S e en Bo a, 34 Sunil R. Lakhani, 35,36,37 Ch is ine Desmed , 38 Gilles Thomas, 39,44 And ea L. Richa dson, 40,41 Colin A. Pu die, 42 Alas ai M. Thompson, 43 Ul an McDe mo , 1 Feng ang Yang, 2 Se ena Nik-Zainal, 1 Pe e J. Campbell, 1 and Michael R. S a on 1 1–43 [Au ho a ilia ions appea a end o pape .] Mi ochond ial genomes a e sepa a ed om he nuclea genome o mos o he cell cycle by he nuclea double memb ane, in e ening cy oplasm, and he mi ochond ial double memb ane. Despi e hese physical ba ie s, we show ha soma ically acqui ed mi ochond ial-nuclea genome usion sequences a e p esen in cance cells. Mos occu in conjunc ion wi h in a- nuclea genomic ea angemen s, and he ea u es o he usion agmen s indica e ha nonhomologous end joining and/o eplica ion-dependen DNA double-s and b eak epai a e he dominan mechanisms in ol ed. Rema kably, mi ochond i- al-nuclea genome usions occu a a simila a e pe base pai o DNA as in e ch omosomal nuclea ea angemen s, in- dica ing he p esence o a high equency o con ac be ween mi ochond ial and nuclea DNA in some soma ic cells. T ansmission o mi ochond ial DNA o he nuclea genome occu s in neoplas ically ans o med cells, bu we do no ex- clude he possibili y ha some mi ochond ial-nuclea DNA usions obse ed in cance occu ed yea s ea lie in no mal soma ic cells. [Supplemen al ma e ial is a ailable o his a icle.] Soma ically acqui ed s uc u al ea angemen s a e common ea- u es o he nuclea genomes o cance cells. These may ange om simple ch omosomal ea angemen s (Campbell e al. 2008) o mo e complex, compound pa e ns, such as ch omo- h ipsis (S ephens e al. 2011) and ch omoplexy (Baca e al. 2013), o mobiliza ion o ansposable elemen s (Lee e al. 2012; Tubio e al. 2014). In ach omosomal ea angemen s a e gene al- ly mo e common han in e ch omosomal ea angemen s, indi- ca ing a highe likelihood o joining a double-s and b eak in a ch omosome o ano he b eak in he same ch omosome despi e he a ailabili yo a much la ge quan i yo nuclea DNA om o h- e ch omosomes (S ephens e al. 2009). In addi ion o he nuclea genome, human cells ha e a ew hund ed o a ew housand mi ochond ia, each ca ying one o a ew copies o he 16,569-bp-long ci cula m DNA (Smei ink e al. 2001; F iedman and Nunna i 2014; Ju e al. 2014). Du ing endo- symbio ic co-e olu ion, mos o he gene ic in o ma ion p esen in he ances al mi ochond ion has ans e ed o he nuclea ge- nome (G ay e al. 1999; Adams and Palme 2003; Timmis e al. 2004). An appa en bu s o m DNA ans e occu ed du ing p i- ma e e olu ion ∼54 million yea s ago (Ghe man e al. 2007) and occasional, p obably mo e ecen , ans e in humans has been obse ed in he ge mline (Tu ne e al. 2003; Goldin e al. 2004; Chen e al. 2005; Milla e al. 2010; Dayama e al. 2014). 44 Deceased. 45 These au ho s con ibu ed equally o his wo k. 46 A ull lis o membe s is p o ided in he Supplemen al Ma e ial. Co esponding au ho : [email p o ec ed] A icle published online be o e p in . A icle, supplemen al ma e ial, and publi- ca ion da e a e a h p://www.genome.o g/cgi/doi/10.1101/g .190470.115. F eely a ailable online h ough he Genome Resea ch Open Access op ion. © 2015 Ju e al. This a icle, published in Genome Resea ch, is a ailable unde a C ea i e Commons License (A ibu ion 4.0 In e na ional), as desc ibed a h p://c ea i ecommons.o g/licenses/by/4.0/. Resea ch 814 Genome Resea ch 25:814–824 Published by Cold Sp ing Ha bo Labo a o y P ess; ISSN 1088-9051/15; www.genome.o g www.genome.o g Cold Sp ing Ha bo Labo a o y P ess on Oc obe 16, 2016 - Published by genome.cshlp.o gDownloaded om Cold Sp ing Ha bo Labo a o y P ess on Oc obe 16, 2016 - Published by genome.cshlp.o gDownloaded om Cold Sp ing Ha bo Labo a o y P ess on Oc obe 16, 2016 - Published by genome.cshlp.o gDownloaded om Al hough m DNA nuclea ans e in a HeLa cell line de i a i e, and hus occu ing in i o, has been epo ed (Shay e al. 1991), de no o nuclea ans e o m DNA in animal soma ic issues has no p e iously been comp ehensi ely s udied o ou knowledge. To in es iga e he possibili y o soma ic mi ochond ial-nuclea DNA usion, we analyzed nex -gene a ion pai ed-end DNA whole-genome sequencing da a om 559 p ima y cance s, 28 can- ce cell lines ( e e ed as 587 cance whole genome below) and no - mal DNAs om he same indi iduals (Supplemen al Table 1). Resul s Disco e y o soma ic m DNA ans e s o cance nuclea genomes F om he 587 pai s o cance and no mal whole-genome se- quencing da a, we sea ched o cance -speci ic clus e s o dis- co dan pai ed-end sequence eads in which one membe o he ead-pai mapped o he nuclea genome and he o he o he mi ochond ial genome, and hen cha ac e ized he nuclea - mi ochond ial genome junc ions o nucleo ide esolu ion using indi idual sequence eads ha b idged he junc ion (Fig. 1A). In 12 samples (o e all posi i e a e 2.0%, 12 ou o 587 samples), we obse ed 25 cance -speci ic mi ochond ial-nuclea DNA junc ions (Table 1; Supplemen al Figs. 1–6). Gi en ha he e a e wo junc- ions o a single in eg a ion e en , we conclude ha he e a e mos likely 16 independen m DNA inse ions (Table 1). In addi- ion o soma ic ans e s, we obse ed se e al no el a e ge mline (inhe i ed) e en s ha we esha ed be ween cance and pai ed no - mal samples (Supplemen al Table 2; Supplemen al Ma e ial). B eas cance PD11372a showed a soma ically acqui ed in- eg a ion o almos he en i e human m DNA sequence (16,556 bp) in o a highly ampli ied 2.75-Mb-long egion o Ch omosome 10q22.3. The in eg a ion e en was s ongly sup- po ed by bo h disco dan and spli ead clus e s (Fig. 1B–D) and was con i med by sho - and long- ange PCR ac oss he nu- clea -mi ochond ial genome junc ions (Supplemen al Figs. 7, 8; Supplemen al Table 3). I was no ound in no mal issue (blood) om he same indi idual o om all he o he cases and did no ma ch any known inhe i ed nuclea m DNA-like sequences (known as num s) (Ghe man e al. 2007; Hazkani-Co o e al. 2010). Consis en wi h i s soma ic o igin, he m DNA used o he nuclea genome ha bo ed sequence polymo phisms iden ical o hose p esen in he mi ochond ia o his indi idual (14,905 G > A; 15,028 C > A; 15,043 G > A; 15,326 A > G; 15,452 C > A, and 15,607 A > G). Fluo escence in si u hyb idiza ion (FISH) expe - imen spe o medon o malin- ixedpa a inembedded issuecon- i med ha he used DNA segmen exis s in he nuclei o cance cells (Fig. 1E). In o al, we ound 10 p ima y cance s (1.8%, 10/559) and wo cance cell lines (7.1%, 2/28) wi h soma ic m DNA in eg a ions in o hei nuclea genomes (Table 1; Supplemen al Figs. 1–6). O he 12 cance s, wo (p ima y cance PD13296a and cance cell line NCI-H2087) had mo e han one mi ochond ial-nuclea DNA ansloca ion e en . All in eg a ions we e suppo ed by bo h dis- co dan andspli eadsand u he con i medbyPCRac oss henu- clea -mi ochond ial genome junc ions (Supplemen al Fig. 7; Supplemen alTable3).Allinhe i edm DNAsubs i u ionpolymo - phisms nea hese b eakpoin s we e de ec ed (Table 1). To u he isualize he ans e e en s, we pe o med high- esolu ion FISH on s e ched DNA ibe s ( ibe FISH) om he melanoma cell line, CP66-MEL (Fig. 2A). Soma ic nuclea in eg a ion o m DNA is equen ly combined wi h o he ea angemen s o he nuclea genome The a e o soma ic nuclea ans e o m DNA may a y acco ding o umo ype. T iple-nega i e b eas cance showed a i e old highe equency compa ed o es ogen- ecep o (ER) posi i e b eas cance s (6.2% and 1.2%, espec i ely; Fishe ’s exac es P = 0.002). T iple-nega i e b eas cance genomes ca y a highe numbe o ch omosomal ea angemen s han ER-posi i e b eas (a e age 254 and 94, espec i ely, in ou da a se ). As a esul , he e was a sugges i e posi i e co ela ion be ween he numbe o ch o- mosomal ea angemen s and m DNA ans e s (Mann-Whi ney U es , one-sided P= 0.05) (Fig. 2B). The leng h o m DNA agmen s ans e ed anged om 148 bp o en i e mi ochond ial genomes (16.5 kb) (Table 1). In e es - ingly, b eakpoin s in m DNA we e en iched nea he mi ochond i- al genome hea y s and o igin o eplica ion (χ 2 es , P= 0.0005) (Fig. 2C). This sugges s ha he gene a ion o m DNA segmen s o be in eg a ed in o he nuclea genome is no andom and may occu in a m DNA eplica ion-dependen manne (Lenglez e al. 2010). O he 25 mi ochond ial-nuclea DNA junc ions, a leas 17 (68.0%) we e clea ly associa ed wi h o he nuclea ch omosomal ea angemen s (e.g., in e sions, ansloca ions, and la ge dele- ions) in he icini y (Table 1; Supplemen al Figs. 1–6). Fo example, wi h espec o PD11372a desc ibed ea lie , genomic agmen s om Ch omosomes 10, 11, and m DNA gene a ed com- plex de i a i e ch omosomes (Fig. 3A). In PD6047a, an m DNA agmen was in ol ed in chains o complex genomic ansloca- ions in ol ing Ch omosomes 6, 7, 11, 22, and X (Fig. 3B). In PD10014a, a local in e sion was combined wi h he m DNA in e- g a ion e en (Fig. 3C), and in PD4252a, a 16.5-kb m DNA in eg a- ion was ound in a posi ion on he X Ch omosome om which ∼20 kb o nuclea DNA had been soma ically dele ed (Fig. 3D). Thus, m DNA is o en in eg a ed in o nuclea genomes in he icini y o , o as pa o , complex ea angemen s. Al hough ge m- line num s end o occu nea ansposable elemen s such as LINEs and SINEs (Mishma e al. 2004), we do no obse e his associa ion o soma ic e en s (χ 2 es , wo-sided P= 0.33) (Supplemen al Table 4). The mechanism and iming o soma ic nuclea ans e o m DNA The e was o e lapping sequence mic ohomology ( om 1 o 4 bp) in 20/25 b eakpoin s (80%) (Fig. 4A,B; Table 1; Supplemen al Figs. 1–6), subs an ially mo e han expec ed by chance (χ 2 es , P=5× 10 −26 ). Thus, DNA sequence mic ohomology plays an impo an ole in mi ochond ial-nuclea DNA in eg a ion e en s, al hough blun -end DNA epai was also obse ed. In wo b eakpoin s, we also ound non empla ed sho -nucleo ide inse ions (1 and 4 bp long) (Fig. 4A; Table 1). O e all, hese ea u es a e cha ac e is ic o DNA double-s and b eak epai by nonhomologous end join- ing (NHEJ) (Has ings e al. 2009). Howe e , hey do no ule ou eplica ion-based mechanisms swi ching empla e be ween nucle- a and m DNA, such as mic ohomology-media ed b eak-induced eplica ion (MMBIR) (Liu e al. 2011). We in es iga ed he iming o soma ic m DNA in eg a ion in o he nuclea genome by assessing cases in which a me as a ic sample had been sequenced in addi ion o he p ima y umo . One such case (PD4252a) showed he mi ochond ial-nuclea in e- g a ion e en in he p ima y bu no in he me as asis (Fig. 4C), in- dica ing ha m DNA ans e o he nucleus can occu a e Nuclea in eg a ion o mi ochond ial DNA in cance Genome Resea ch 815 www.genome.o g Cold Sp ing Ha bo Labo a o y P ess on Oc obe 16, 2016 - Published by genome.cshlp.o gDownloaded om neoplas ic ans o ma ion and du ing he cou se o subclonal e o- lu ion o he cance . The o he (PD6728b) showed i in bo h he p ima y and me as asis (Fig. 4C), sugges ing ha his e en oc- cu ed in he common ances al cance clone o in no mal soma ic cells p io o neoplas ic change. Nuclea ans e o m DNA is unexpec edly equen in human soma ic cells To ob ain a pe spec i e on he equency o mi ochond ial-nuclea DNA ansloca ion, we compa ed i s a e o ha o in anuclea 1 0 20 40 60 80 100 120 140 160 180 200 220 240 2 0 20 40 60 80 100 120 140 160 180 200 220 240 3 0 20 40 60 80 100 120 140 160 180 4 0 20 40 60 80 100 120 140 160 180 5 0 20 40 60 80 100 120 140 160 180 6 0 20 40 60 80 100 120 140 160 7 0 20 40 60 80 100 120 140 8 0 20 40 60 80 100 120 140 9 0 20 40 60 80 100 120 140 10 0 20 40 60 80 100 120 11 0 20 40 60 80 100 120 12 0 20 40 60 80 100 120 13 0 20 40 60 80 100 14 0 20 40 60 80 100 15 0 20 40 60 80 100 16 0 20 40 60 80 17 0 20 40 60 80 18 0 20 40 60 19 0 20 40 20 0 20 40 60 21 0 20 40 22 0 20 40 x 0 20 40 60 80 100 120 140 umou blood 1 Mb 10 kb 100 bp 1 bp 1 bp 100 bp 10 kb 1 Mb PD11372a CTCCTGGGTG AGAAA CTCCTGGGTG TTGGCCTCAC GATAT TTGGCCTCAC Fusion Ch 10 m DNA TACTGTGGC CC AGACCTCTT ACACT GC AGACCTCTT TACTGTGGC CC CTCAG x 60 x 27 x 25 x 76 ch 10 (+) 81,670,932 MT (-) 15,157 MT (-) 15,171 ch 10 (+) 78,920,385 x 24 x 28 16,556 bp * * * * * * * * * * * * * * * * * * * * * * * ** ch 10 posi ion (Mb) 0 20406080100120 * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * ** * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * ** * * * * * * * * ** * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * ** * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * ** * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * ** * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * 0 2 4 6 8 10 Copy numbe * ** * ** ** * * * * * * * * * * * * * * ** * ** * * ** ** * * * * ******* **** * * * * ** * * ** ** * * * * ** * ******* **** *** * * * * ** * * * * * * * * * * * ** * * **** * * **** *** * * ** ** **** **** *** ** ** * * * * ** * *** **** * * * * * * * * * * * * * * * * * * * * * * ** * ** **** * * * * * * * * * * * * * * * * * * * * * * * ** * * ** **** * * * * * ** * * ** * ** ** **** * * ** * * * ** ** * ** * ** * * * * * * * * * * * * * * * * * * * * * * * * * ** * * * * * ** * * *** * ** * ** * * * * * * * * ** * * * * * * * * * * * * * * * * * * * * * * * * * * * * * ** * * * * * ** * * * * * * * ** ** * * **** **** * * * * * * * * * * * ** ** ** ** ** * * * * ** ** * ** * *** * ** ******** * ** ** **** ** *** *** * * * * * * * * * * * **** * * * ***** * *** * ********** * ** * * * * ** * * * * * * * * ** * * * * m DNA PD11372a dele ion ype andem duplica ion ype in e sion ype (head-head) in e sion ype ( ail- ail) m DNA ch 10: 80Mb ch 10: 78Mb me ged E DC BA mi ochond ial DNA (inse ed segmen ) (2) DRs (1) SRs (Nu) nuclea DNA nuclea DNA Mapping o he e e ence genome (using BWA) cance genome (3) SRs (MT) nuclea (Nu) genome mi ochond ial (MT) genome (2) DRs (1) SRs (Nu) mapped in Nu. genome as ma e-unmmaped (3) SRs (MT) mapped in MT genome as ma e-unmmaped Figu e 1. Disco e y o soma ic nuclea m DNA ans e om PD11372a. (A) The s a egy o de ec ion o nuclea m DNA ans e e en s. See Me hods o a de ailed desc ip ion. (SRs) Spli - eads, (DRs) disco dan eads, (Nu) nucleus, (MT) mi ochond ia. (B) G aphical ep esen a ion o disco dan ead clus- e s in PD11372a and i s pai ed-no mal issue (PD11372b). The ed a ow indica es umo -speci ic disco dan - ead clus e s in Ch 10. Ch omosome ideo- g ams a e shown in he ou e laye . The dis ance be ween each disco dan ead and one p io o i ( he in e - ead dis ance) is plo ed on he e ical axis on a log-scale in he middle ( umo ) and inne laye (blood). Blue do s shown in he middle laye ep esen known num s. (C) m DNA in eg a ion in PD11372a. B eakpoin sequences a e shown.Red ec anglehighligh s mic ohomology. Numbe s o disco dan spli eads a ep esen ed. Inhe i ed m DNA subs i u ion polymo phisms a e shown by ed as e isks. (D) Rea angemen a chi ec u es o Ch omosome 10 o PD11372a. DNA copy numbe s a e shown by black do s. The copy numbe o 2.75-Mb-long egion used wi h m DNA is colo ed in ed. Reads suppo ing ea angemen s (la ge dele ions, andem dupli- ca ions, ail- ail and head-head in e sions) a e shown by a cs and e ical lines. Ch 10-m DNA usions a e shown wi h ed a ows. (E) Nuclea FISH con i ms he mi ochond ial-nuclea DNA usion in he nucleus. (Red) Ch 10 (80 Mb), (blue) Ch 10 (78 Mb), and (g een) m DNA. Ju e al. 816 Genome Resea ch www.genome.o g Cold Sp ing Ha bo Labo a o y P ess on Oc obe 16, 2016 - Published by genome.cshlp.o gDownloaded om Table 1. Summa y o soma ic mi ochond ial-nuclea DNA usions iden i ied om 12 cance samples Tissue Sample Le junc ion Righ junc ion F ag. size (bp) Mic o- homology (bp,bp) Va ian s (#D/#P) a Con ex o ea angemen Nuclea MT MT Nuclea P ima y PD11372a 10+:81,670,932] [M−:15,157 M−:15,171] [10+:78,920,385 16,556 (0,1) 6/6 m DNA inse ion wi h complex ea angemen s PD4252a X+:45,631,665] [M+:14,450 M+:14,496] [X+:45,652,120 16,616 (2,1) 2/2 m DNA inse ion wi h la ge ch . dele ion PD6047a X+:14,944,764] [M−:12,735 M−:16,128] [7+:96,923,229 13,177 (1,1) 6/6 Mul iple in e ch omosomal ansloca ions PD10014a 17−:75,618,348] [M−:13,365 M−:9055] [17+:75,688,733 4311 (2,3) 0/0 m DNA inse ion wi h Ch 17 in e sion PD13296a 4+:102,463,870] [M−:14,705 M−:13,235] [4+:102,464,084 1471 (4,0) 0/0 m DNA inse ion wi h la ge ch . dele ion 6+:103,639,248] [M+:14,692 M+:14,972]TA AT [6+:103,690,941 281 (2,0) 2/2 m DNA inse ion wi h la ge ch . dele ion PD6728b 2+:138,664,890] [M−:13,199 M−:13,052] [2−:139,012,040 148 (4,2) 1/1 m DNA inse ion wi h complex ea angemen s PD11397a 19−:12,650,382] [M+:16,233 M+:96] [17+:40,005,738 433 (0,2) 1/1 Mul iple in e ch omosomal ansloca ions PD7404a 1+:44,914,376] [M+:3732 –– >200 (1,–) 0/0 – PD6733b 6−:45,823,498] [M+:16,107 –– >200 (0,–) 1/1 – PD11768a 1−:144,944,326] [M+:16,104 –– >200 (4,–) 1/1 – Cell line CP66-MEL 3+:47,419,506] [M−:7048 M−:16,193] [3+:47,419,447 7425 (1,1) 1/1 m DNA inse ion NCI-H2087 10+:26,775,605] [M+:1690 –– >200 (1,–) 1/1 – 20−:33,836,717] [M−:5666 –– >200 (1,–) 1/1 – 17−:7,481,787] T [M−:3452 –– >200 (1,–) 1/1 b Mul iple in e ch omosomal ansloca ions 17−:31,744,235] [M+:4346 –– >200 (3,–) 1/1 – a Inhe i ed m DNA polymo phisms in he icini y o b eakpoin s. (#D) Numbe o de ec ed, (#P) numbe o p esen . b A soma ically acqui ed he e oplasmic mu a ion in mi ochond ia. Nuclea in eg a ion o mi ochond ial DNA in cance Genome Resea ch 817 www.genome.o g Cold Sp ing Ha bo Labo a o y P ess on Oc obe 16, 2016 - Published by genome.cshlp.o gDownloaded om in e ch omosomal ansloca ion, aking in o accoun he sizes and copy numbe s o he mi ochond ial and nuclea genomes. Ou sequencing da a sugges ha each cance cell ca ies ∼500 copies o ci cula m DNA (median alue 495) (Fig. 5A), amoun - ing in agg ega e o ∼8 million base pai s (bp) o m DNA (500 cop- ies × 16.5 kb) enclosed by he mi ochond ial double memb ane in he cy oplasm o each cance cell. The a e age equency in he cance s analyzed o mi ochond ial-nuclea DNA usion was 5.1 × 10 −3 junc ions pe million bp o m DNA, only hal he a - e age a e o in anuclea in e ch omosomal ansloca ion (1.2 × 10 −2 junc ions pe million bp) and simila o ha o Ch omosomes 2, 4, and 13 (Fig. 5B). Gi en he mul iple physical ba ie s o con ac be ween he wo genomes, he esul s indica e ema kably high a es o m DNA escape, con ac , and/o in eg a- ion wi h nuclea DNA in human cance cells. These appea o be conside ably highe han in he ge mline ac oss human e olu- ion bu compa able o hose obse ed in Saccha omyces ce e isiae (Tho sness and Fox 1990) and o chlo oplas DNA mig a ion in o he nucleus in obacco plan s (Me hods; Supplemen al Ma e ial; Huang e al. 2003). Discussion Despi e mul iple physical ba ie s, he e a e plausible mechanisms by which m DNA and nuclea DNA could come in o con ac (Fig. 5C). F ee m DNA can be eleased in o he cy oplasm om de- g adingmi ochond iao a e mi ophagy (Zhang e al. 2008; Eiyama e al. 2013; Higgins and Coughlan 2014). Deg ada- ion o mi ochond ia may be accele a ed in cance cells due o hypoxia and in- c eased ene gy demands (Zhang e al. 2008; Eiyama e al. 2013; Higgins and Coughlan 2014). E en wi hou a bespoke molecula p ocess o anspo a ion, m DNA could hen, in p inciple, mig a e o he nucleus du ing mi o ic me aphase o anaphasewhen henuclea memb ane has b oken down. When hese e en s a e coupled wi h concu en double-s and b eaks (DSBs) and/o eplica ion o k s alling o nuclea ch omosomal DNA, m DNA could be picked up and in eg a - ed in o he nuclea genome as pa o he p ocess o ejoining DSBs (NHEJ) (Has ings e al. 2009) o used as an al- e na i e DNA empla e in eplica ion (MMBIR) (Liu e al. 2011). Mic onuclei in cance cells, which can be gene a ed bye o sinseg ega iono mi o icnuclea ch omosomes, may con ibu e o he e en s.Ch omosomesinmic onuclei e- quen ly unde go de ec i e and delayed DNA eplica ion, esul ing in ex ensi e agmen a ion wi h subsequen jumbled ejoiningcompa ed o hei o iginalo de and o ien a ion (C as a e al. 2012; Fo - men e al. 2012). Thus, m DNA ag- men s inco po a ed in o mic onuclei could end up used o sha e ed nuclea ch omosomes. I is wo hy o no e ha m DNA escaping o he nucleus canbe ac i elyused o DNA epai in Saccha omyces ce e isiae (Ricche i e al. 1999; Yu and Gab iel 1999), pa icula ly when e o - ee DSB DNA epai is no possible. Whe he his applies in mammalian cells is unknown. Some o he soma ic nuclea m DNA in eg a ions we iden i- ied a e di ec ly adjacen o nuclea genes. Fo example, nuclea - m DNA usion in PD11372a occu ed in he i h in on o he KCNMA1 gene, a po assium channel equen ly ampli ied in p os- a e and b eas cance s (Oegge li e al. 2012). Howe e , we do no ind ob ious en ichmen o he nuclea -m DNA usion b eak- poin s nea human nuclea genes. RNA-seq om he NCI-H2087 cell-line indica es ha m DNA agmen s in he nucleus o he cell line a e no exp essed as pa s o mi ochond ial-nuclea usion ansc ip s.Thus, hemajo i yo henuclea m DNA ansloca ion e en s a e likely o be passenge e en s, simila o mu a ions o all o he ypes in mos cance genomes. Howe e , we do no ex- clude hepossibili y ha someo hese e en smayha e unc ional consequences in human cance by gene a ing usion mRNA an- sc ip s (Shay e al. 1991) and/o unca ing cance genes by m DNA inse ion wi hin exons. ch 3 (downs eam)ch 3 (ups eam) m DNA (7.4kb) A CP66-MEL (melanoma cell-line) 250 500 1000 B 78 133 125 Cance issue ypes B eas (ER + e) O he b eas B eas ( iple − e) O he ypes m DNA ans e s -+ cance samples 0 1 2 3 4 0 2 4 6 8 10 12 14 0.5-3kb 3-5.5kb 5.5-8kb 8-11.5kb 11.5-14kb 14-0.5kb Ra io (obs/exp) F equency o e en s Loca ion o b eakpoin s in mi ochond ial genome Expec ed Obse ed Ra io (obs/exp) P = 0.00052 * D-Loop RNAs 16s12s CO1 ND5ND4 ND6 Replica ion o igin ( H s and) Replica ion o igin ( L s and) C CYB Figu e 2. Fea u es o soma ic m DNA nuclea ans e in 12 cance samples. (A) Fibe FISH isualizes he mi ochond ial-nuclea DNA usion om he CP66-MEL cell line. (B) Posi i e co ela ion be ween m DNA ans e and numbe s o nuclea ch omosomal ea angemen s (la ge dele ion, andem duplica- ion, in e sion, and ansloca ion) in cance genomes. Median alues a e shown. (C) m DNA b eak- poin s a e en iched in he 14 kb- o 500-bp egion o he MT genome. (Top) Blue and ed ba s ep esen he expec ed and obse ed numbe s o b eakpoin s in each in e al o MT genome, espec- i ely. G een line shows a io be ween obse ed and expec ed numbe s. A χ 2 es was applied o es en ichmen . (Bo om) Schema ic s uc u al ea u es o he MT genome co esponding o he in e als a e shown. Ju e al. 818 Genome Resea ch www.genome.o g Cold Sp ing Ha bo Labo a o y P ess on Oc obe 16, 2016 - Published by genome.cshlp.o gDownloaded om This s udy has shown ha usion o m DNA o nuclea DNA occu s in human soma ic cells a a a e simila o ha o ansloca- ion be ween nuclea ch omosomes. Physical mig a ion o m DNA in o he nucleus may be much mo e equen in s em cells han ones in a e minally di e en ia ed s age (Schneide e al. 2014). Fu he s udies will need o add ess he mechanisms by which he appa en physical ba ie s o con ac be ween mi ochond ial and nuclea DNA a e so e ec i ely o e come. Me hods Samples and sequencing da a We analyzed 559 p ima y umo s and 28 cance cell-lines in his s udy. Pai ed-no mal samples o all he cance s we e also included in his s udy in pa allel. Whole-genome sequencesusedin hiss udywe egene a - ed by Illumina pla o ms (ei he Genome Analyze o HiSeq 2000). Cance ge- nomeswe esequenced oa leas 25×co - e age. Wi h espec o TCGA da a, we downloaded aligned BAM iles h ough UCSC CGHub (h p://cghub.ucsc.edu). Sequencing eads we e aligned on he human e e ence genome build 37 (GRCh37) and human e e ence m DNA sequence ( e ised Camb idge e e ence sequence, CRS) (And ews e al. 1999), mainly by he BWA alignmen ool (Li and Du bin 2009). SAM ools (Li e al. 2009) was used o manipula ing se- quence eads. Calling mi ochond ial-nuclea DNA usion e en s We employed a pipeline o iden i ica- ion o pu a i e m DNA ansloca ion o ch omosomal DNA (Fig. 1A). F om pai ed-end whole-genome sequencing da a o umo s, we ex ac ed disco dan eads (DRs), whe e one end aligned uniquely o m DNA and he o he end o nuclea DNA. In all cases, bo h ends mus ha e a mapping quali y g ea e han ze o. Those disco dan eads a e clus e ed oge he using he ollowing c i e ia: eads sha ing (1) close alignmen posi ions (<500 nucleo ides) o bo h ends on nuclea and m DNA, and (2) he same o ien a ions.In o de o emo e alse posi i es, we emo ed clus e s sup- po ed by less han i e disco dan eads. In o de o emo e po en ial ge m- line calls, se e al il e s a e applied o he umo candida e clus e . The clus e s om umo cells we e emo ed i hey o e lap wi h clus e s iden i ied om ma ched and/o unma ched no mal is- sues by mo e ole able c i e ia (suppo ed by mo e han one disco dan ead) om (1) i s pai ed-no mal issue, and (2) om he o he 586 unma ched no mals. Fil e ed clus e s we e u he e ined wi h known ge mline human num s, a combined se om he human e e ence genome (hg19) de ec ed by BLAT (Ken 2002) (n= 123) and om Simone e al. (2011) (n= 766). Finally, 25 clus e s we e selec ed as soma ic candida es. Nucleo ide- esolu ion b eakpoin s o he ansloca ion junc ions To ob ain nucleo ide- esolu ion b eakpoin s, we sea ched o spli - eads(SRs) wi honeo he endsspanning hejunc iono he ans- loca ion. We ex ac ed “o phan”o “ma e-unmapped” eads (one end o a ead is unmapped by he BWA aligne ) in he icini y (<1000bp)o disco dan - eadclus e sonnuclea andmi ochond i- al genome sequences. Sequences om he unmapped end a e hen e-aligned by BLAT (Ken 2002), which enablesspli - ead mapping. A 10 0 5 5 10 15 20 25 30 35 3 40 45 4 50 55 60 65 6 70 75 80 85 8 90 95 100 105 110 115 1 120 125 130 135 11 0 5 5 10 15 5 20 25 5 30 35 40 45 5 50 55 5 60 65 5 70 75 5 80 85 5 90 95 5 100 105 110 115 5 120 125 130 135 5 MT 0 PD11372a D B ch X(+):45,631,665 ch X(+):45,652,120 m DNA inse ion (16.5kb) nuclea DNA dele ion (20 kb)  ch omosome X 0 20 40 60 80 100 120 140 p11.3 Copy numbe 45.61 45.62 45.63 45.64 45.65 45.66 45.67 024 o oooo o oooooooo o ooooooooooooooo o o o o oo oo o ooo o oooooooooo oooooooooooooo o ooooooooooooooooooooo oo oooooooooooooo ooooooo ooo ooooooooooooooo o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o 0 8 abe an ead clus e s PD4252a PD6047a 2 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 3 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 4 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 5 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 6 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 7 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 8 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 11 0 10 20 30 40 50 60 70 80 90 100 110 120 130 12 0 10 20 30 40 50 60 70 80 90 100 110 120 130 13 0 10 20 30 40 50 60 70 80 90 100 110 16 0 10 20 30 40 50 60 70 80 90 22 0 10 20 30 40 50 x 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 MT 0 m DNA (-) ch 7(+):96,923,229 ch X(+):14,944,764 13.1 kb m DNA(-): 13,365 - 9,055 (4.3kb) ch 17(+):75,688,733 local in e sion C ch 17(+):75,564,373 ch 17(-):75,655,898 - 75,618,348 (37.6kb) m DNA inse ion PD10014a Figu e 3. Concu ence o soma ic m DNA nuclea ans e s wi h o he s uc u al a ia ions. The com- plex web o ea angemen s in he icini y o mi ochond ial-nuclea DNA usions om ou examples. (A) In PD11372a, m DNA in eg a ion wi h complex ea angemen s be ween Ch 10 and 11. (B)In PD6047a, m DNA in eg a ion wi h complex ea angemen s among Ch 6, 7, 11, 22, and X. (A,B) DNA copy numbe s a e shown by black do s wi h a log scale. Red lines ep esen ansloca ions in ol ing m DNA. (C) In PD10014a, m DNA in eg a ion combined wi h a local in e sion (yellow). (D) In PD4252a, m DNA in eg a ion wi h a local dele ion. DNA copy numbe s a e shown wi h blue do s and lines. Abe an ead clus e s (disco dan and spli eads) a e shown by g een and ed a ows, espec i ely. Nuclea in eg a ion o mi ochond ial DNA in cance Genome Resea ch 819 www.genome.o g Cold Sp ing Ha bo Labo a o y P ess on Oc obe 16, 2016 - Published by genome.cshlp.o gDownloaded om Valida ion by PCR A PCR alida ion assay o he soma ic m DNA ans e was pe - o med using genomic DNA om bo h cance and pai ed-no mal issues. P ime s we e designed o ampli y all he b eakpoin s (Supplemen al Table 3). The sho - agmen PCR eac ions we e pe o med as p e iously desc ibed (Tubio e al. 2014). Wi h espec o long- ange PCR, elonga ion ime was inc eased 1 min pe 1 kb. Gene a ion o FISH p obes Human bac e ial a i icial ch omosomes (BAC) and osmid clones used in his s udy we e ob ained om he clone a chi e eam o he Wellcome T us Sange Ins i u e. Plasmid DNA was p epa ed using he PhaseP ep BAC DNA ki (Sigma-Ald ich). Human m DNA was isola ed om lymphoblas- oid cells using a Mi ochond ial DNA Isola ion ki (Abcam). P obes o use in FISH we e made as desc ibed be o e (G ibble e al. 2013). Pu i ied m DNA and plasmid DNA we e i s ampli ied using a GenomePlex Whole Genome Ampli ica ion (WGA) ki (Sigma-Ald ich) ollowing he manu- ac u e ’s p o ocols, hen labeled using a WGA eampli ica ion ki (Sigma- Ald ich) wi h a cus om-made dNTP mix. P obes o in e phase FISH we e labeled di ec ly wi h Aminoallyl-dUTPs - ATTO- 488, -Cy3, -Texas Red, and -Cy5 (Jena Bioscience); p obes o ibe -FISH we e la- beledwi hBio in-16-dUTP,Digoxigenin- 11-dUTP (Roche), and DNP-11-dUTP (Pe kinElme ). Valida ion by ibe -FISH wi h single- molecule DNA ibe s gene a ed by molecula combing Single-molecule DNA ibe s om he cance cell line, CP66-MEL, we e p e- pa ed by molecula combing (Michale e al. 1997) ollowing he manu ac u e ’s ins uc ions (Genomic Vision). B ie ly, he cells we e embedded in a low-mel - poin aga ose plug (1 million cells pe plug), ollowed by p o einase K diges- ion, washing in 1 × TE (10 mM T is, 1 mM EDTA, pH 8.0) and be a-aga ose di- ges ion s eps. The DNA ibe s we e me- chanically s e ched on o saline-coa ed co e slips using a Molecula Combing Sys em (Genomic Vision). Fo ibe -FISH, ∼500 ng o labeled DNA om each p obe and 4 μg o human Co -1 DNA (In i ogen) we e p ecipi- a ed using e hanol, hen esuspended in a mix (1:1) o hyb idiza ion bu e (con aining 2 × SSC, 10% sa kosyl, 2 M NaCl, 10% SDS, and blocking aid [In i- ogen]) and deionized o mamide ( inal concen a ion 50%). Co e slips coa ed wi h combed DNA ibe s we e dehyd a ed h ough a 70%, 90%, and 100% e hanol se ies and aged a 65°C o 30 sec, ollowed by dena u a ion in an alkaline dena u e solu ion (0.5 M NaOH, 1.5 M NaCl) o 1–3 min, h ee washes wi h 1×PBS (In i ogen), and dehyd a ion h ough a 70%, 90%, and 100% e hanol se ies. The p obe mix was dena u ed a 65°C o 10 min be o e being applied on o he co e slips, and he hyb idiza ion was ca ied ou in a 37° C incuba o o e nigh . The pos -hyb idiza ion washes consis ed o wo ounds o washes in 50% o mamide/2 × SSC ( / ), ollowed by wo addi ional washes in 2 × SSC. All pos -hyb idiza ion washes we e done a 25°C, 5 min each ime. Digoxigenin-11-dUTP (Roche) labeled p obes we e de ec ed using a 1:100 dilu ion o monoclonal ch 4: GGCGA AACCC CATT TCTACT Fusion: GGCGA AACCC CATT GGTCGT m DNA: TTTTT CATAT CATT GGTCGT TAAT A ch 4(+)102,463,870 m DNA(-):14,705 PD13296a (2 m DNA nuclea in eg a ion e en s) m DNA(-):13,235 ch 4(+):102,464,084 4bp mic ohomology blun -end DNA joining GCTACGATTT CTTTTGATGT : m DNA GCTACGATTT AAATAACCAC : Fusion GTTATCTTCA AAATAACCAC : ch 4 In eg a ion #1 In eg a ion #2 ch 6(+):103,639,248 m DNA(+) 14,692 m DNA(+) 14,972 ch 6(+):103,690,941 ch 6: TTGTAAGA AC TAATAGAATG Fusion: TTGTAAGA AC AACCACGACC m DNA:CACGGACT AC AACCACGACC GTAAATTATG GCTGAATCAT : m DNA GTAAATTATG TAAT AAAATATTTG : Fusion CTGGGTCCTA AAAATATTTG : ch 6 2bp mic ohomology non- empla e 4bp inse ion PD6728b B ch 2(+):138,664,890 m DNA(-) 13,199 m DNA(-) 13,052 ch 2(-):139,012,040 ch 2: TCATCT TGCT TGCGTTTTGC Fusion: TCATCT TGCT GCGAACAGAG m DNA:GCAGAC TGCT GCGAACAGAG 4bp mic ohomology GGGGTGGGGC CT TCTATGGC : m DNA GGGGTGGGGC CT GACTGCAG : Fusion CTTGGTCTTG CT GACTGCAG : ch 2 2bp mic ohomology C PD4252 Fe ilized egg Blood (PD4252b) MRCA LN me as asis (PD4252c) P ima y locus (PD4252a; subclonal) m DNA ans e PD6728 Fe ilized egg Blood (PD6728a) MRCA LN me as asis (PD6728c; clonal) P ima y locus (PD6728b; clonal) m DNA ans e T ans o ma ion T ans o ma ion Figu e 4. Nucleo ide- esolu ion b eakpoin sequences and he iming o soma ic m DNA nuclea in e- g a ion. (A) B eakpoin sequences o nuclea -m DNA usions in PD13296a. Red ec angles highligh se- quence mic ohomology and non empla e nucleo ides inse ion. (B) B eakpoin sequences o nuclea - m DNA usions in PD6728b. Red ec angles highligh sequence mic ohomology. (C) Phylogene ic ees showing he iming o soma ic m DNA nuclea ans e s in PD4252 and PD6728 samples. (MRCA) Mos ecen common ances o cell. Ju e al. 820 Genome Resea ch www.genome.o g Cold Sp ing Ha bo Labo a o y P ess on Oc obe 16, 2016 - Published by genome.cshlp.o gDownloaded om mouse an i-dig an ibody (Sigma-Ald ich) and a 1:100 o Texas Red- X-conjuga ed goa an i-mouse IgG (Molecula P obes/In i ogen); DNP-11-dUTP (Pe kinElme ) labeled p obes we e de ec ed using a 1:100 dilu ion o Alexa 488-conjuga ed abbi an i-DNP IgG and 1:100 Alexa 488-conjuga ed donkey an i- abbi IgG (Molecula P obes/In i ogen); bio in-16-dUTP (Roche) labeled p obes we e de ec ed wi h one laye 1:100 o Cy3-a idin (Sigma-Ald ich). A e de ec ion, slides we e moun ed wi h SlowFade Gold moun ing sol- u ion con aining 4′,6-diamidino-2-phenylindole (Molecula P obes/In i ogen). Images we e isualized on a Zeiss AxioImage D1 mic oscope. Digi al image cap u e and p ocessing we e ca ied ou using he Sma Cap u e so wa e (Digi al Scien i ic UK). Nuclea in e phase FISH Nuclei ex ac ion om pa a in-embed- ded issue o pa ien PD11372a and in e - phase-FISH ollowed Pa e nos e e al. (2002), wi h he excep ion ha 60-μm- hick sec ions we e used in ou s udy. The pos -hyb idiza ion washes consis ed o wo ounds o washes in 50% o mam- ide/2 × SSC ( / ), ollowed by wo ad- di ional washes in 2 × SSC. Slides we e moun ed wi h SlowFade Gold moun - ing solu ion con aining 4′,6-diamid- ino-2-phenylindole (Molecula P obes/ In i ogen). Images we e cap u ed and p ocessed as desc ibed abo e. Co ela ion be ween soma ic m DNA in eg a ion si e and ansposable elemen s We pe o med a s udy simila o he p e- ious epo (Mishma e al. 2004). We calcula ed he dis ance be ween each m DNA-inse ion si e (b eakpoin ) and i s nea es ansposable elemen s (ei- he o SINE, LINE, LTR, simple epea , o DNA ansposon by Repea Maske , downloaded om he UCSC Genome B owse , June 6, 2013). Then, each m DNA-inse ion si e was ca ego ized in o one o ou g oups: (A) b eakpoin wi hin a ansposable elemen ; (B) b eak- poin wi hin 15 bp om a ansposable elemen ; (C) wi hin 15–150 bp; and (D), >150 bp. In o de o unde s and he posi- ional en ichmen o b eakpoin s om ansposable elemen s, we andomly gene a ed in silico b eakpoin posi ions 40 imes as many ( o al n= 1000) as we obse ed om each ch omosome in he eal da a se . In silico b eakpoin s loca ed wi hin gaps o he human e e ence ge- nome we e emo ed and eplaced by newly gene a ed inse ions. Fo hese in silico-gene a ed b eakpoin s, he dis- ances om he nea es ansposable elemen s we e calcula ed and hen ca e- go ized in o one o he ou g oups (A, B,C, and D). Finally, he di e ence in he equency o b eakpoin s in each g oup be ween he obse ed and in silico-gene a ed da a se was compa ed using a χ 2 es . Assessmen o m DNA copy numbe s To unde s and m DNA copy numbe s in a cance cell, we com- pa ed a e age ead dep h o co e age be ween 22 au osomes and m DNA. Wi h espec o he umo sequences by whole-genome sequencing, a e age haploid au osomal co e age (RD au osome ) was ob ained om he ead dep h o 2.685-Gb-long au osomal e- gions (excluding ch omosomal gaps). Likewise, a e age m DNA co e age (RD m DNA ) was ob ained om he ead dep h o he 500 1000 2000 Cance issue ypes B eas Os eosa coma O he ypes P os a e A Es ima ed ci cula m DNA copy numbe s (in cy oplasm) pe cance cell 0.00 0.01 0.02 0.03 ch omosomes T ansloca ion a e (# o e en s pe Mb) ch 2 ch 17 ch 19 m DNA ch 4 ch 13 B mi ophagy mi ochond ial deg ada ion escape o m DNA nuclea memb ane b eaks down beginning o mi osis DNA double-s and b eaks (ch omosome sha e ing) and/o eplica ion o k s alling m DNA mig a ion o he nucleus (mic onucleus) ( a e> 2x10 pcpg) cell memb ane -4 nucleus (mic onucleus) m DNA in eg a ion DSB epai (NHEJ, MMBIR) C Figu e 5. F equency and po en ial mechanisms o soma ic m DNA nuclea ans e in human can- ce . (A) Es ima ed ci cula m DNA copy numbe s (in he cy oplasm) pe cance cell om 587 cance is- sues sequenced. The a io o ead dep hs be ween au osomes and m DNA was used (see Me hods). (B) Simila equency o soma ic nuclea m DNA in eg a ions compa ed o he equency be ween au o- somes (ch omosomal ansloca ion). (C) A model o soma ic m DNA ans e o he nuclea genomes. Nuclea in eg a ion o mi ochond ial DNA in cance Genome Resea ch 821 www.genome.o g Cold Sp ing Ha bo Labo a o y P ess on Oc obe 16, 2016 - Published by genome.cshlp.o gDownloaded om 16.5-kb mi ochond ial genome. Finally, m DNA copy numbe in a diploid cell (C m ) is calcula ed as shown below: Cm =2×RDm DNA RDau osome . Assessmen o ansloca ion a e o au osomes and mi ochond ia We iden i ied s uc u al a ia ions among nuclea ch omosomes (la ge dele ions, andem duplica ions, in e sions, and in e ch o- mosomal ansloca ions) using he BRASS II algo i hm (Nik- Zainal e al. 2012), which iden i ies ea angemen s by clus e ing disco dan ead pai s ha poin o he same junc ion and con i ms b eakpoin s by local assembly o unmapped eads. The sensi i i y and speci ici y o he BRASS II algo i hm is equi alen o hose al- ues o he algo i hm used o mi ochond ial-nuclea DNA usions (da a no shown). We ex ac ed in e ch omosomal ansloca ions o calcula e he a e o such e en s. The a e o each haploid au o- some (R ,ch) is calcula ed as shown below: R ,ch(e en s pe megabase)=N ,ch/(2×Lch)/Nsam, whe e N ,ch is he o al numbe o soma ic in e ch omosom- al ansloca ion junc ions in ol ing a speci ic ch omosome, Lch is heleng ho henon edundan egiono hech omosomeinmeg- abases, and Nsam is he o al numbe o samples analyzed. To ob- ain he unique egion leng h (Lch), we excluded edundan (o highly epe i i e) sequence leng hs om he ungapped leng h o each ch omosome. Genomic egions classi ied in one o mo e o he h ee c i e ia shown below we e de ined as edundan , whe e ansloca ion e en s could no be easilyde ec ed due o ambiguous ead alignmen : (1) simple epea s, loca ed by Tandem Repea s Finde (Benson 1999); (2) segmen al duplica ions wi h mode a e o high sequence simila i y (≥95%) (Bailey e al. 2002), o (3) epe - i i e sequences including up o 10 di e en classes o epea s (such as SINE, LINE, LTR, DNA ansposons, andmic osa elli es), loca ed by he Repea Maske p og am (h p://www. epea maske .o g), wi h a low di e gence le el (di e gence < 5%). These non edun- dan sequence egions we e downloaded om he UCSC Genome B owse (h p://genome.ucsc.edu). Simila ly, he a e o mi ochond ial-nuclea DNA ansloca- ions (R ,m ) was calcula ed as below: R ,m (e en s pe megabase)=N ,m /(Cm ×Lm DNA)/Nsam, whe e N ,m is he o al numbe o junc ions o soma ic mi o- chond ial-nuclea DNA usions iden i ied, C m is he median alue o mi ochond ial genome copy numbe s in a diploid cance cell calcula ed abo e (495 copies), and L m DNA is he leng h o he mi- ochond ial genome in megabases (0.016569 Mb). Assessmen o he a es o nuclea m DNA usion and m DNA escape o he nucleus Fusion o m DNA o he nuclea genome equi es a leas wo e en s, each o which could in luence he a e o mi ochond ial- nuclea DNA usion.Theseincludeescapeo m DNA o henucleus and in eg a ion o nuclea DNA. Acco ding o his model, he o e - all numbe o such usion e en s can be calcula ed using he a es o hese p ocesses (ρ escape and ρ in eg a ion , espec i ely): N =Nsam ×Ngen × escape × in eg a ion, whe e N is he numbe o o al soma ic mi ochond ial-nu- clea DNA usion e en s (n= 12), Nsam is he o al numbe o can- ce issues (n= 587), and Ngen is he numbe o a e age cell gene a ion om he e ilized egg. Using a easonable assump ion ha Ngen= 1000, we ob ain he a e o soma ic m DNA usion o he nuclea genome (ρ escape ×ρ in eg a ion ) obe2×10 −5 pe cell pe cell gene a ion (pcpg). Wi h one mo e e y conse a i e assump- ion ha ρ in eg a ion is 0.1, we ob ain ρ escape o be 2 × 10 −4 pcpg, o a leas one escape e en pe 5000 cell gene a ions. We hypo h- esize ha he eal ρ in eg a ion alue is hough o be much lowe han 0.1, which esul s in a highe ρ escape . Fo example, du ing he gene a ion o knockou mice, homologous ecombina ion al- lows one ixa ion e en pe 1000–10,000 mic oinjec ed DNA cop- ies (B ins e e al. 1985). The in eg a ion a e may, howe e , be highe han he a e in cance cells wi h de ec i e homologous e- combina ion-based epai and inc eased a ailabili y o nuclea double-s and b eaks, which can be joined o by NHEJ o MMBIR. The m DNA usion o he nuclea genome in he ge mline ( he a e o num s inse ion) is a ound 5 × 10 −6 pe ge m cell pe indi idual gene a ion in p e ious phylogene ic s udies (Hazkani- Co o e al. 2010). The a e is equi alen o ∼5×10 −8 pcpg, gi en ha he numbe o ge m cell di isions pe human gene a ion is ∼100 (401 in males and 31 in emales [D os and Lee 1995]). Da a access Sequence da a o sample pai s wi h posi i e m DNA nuclea ans- e ha e been submi ed o he Eu opean Genome-phenome A chi e (EGA; h ps://www.ebi.ac.uk/ega/home). The s udy acces- sion numbe is EGAS00001001234. Sample accession numbe s a e a ailable in Supplemen al Table 1. Lis o a ilia ions 1 Cance Genome P ojec , Wellcome T us Sange Ins i u e, Hinx on, Camb idge CB10 1SA, Uni ed Kingdom; 2 Cy ogene ics Facili y, Wellcome T us Sange Ins i u e, Hinx on, Camb idge CB10 1SA, Uni ed Kingdom; 3 Camb idge Uni e si y Hospi als NHS Founda ion T us , Camb idge CB2 0QQ, Uni ed Kingdom; 4 Depa men o Haema ology, Uni e si y o Camb idge, Camb idge CB2 0XY, Uni ed Kingdom; 5 Cance Resea ch UK (CRUK) Camb idge Ins i u e, Uni e si y o Camb idge, Camb idge CB2 0RE, Uni ed Kingdom; 6 BioCa e, S a egic Cance Resea ch P og am, SE-223 81 Lund, Sweden; 7 CREATE Heal h, S a egic Cen e o T ansla ional Cance Resea ch, SE-221 00 Lund, Sweden; 8 Depa men o Oncology and Pa hology, Lund Uni e si y Cance Cen e , SE-221 85 Lund, Sweden; 9 B eak h ough B eas Cance Resea ch Uni , Resea ch Oncology, King’s College London, Guy’s Hospi al, London SE1 9RT, Uni ed Kingdom; 10 Labo a o y o In e na ional Alliance on Genomic Resea ch, RIKEN Cen e o In eg a i e Medical Sciences, 230-0045 Yokohama, Japan; 11 Na ional Cen e o Genome Medicine, Ins i u e o Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan; 12 Depa men o Labo a o y Medicine, Helen Dille Family Comp ehensi e Cance Cen e , Uni e si y o Cali o nia, San F ancisco, Cali o nia 94158, USA; 13 Ne he lands Cance Ins i u e, 1066 CX Ams e dam, Ne he lands; 14 Depa men o Gene al Su ge y, Singapo e Gene al Hospi al, Singapo e 169608; 15 Depa men o Molecula Oncology, B i ish Columbia Cance Agency, Vancou e V5Z 1L3, Canada; 16 Depa men o Radia ion Oncology and Depa men o Labo a o y Medicine, Radboud Uni e si y Medical Cen e , 6525 HP Nijmegen, Ne he lands; 17 Depa men o Medical Oncology, E asmus MC Cance Ins i u e, E asmus Ju e al. 822 Genome Resea ch www.genome.o g Cold Sp ing Ha bo Labo a o y P ess on Oc obe 16, 2016 - Published by genome.cshlp.o gDownloaded om