{"paper_id":"0c8ef8a4-a6c6-48df-806e-19567e79edb0","body_text":"1\nTitle: P opula ti on-lev el g enome seque ncing r ev eals di s tin ct Myc obac t er ium tuber culo sis \nin tr ahost mut a ti onal tr ajec t or ies in s i mian im munode ficien cy vi rus c o-inf e ct ed and antir e tr ovir al \ntr eat ed non-human prima t es  \n \nAuthor s:  Michael C. Chao\n1# , Michael R. Chase 1 , Shok o W ak aba y ashi 1 , Andr ew Vick er s 1 , B y r on \nR oman 1 , F orr es t Hopkins 1 , P et er H. C ulviner 1 , Ma ximillan G. Marin 2 , P auline Maiello 3,4 , Coll in R. \nDietrich 4,5 , Zandr ea Ambr ose 3,4 , Phila na Ling Lin 4,5 , Qingyun Liu 6 , Sar ah M. F ortune 1  \n \nAffiliations \n1  Departmen t of Immunology and Inf ectious D isease, Har v ar d T .H. Chan Sc hool of Publi c Health, \nBos ton, Massachuset ts, Uni t ed St at es  \n2  Departmen t of Dat a Scienc e, Dana-F arber Cancer Ins t itu t e, Bost on, USA  \n3  Departmen t of Mi cr obio logy and Molecular G enetics, Uni v er sit y of Pittsb ur gh School of \nMedicine, Pit tsbu r gh, P enns ylv ania, U nit ed Sta t es  \n4  Cen t e r f or V ac cine R es ear ch, Univ ersity of Pi t tsbur gh, Pit tsbur gh, P enns ylv ania, Unit ed St at es  \n5  Departmen t of P edia t rics, Childr en's  Hospit al of  Pit tsbur gh of the Uni v er si ty of Pittsbur gh \nMedic al Ce n t e r , Univ er s ity o f Pit tsbu rgh School of Medici ne, Pit tsbur gh, P enns ylv ania, Unit ed \nState s  \n6  Departmen t of Genet ics, Univ ersity of North Car ol ina, Chapel Hill, North Car olina, Unit ed \nState s  \n#Addr ess c o rr espond ence: mchao@hsph.har v ar d.edu   \n \nRunning title: M. tuber cu losis i n tr a-hos t sequen cing \n \n \n \n \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2026. ; https://doi.org/10.64898/2026.04.03.714442doi: bioRxiv preprint \n\n 2\nAbstract \nWhole g enome sequencing o f Mycobact erium tubercul osis  (M tb) pop ula tions  from clini c a l  \nsamples has incr easingly ide n t ified genes under going selection wi thin an d betw een hos ts tha t  \ndriv e d iff eren t ial in f ect ion and t r ea t men t outc omes. How ev er , the i n tr a hos t Mtb mut ational  \nlandsc ape—especia lly in the c o n text of human i mmunode ficiency vi rus c oinf e ction and  \nan tir et r ovir al ther apy (AR T)—r emain s less clea r , as do the po t ential impacts of such mut ations  \non Mtb inf ec tion dynamics. Her e, w e perf ormed whole g enome sequencin g of Mtb popula t ions  \nisolat ed from appr o xima t ely 480 inf ec t ed tis sues acr oss 20 non-human prima t es, includ ing \nanimals c o-inf ected with simian i m munode fici ency vir us (SIV) with or without vi r ologic a l  \nsuppr ession by AR T . W e iden tified 11 6  mut a tions tha t emer g ed during inf e ction, includ ing those  \ntha t ar e  ov er r epr es ent ed within indi vidual tissu es and a subset that ar e shar ed acr oss tissue s  \nduring Mtb dissemina tion. W e furth er find di ff er e n tia l mut a tion tr ajecto ries acr oss tr eatmen t  \ngr oups, with higher mut a tion r at e  and bact er ial ou tgr owth in SIV-inf ect ed  hos ts and  increase d  \npr ev alence  of o xi da ti v e damag e-asso cia ted mut a t ions in c oin f ec t ed anima ls on A R T . Finall y , w e  \ndemons t rate  a  c ommon p atte r n  o f  m u tat i o n  i n  M t b  l i p i d  m e t abolism an d polyk etid e s yn thas e  \ng enes and iden ti fy a subset of NHP - deriv ed mut a t ions that ha v e also in dependen t ly arisen i n  \nhuman clinic al isol a t e s. T og ether , our popula ti on-based sequencing unc ov e r s Mtb div er si fi c a ti on  \nduring early inf ec tion, c aptu r es discret e bact e rial dissemination ev e n ts a nd inf er s di f f e r entia l  \nimmune pr essur es f aced by Mtb i n th e set ting of SIV-Mtb c oin f ect ion and AR T suppr ession.   \n \nImportance \nT uber culosis r emains a leading c ause  of d ea th  w orl dwide, espec ially  in  p eople living with  HIV . \nHow HIV inf ection and a n ti r etr ovi r al ther apy impacts Mycobac t eriu m tuberculosis  (Mtb)  \nin tr ahost ev o luti on r emains un clea r . Using whole g enome sequ encin g fr om hundr eds o f  \ninf ec t ed tissu es fr om non-human pr ima t es, w e find th a t simian immun ode fi ciency v irus c o-\ninf ec t ed ho s ts and  those  r ece iving a n tir e tr ovir al ther apy e x act d iff e r en t  i m mune pr essur es  on  \nMtb leading t o diff er e n t mut a t ion r a t es and types of DNA damag e tha t ar e incurr ed. Mt b  \nmut a tions w e r e en riched in g enes i n v olv ed  in lipid met abol isms and so me of these a r e also  \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2026. ; https://doi.org/10.64898/2026.04.03.714442doi: bioRxiv preprint \n\n 3\nseen in human TB str ains. This w or k  highligh ts th e r ol e of  immune pr ess ur e t o al t er bact e rial  \npa thw a y s tha t  ma y enable Mtb adapta tion t o th e hos t.  \n \nBackgr ound \nT uber culosis (TB) r emains the t op s i ngle inf ect ious c ause of  mort ali ty worldwide, with  \npeople l iving with HIV (PLHIV) bearing an outsiz ed c o s t o f the ongoing epidemic 1 . Studies ha ve  \nshown signific an t impr ov emen t in pr ev enting TB mort ality in this po pula tion t hr ough the  \nwidespr ead use  of  an t ir etr ovir a l therapy (AR T)—especially  when st arted early—but ind ividual s  \non long-t erm AR T s till  r et ain  an elev at ed r isk o f dev e loping of ac tive TB dis ease despi t e vi r ologi c  \nc ontr ol and st able CD4 c ounts\n2–5 . Though s tudies ha v e ide n ti fied d y sfu nctional adapti v e and  \ninna t e  immune r esponses assoc iated with HIV /Mtb c oinf e ction  (e. g.,  r eview ed in  6 ) ,  t he \nmechanisms acc oun ting f or  inc r eas ed TB disease p r ogr ession in PLHIV r emain incomplet ely  \nundersto o d .  T o  model early Myc obact erium tuberculosis  (M tb) inf ec tion out c omes r el a ted to  \nHIV c oinf ect ion, a r ece n t study in  non-human prima t es (NHP) es t abl ished chr oni c simian  \nimmunode ficiency  virus  (SIV) inf e ct ion fi r s t—includ ing a subset o f an imals with virologic  \nsuppr ession  using AR T—be f or e cha ll enging animals with a g eneti c ally  barc oded isog enic  libr a ry  \nof Mtb\n7 . This w o rk f ound  that SIV-positive animals developed  mor e sev e r e  TB disease, in cluding  \nhigher bact erial burden and gr ea t er i nflamma tion; and tha t early AR T during SIV inf ection c oul d  \nsignific a n tly r educe th ese eff ects. Ho w ev er , PET -CT i ma ging ,  pa thology scoring and sequencing \nof bar c oded Mtb str ains also showed tha t AR T did not r es t o r e Mtb di ssemina tion t o le v els  \nobser v ed in non-SIV inf ect ed c o n tr o ls —especially to e xtr apulmonary si t es —su g g es ting tha t AR T \nanimals e xperienced  con t inuing def e cts in  cer t ain aspe cts o f ear ly M tb con tr ol\n7 . How ev e r , th e  \nimpact of SIV and AR T on th e bac t eri al intr ahos t e v olu tiona ry lands c ape i n this s tudy r emained  \nunclear .  \n  Whole g enome sequencing of Mtb c lini c al s tr a ins is in cr easingly iden t if yi ng g enes and \npa thw a y s under going selection in  hos ts that dri v e di f f e r ential in f e ction and trea tment  \nout c omes\n8–10 . These mut a tional signa tur es ha v e been pow erfu l t oo ls f or s t udying long t erm c o-\nev oluti on of Mtb wi th human hos ts 10  and identif ica tion of nov e l mut a ti o ns tha t c ontribu t e t o  \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2026. ; https://doi.org/10.64898/2026.04.03.714442doi: bioRxiv preprint \n\n 4\nthe emer g ence o f antibio tic r esi s t anc e 11,12 , man y of which  ha v e be en tr ans la t ed  int o d iagnos tic s  \nf or det ec tion  of multi- and e xtremely- drug r esis t a n t inf ec tions. Furt her , se quencing of i n tr aho st  \nMtb popula t ions c an ide n ti fy unfix ed an tibio tic-r esi s t anc e mut a tions tha t emer g e during  \ntr ea tment\n13–15  and c an ser v e  as a r e cor d of ho s t immune pr essure acting on Mtb 16,17 . How ev er ,  \nsequencing is oft en limi t ed t o anat omic al ly accessib le sit e s such the airw a y , with s tudie s  \nsug g es ting ther e is gr eat er  Mtb  di v ersity p r ese n t in th e human lung and extr apulmonary t issues  \nc ompar ed t o sputum\n18,19 .  \nIn this s tudy , w e  h ypothe siz ed tha t  s equencing Mtb g enomes fr om ana to mic ally d iv er se  \ntissues in NHP s w ould c ompr ehensi vely ide n ti fy i n tr ahost mut a t ions, uncov er distinc t immune \npr essur es  in  the  cont e xt  of  SIV c o inf ect ion wi th and  withou t AR T and  a lso highligh t  bio logic  \npr ocesses that ma y be i mport an t f or  Mtb adapt a tion t o the host milieu. Her e, w e perf ormed \nwhole g enome sequencing of Mtb p opula ti ons isol a ted fr om ov er 480 inf ect ed t issues f r om 20 \nnon-human pri ma t es, finding 116 M tb mut a tions that ar ose within the h os t. These mut a tions  \nshow tha t the Mtb mut a t ion r a te i s signific a n tly incr eased in SIV-inf ect ed NHP s, while the \npr oporti on of o xid a ti v e damag e-associa ted mut a ti ons is incr eas ed in anim als on AR T c ompar ed  \nt o n on-SIV c on tr o ls. W e fur ther  sho w tha t  Mtb lipid  met abolism and b i os yn theti c g enes  ar e  \nenriched f or int erho s t mut a t ions, which w e pr opose r eflec t oppor tuniti es  f or Mtb adaption t o  \nhos t nutr ient condit ions.  \n \nResults \nWhole genome sequencing defines t h e in tra hos t M tb mutati onal lan dscape \nIn this w ork, w e cur at ed a set o f Mt b g enomic DNA samples isola t ed fr o m a prior non-\nhuman prima t e (NHP) s tudy\n7 , which i ncluded naï v e or chr oni c ally  SIV-inf ect ed animals (with or  \nwithout AR T) tha t w er e cha lleng ed wi th a bar c oded libr a ry of Mtb. In prior w ork, the viable Mtb  \npopula ti ons from all in f ec t ed t issues (including lung , thor acic  lymph node  and e xtr apulmonar y  \nsit es)  w er e  plat ed for g enomic DNA  e xtr acti on and ampli c on-based seq uencing w as used t o  \nde fine th e bar c oded Mtb str ains that w er e inf ec ting each tissue\n7 . In this s tudy , w e perf ormed  \nwhole g enome sequencing of this DN A fr om a subset of 482 in tr aho s t Mtb  popula t ions is olat e d  \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2026. ; https://doi.org/10.64898/2026.04.03.714442doi: bioRxiv preprint \n\n 5\nfr om 20 NH P s (Supplemen t ary T able 1), including 7 tha t w e r e sole ly inf ec t ed with Mtb (TB); 4 \nchr onic a lly in f ec t ed with SIV prior to Mtb challeng e (SIV+TB); and 9 SIV-inf ect ed but v ir ally  \nsuppr essed with A R T (SIV+AR T+TB) only 3 da y s aft e r inf ecti on (Figur e 1A). \nMtb sequen ces wer e  mapped t o  a r e cen tl y g ener at ed  Mtb  Er dman r ef ere nce g enome\n20 , \nwhich w as deri v ed f r om a r epr es en ta tive colon y f r om the l ibr ary us ed to inf ec t thes e NHP s.  \nUsing the haplotype mut a t ion c a lling  t ool Mu t ect2, w e d e fined o v er 100,000 single nucleotide  \npolymorphisms (SNP s) and small ins ertion and  delet ions ( indels)  acr oss all samples, with the  \nv as t majority a t low f r equency  and li k ely r ep r ese n t sequ encing artif ac ts ( Supplemen t ary Figure  \n1A). Subsequen t fil t e ring of low c over ag e sit es  and c onside ring only  v aria n t s a t >10% pr ev alenc e  \nwithin tissues (see Methods) y ielde d 143 unique, high -c onfidence c and ida t e mut ations th at  \nw er e not pr ese n t in our ref er en ce g enome (Supplemen t ary T able 2). As  some v arian ts c oul d  \nha v e arisen during libr ary c ons t ruct io n in vitr o  (e. g., see ‘B C1’  e x a mple in F igur e 1B), w e further  \nc ompar ed the tissu es that c o n t ain ed a g enomic v arian t ag ains t pr evious ly g ener a t ed barc oding  \nda t a fr om the same samples t o down select 116 mut a tions that w er e lik el y in viv o-deri v ed (se e  \nMethods), as they ar e only f ound in a subset of sit es inf ec t ed by the same bar c oded Mtb s tr a in  \n(e. g., BC2 , BC3 and BC4 in Figur e 1B ). This l e ft  27 mut a ti ons th a t we c an not rul e ou t ar e  pr e-\ne xis ting in th e in f ect ing libr a ry (Supplemen t ary  T able 2), s o w e  el ected t o  e x clude these  fr om  \ndowns tr eam analy ses.  \nOf the in tr aho s t mut a tion s, 57% w er e pr edicted t o chang e pr o t ein c od ing (53 missense  \nmut a tions, 11 fr ameshift indels and  2 in-fr ame in t ernal delet ions), 31 w er e pr ed ict e d t o b e  \ns ynon ymous and 19 mut a tions  w ere l oc at ed wi thin int er g eni c regions (Figur e 1B).  Cumula tively ,  \nin tr ahost mut a t ions o ccurred acr o ss  the M tb g enome, but c ert a in g eno mic r egions harbo r ed  \nmor e mut a tions  than  w ould  be  e x pect ed  giv en  c omplet e ly r andom s ampling (Figur e 1C). \nSpecifi c all y , w e obser v ed loc al mut at ional ma xima betw een nucleotides 1 –1.2 Mb, 1.8–1.9  Mb  \nand 3.5–3.7  Mb, with the highes t m utat i onal densit y f ound betw een 1.8–1.9 Mb,  a r egion tha t  \nincludes se v er al poly k etide s y n thase g enes ( pk s7 , pk s 8  and p k s11 ). \n \n \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2026. ; https://doi.org/10.64898/2026.04.03.714442doi: bioRxiv preprint \n\n 6\n \nSIV-inf ect ed and an t iretroviral trea t ed NHPs harbor Mtb with divergen t m ut at iona l traject or ies \nW e ne xt c alculat ed r el a ti v e Mtb muta tion r at es ac r oss t r eatmen t gr oups using the t o t a l  \nnumber of unique mut a tions identif ied in each animal (see Methods ). Giv en the e x act Mt b  \ng ener a tion time in NHP s is unknow n (though it is e xpect ed t o be signif ic antly slow er than in  \na x enic  cultu r e\n16 )  and th a t  mut a tion  r at es ma y be  furt her a f f e ct ed  by SIV c oi nf ection  and AR T , w e  \nelected t o use a fi x ed g ener a t ion time f or our c alcu la t ions in or de r t o d e fi ne a r ela ti v e mut a t io n  \nrate  p e r  u n i t  t i m e  a c r oss c ond itions  ra ther than deri v e absolu t e r at es  per g ener a tion. In Figur e  \n2A, w e f ound tha t SIV-c oinf ect ed NHP s (but not those on AR T) had  signific antly highe r  \nmut a tional  r a tes c ompar ed t o  Mtb  o nly animals. Ther e  ar e  sev er a l poss ibl e dri v er s  of  inc r eased  \nr ela t iv e muta tion ra t e.  Firs t, in SIV-inf ect ed  animals, ther e w as a s t atisti c al t r end (p=0.09) o f  \nincr eased  Mtb r epli c ation o v er the cour se of  inf e ction  (Figur e 2B), as ev a lua t ed  by quanti fying  \nchr omosomal equiv alen ts (CE Q )—t he number of Mtb g enomes in tissue homog ena tes  \nr epr ese n ting the t o t al li v e and dead bact eria bur d en ov er the c our se of in f ect ion. Further , there  \nis signific a n tly mor e li v e Mtb (CFU) in SIV  c oinf ec t ed animals a t necr ops y (Figur e 2C),  su g g es ting \ndecr eased c apaci ty t o c lear Mtb, w hich ma y also c o n tr ibute to gr eat er  r et e n tion  of mut ant  \ns tr ains in these immunoc ompr omised hos ts.  \nSec ond, inc r eased mut ation  rate s  co u l d  a l s o  b e  d r i ve n  b y  e l e v a ted DNA d amag e due t o  \nhos t immune a t t ack, with prior w ork sug g es ting tha t the majority of in tr ah os t Mtb mut a tions in  \nla t e n tl y in f ec t ed NHP s and in  sputu m of acti v e TB p a tie n ts  ar e muta tion s associat ed  o xida t ive  \ndamag e\n16,17 —namely , C>T /G> A tr an sitions and A>G/C>A tr ans v er sions  r e fle cting cy t osine  \ndeamina tion 21  and 8-o x o-guanine f orma tion 22 , r espect iv e ly . If our obser v e d o xida ti v e mut a t ions  \nar e associat ed with hos t immune atta c k ,  we  wo u l d  h ypothesiz e tha t t issues  mark ed by  o xida tiv e  \nmut a tions also e x er t gr ea t e r immun e pr essur e on Mtb, leading t o bet te r bact eria l c ontr ol. T o  \nt es t  th is, w e fir st down-selec t ed 54  ‘ origin’ tissues  (Figur e 3, bo x ed  no des) wher e  g enomic  \nmut a tions lik e ly ar o se (s ee Method s), as mut a t ions f ound  in  some tissu es could ha v e  hi t chhik e d  \nthr ough Mtb dissemina tion. W e find tha t origin si t es with o xidativ e mut ati ons ha v e signifi c an tl y  \nlow er Mtb bur dens c ompar ed t o si t e s associa ted with non-o xida tive mut a tions, c onsi s ten t wit h  \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2026. ; https://doi.org/10.64898/2026.04.03.714442doi: bioRxiv preprint \n\n 7\nincr eased  immune pr essur e (Figur e 2 D). With this  lin k t o ho s t damag e, w e ne xt c ompar ed the  \npr ev alence of o xidati v e mut a tions a cr oss tr e a tmen t condit ions, finding t ha t SIV+AR T+TB (but \nnot SIV+TB) animals tr ended (p = 0. 09) t ow ar ds mor e o xida tiv e mut a t io ns c ompar ed t o Mtb  \nonly animals (Figur e 2E). This si gnal w as driv en by a majority of AR T anim als harboring elev at ed  \nfr equenc ies of C>T /G>A and C>A/ G>T mut a tions (Figur e 2F). Further , ther e is signif ic a n t  \nc orrela t ion betw e en the p r ev alence  of o xida t iv e damag e-associa ted mut ations with in vi v o Mtb  \nmut a tion ra t es  in  SIV+AR T+TB anima ls, but no t f o r th e TB on ly o r SIV+TB animals (Figur e 2G) ,  \nsug g es ting tha t Mtb is e xper iencing  incr eased  o xida t iv e damag e in a s ubset of SIV+AR T+TB \nanimals. This incr eased o xida tive damag e ma y be r ec or ded as earlier ap pear ance of mut a tion s  \na t origin s it es; and c ons ist en t  with th is, w e find tha t lung gr anulomas with  in tr ahost mut a t ion s  \nw er e det ec t ed signifi c antly ear lier b y PE T-C T in the SIV+AR T+TB ani mals c ompar ed t o the TB \nonly gr oup (Supplemen t ary Figur e 1B ). T og ether , the da t a sug g es t tha t de s pit e similar bac t er ial  \nout c omes (Figur e 2A-C), AR T vir al suppr ession in some hos ts d oes n ot r est o r e the h os t  \nen vir onmen t t o tha t  of n on-SIV a nimals, ins t ead f av oring an immu ne milieu assoc iat e d  \nincr eased o xid a ti v e immune pr essur e . \n \nIn traho s t mutatio ns unc over dissemin ation even ts wi thin  barc o ded Mtb dis semination  network s  \nB y int egr a ting our  whole g enome s equencing with p r evious  barc oding da t a, w e  w ere  \nable t o link  79 i n tr ahost mut a t ions  with the  Mtb  barc oded  s tr ain  that h arbor s th ese v a riants  \n(see Methods). Of these, 67 mut a tio ns w er e f ound in a single tissue (c o nsidering all sampled \nlung , lymph  node and e xtr apulmon ary sites) while 13 v arian ts w e r e s har ed acr oss tissu es  \n(Figur e 3), sug g es ting they lik e ly aros e once  and w e r e spr ead  thr ough subs equen t  dissemina t ion  \nev en ts. As only some tissues c o lonized by the same bar c oded Mtb s tr ai n ma y harbor a giv en \nin tr ahost mut ation, w e  used  in v iv o  v ariants as  addit ional ‘barc odes’ wi th which t o  inf er th e  \nor der o f dis semina tion  ev e n ts in eac h Mtb dis semina tion  netw or k. As an  annot at ed e x ample, \nw e identif ied 4 in viv o  v aria n ts o ccu rring in the  same Mtb barc oded strain (Q25_C G TCAG T C ) \nbackgr ound in NHP  232 18 (Fi gur e 3,  bot t om righ t). B y mapping the in tr ahos t v ariants pr ese n t  \nand absen t in these tissues, w e  ar e able t o  inf e r a t  least 3 distinc t disse mina tion ev e n ts: f ir s t ,  \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2026. ; https://doi.org/10.64898/2026.04.03.714442doi: bioRxiv preprint \n\n 8\ntw o tissues r ep r ese n t dissemina t ion of the wildtype par e n t str ain; sec ond,  a T C CC AC> T deletion \na t posi tion 182022 (r ed r ing) emer g ed and w as then d issemina ted to 1 2 tissues; and th ir d, a  \nnew 277532_ C>T v arian t (bla ck r i ng) ar ose on t op of the T CCC AC> T deletion and  both  \nmut a tions w e r e then diss emina t ed to 7 other  si t es. Finall y , tw o v a riants —one (2554553_ C>T ,  \nor ang e ring) th a t appea r ed in an oth erwise wi ldtype  backgr ound and  one  (2806057_G>T , blue \nring) tha t appear ed on t op of the T CCC AC> T deletion—emer g ed independen tly in s ingle tissues  \nlik ely af t er these pr ior d issemina ti on ev en ts.  \nSimilar br anched netw ork s wer e in f e r able acr oss othe r animals (e. g., NH P  22517 (gr ey),  \nNHP 3351 9 (gr een), NHP 24018 (gr een) and NHP 22117 (y ellow)), suppo rting a model where  \nMtb dissemina t ion is s truc tur ed as p ar allel subnetw o rk s of diss emina tion  r a ther than  a single  \nr adia tion  ev ent. Furthe r , w e find 7 i ns t ances  (Figur e 3, arr ow s) whe r e we c an inf er  directio nal  \ndissemina tion (i.e., wher e a mut ation  appear s unfix ed a t an or igin si t e and  then hi t chhik e s as a  \nfix ed mut ation  t o  downs tream tissue s). Of these, 5 d issemina tio n ev e n ts  i n v olv e  Mtb  spr eading  \nfr om and t o lung si t es (Figur e 3, r e d arr ow s), while  2 c ases in v o lv e  disse mina tion t o  lung si t e s  \nfr om a thor acic lymph node (Figur e 3 , blue arr ow s), sug g es ting tha t Mtb can in princip le r eturn  \nt o the lung aft er d issemina tio n t o oth er ana tomic al si t es.  \n \nMtb mut a tions in NHPs and huma ns are enriched in l ipi d met abol ism and bios yn t hetic processes  \nW e ne xt c orr elat ed th e NHP in tr ahos t mut a tions ag ains t two tr e a tmen t-naïv e human  \ns tudies  that als o ide n ti fied  intr ahos t  Mtb mut ations: L iu et  al.\n17  (321 mut a ti ons f ound  in 25 7  \ng enes in  sputum fr om non-HIV inf ec t ed p a tie n ts  with a cti v e TB);  and Lie berman et al. 18  ( 5 18 \nin tr ahost mut a t ions in  403 g enes fr o m an aut ops y s tudy o f HIV-c oinf ec t e d individua ls). In all  3  \nda t asets, in tr aho s t mut a tions w e r e mos tly f ound once in diff er e n t g en es, which pr ecluded  \nlooking at the r atio  of  non-s ynon ymous t o  s ynon ymous mut a t ions on a g e ne basis; and  only  th e  \nir on tr anspor t e r , irtA, w as mut a t ed i n all thr e e da t ase ts (Figur e 4A). Despit e la ck of on e-t o-one  \ng ene ov erlap, ther e w as a signifi c a n t enrichment of intr ahos t mut a t io ns in My c obr osw e r\n23 -\npr edic t ed l ipid met abol ism g enes ( Figur e 4B) in the NHP da t aset  (15% of mut a ti ons) an d  \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2026. ; https://doi.org/10.64898/2026.04.03.714442doi: bioRxiv preprint \n\n 9\nLieberman et al. (9% of mut a t ions) d a t asets, with a st atisti c al t r end (p=0. 05) f or Liu et al. (10%  \nof mut a tion s).  \nFurther , w e find tha t in tr a-NHP mut a t ed g enes had signif ic a n tly mor e pr edic t e d  \nin t er ac tions than e xpected by chanc e (p = 0.005) using the STRING da t abase 24  and th a t the  \nput a ti v e g en e int er ac tions c lust ered by My coBr ow ser  func tional ca t egories, with lipi d  \nmet abolism g enes (especial ly pk s  g e nes) centr ally l oca t ed (Supplemen t a r y Figur e 1C). In t erPr o \nanaly sis identif ied sever al pr o t ein  domains tha t w er e enrich ed in  the NHP da t ase t  \n(Supplemen t ary  Figur e 2), which  inc l uded lip id met abolism domains ( e. g., acyltr ansf er ase (FDR<  \n1x 10\n-5 )) and po lyk etid e s ynthase-associat ed domains (FDR < 3 x 10 -3 ). The g enes associat e d  \nwith these t e rms w er e highly ov erlap ping and included pk s7, pk s8, pk s12, mbtD , pk s1 and pk s2 , \nwhich ar e inv olved in the bios y n th esis of d iv er s e cel l w all lipid s tru ct ur es and se c ondar y  \nmet abolit es. As in NHP s,  w e did obse r v e enrichmen t of po lyk etid e domains t erms in the human \nda t asets as  w ell  (Supplemen t a ry Figu r e 3), sug g es ting these  bios ynthetic  g enes as a whole  ar e  \nc ommon t ar g ets of mut a t ion in v iv o.  \n \nIn traho s t mutatio ns are enriched wit h in gran ulomas a nd are associ at ed wi t h dissemina tion  \nW e ne xt assessed whether in vivo  deriv ed mut ations c an conf er i n trahos t fi tness  \nbene fi ts dur ing inf e ction, f o cusing on mut a tion s th a t chang e  pr o t ein  sequence (missens e, \nindels), which ar e mor e lik e ly t o  impact g ene funct ion and Mtb fitness c ompar ed t o  \ns ynon ymous mut a tions. T o do this, w e c onsid er ed M tb fi tness acr os s t w o ana t omic a l sc a les:  \neither lo c al ly within an ind ividual t issue or s y st emic a lly through dissemina tion t o multip l e  \ntissues. Fir st, t o  assess ind ividual  tiss ue fitn ess, w e c ompar ed the proport i on of wild type a llel es  \nt o non-s ynon ymous and s ynon ymous mut a tions, f inding tha t the pr ev alence o f non-\ns ynon ymous mut a tions w er e signif ican tly higher than tha t o f s ynon ymous alleles (Figur e 4C),  \nsug g es ting tha t a subset of these mut a ti ons ma y be loc a lly ben e f icial f o r Mtb. Sec ond, t o  s tud y  \ndissemina tion, w e c ompar ed th e fr ac tion of tissue s inf e ct ed by a bar c o de d Mtb s tr ain tha t  also  \nc ont ained a non-s ynon ymous or s ynon ymous g enomic v arian t, with the h ypothesis th a t  \ndissemina tion-pr omoting mut a tions will be enrich ed within dis semina t e d netw ork s. Her e, we  \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2026. ; https://doi.org/10.64898/2026.04.03.714442doi: bioRxiv preprint \n\n 10\ndid not obser v e a st atistic a l di ff er en ce betw een s yn on ymous and non-s ynon ymous mut a tions,  \nthough ther e is a wide  r ang e of dissemina tion  in bo th gr oups (Figur e 4D). Int er e s tingly ,  \nmut a tions th a t wer e asso ciat ed wi th  highly disseminat ed Mtb  s tr ains  ar e  dis tin ct fr om those  \ntha t ar e  enrich ed in ind ividual tissue s, which ma y su g g es t tha t addi tional  f act or s beyond lo c a l  \nfitness c o n tribu t e t o d issemina tion.  \n \nNHP derived Mtb mut atio ns also arise  in huma n clinical s trai ns  \nW e finally ass essed whethe r i n tr ahost NHP mut a tions  c an  be found acros s human Mtb  \nclini c al str ains. T o do th is, w e lev er aged a r ece n t ph ylog ene tic anal y sis tha t  mapped ov er 55,000 \nMtb cl ini c al g enomes t o  an ance s tral g enome\n25  and l ook ed  f or identic a l mut a ti ons as  thos e  \nobser v ed in ou r NHP da t aset. W e find 22 mut a tions (12 non-s ynon ymous, 9 s ynon ymous and 3 \nin t e r g enic ) th a t w er e  also  pr ese n t  in  a t  least one  human Mtb clin ic al  str ain (T able 1). M os t  o f  \nthese mut a tions appear in a single clinic al is olat e, but some mut a tions (e. g.,  pk s7  fr ameshift a t  \nc odon 1174 and non-s ynon ymous mut a tions  in r v0785, r v0987 and r v3594) ar ose \nindependently acr os s diff e r en t inf ec tions and w er e suc cessfully tr ansmit t ed betw e en peopl e  \n(e. g., a missense mut a tion in rnj, a gene whose l oss has  been ass ociat ed  with drug toler ance\n26 , \nar ose once but w as subsequen t ly s har ed acr oss 10 individuals ). W e al so find an int er g eni c  \nmut a tion 25 nucleo tides upstr eam of the E SX-1 r egula t or whiB6,  whic h is an ar ea of  high  \nmut a tional d iv er s ity ac r oss c lini c al str ains 25 . Finally , w e  show tha t  intr a-NHP mut a tions tha t  \nma t ch mut a tions in clin ic a l s tr ains  are signifi c an tl y enri ched f or C>T /G>A mut a tions (Figur e 4E), \nfurther  support ing a model in which i m mune o xida tive damag e is dri v er o f Mtb div e r sif ica tion  in  \nthe hos t.   \n \nDiscussion  \nDe fin ing the in tr ahost Mtb mut a tio nal r eper t oire within trea tmen t-nai v e hos ts ma y  \ninf orm the ev olu tiona ry pr e ssur es s haping Mtb adapt a t ion t o the  host and also r e c o r d how \nimmune en vir onmen ts ma y dif f er in individuals who ha ve othe r c omorbi dities such as HIV c o-\n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2026. ; https://doi.org/10.64898/2026.04.03.714442doi: bioRxiv preprint \n\n 11\ninf ect ion. Her e, sequencing of Mtb fr om inf ect ed NHP hos ts finds th a t  SIV c oinf ection w a s  \nassociat ed with  signifi c antly incr ease d Mtb mut a t ional d iv er s ity c ompar ed  t o M tb onl y animals,  \nlik ely du e t o tw o non-mutually e x clus iv e mechanisms associat ed with immunosuppr ession: fir st, \ngr ea t er over all Mtb r epli c ation c ap acity in SIV-inf ect ed h os ts th a t c r e a t e oppo rtuni ties f or  \nmut a tions and s ec ond, in cr eased  r e t ention o f dele t eri ous spont aneous  mut a tions tha t ma y  \notherwise be c lear ed by the host. Con v er sel y , incr eased o xidative immune  pr essur e is not l ik ely  \ndriving mut a tion, as ch r onic SIV coi nf ection  is known t o impair immune c ontr ol  in th is NHP \nmodel\n28,29  and  w e obser v e f ew er o xi da tive damag e associat ed mut ations  in SIV animals r elative  \nt o Mtb on ly c ontr ols, a f inding tha t h as also been repor t ed in HIV-positive pa tie n ts 17,18 . \nFurther , w e also  ev aluat ed th e impact  of AR T on Mtb e v olut ion with in SIV-inf ect ed  hos ts.  \nPrior bar coding analy ses  sug g es t ed th a t SIV+AR T+TB animals r emained mor e permissiv e  f or Mt b  \ndissemina tion t o e xtr apulmonary si tes, sugg es ting a lev el of c on t inuing immune dy s function  \ndespit e  vir o logic con t r ol\n7 .  Her e, w e f i nd Mtb from a majority of A R T animals had an inc r eased  \npr oporti on of  o xida t iv e damag e asso cia ted mut a t ions relativ e  to TB only  animals, and tha t t his  \nsignal w as posi tively c or r el a t ed  with higher ov er all  mut a t ional ra t es.  W e speculat e tha t  a subse t  \nof SIV+AR T+TB ani mals c ould be moun ting a dy sr egulat ed heigh tened infla m ma t ory response t o  \nMtb inf ecti on. Pr evious  w or k has  shown that a lv eo lar macr ophag e s fr om HIV-neg a tiv e  \nindividuals on pre-e xposur e AR T pr oph yla xis moun t an alt ered immune r esponse t o subsequent  \nMtb e xposur e\n30  and as w e did not ha v e samples fr om an  SIV -n egat i ve  A RT  o n l y  g r oup,  w e \nc annot d iscern if a n ti r etr ovir al e xpos ur e alone or a c ombina ti on of SIV a nd AR T is r espons ible  \nf or this sh ift in Mtb o xidati v e damag e.  \nOur findings also c o ntex tualiz e bac terial ev o luti on and dissemina t ion wi thin the ho s t.  \nFir s t, as gr anulomas ar e  typ ic a lly  c o l oniz ed by a  single  bacterial  f ounder  and tha t  di ff er e n t  Mtb  \nisog enic  s tr ains  ha v e  het e r og eneous  dissemina t ion c apaci ty e v en wi thin  the same hos t\n31 , we  \nw er e ab le t o  in c orpor at e  intr ahost mut a t ions as  se c onda ry ‘barc od es’ t o  fur ther  s tud y  \notherwise  cl onally  barc oded s tr ains  and inf er  c omple x dissemination  pat t erns  wher e  Mtb  is  \nlik ely  spr ead ing in par all el subne twork s  s tr ains  originating fr om di ff er e n t t issues. This  is in  \nc ontr as t t o a single ev e n t di ssemina ti on model, wher e all futu r e dissemin a t ed si t es a r e seede d  \nfr om a single early lesion. W e do n ot e that a majority of the dissemination netw or k s harbor  \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2026. ; https://doi.org/10.64898/2026.04.03.714442doi: bioRxiv preprint \n\n 12\nmut a tions a t a single tissue, which c ould be c ons ist ent with a single ev en t model, but giv en \nther e ar e f ew in tr ahost mut a tion s det ect ed i n g ener al, w e c anno t rule out par alle l  \ndissemina tion in these  c ases  as w ell.  \n Sec ond, our g enomic mapping  f ound tha t in tr aho s t mut a t ions w er e enr iched in Mtb  \nlipid c at abol ism and bios y n thet ic g e nes (Figur e 5), whi ch ma y re fle ct th e import ance  of  bet a-\no xida tion o f host lip ids f or M tb nut rien t  acquisi tion du ring inf ec tion\n32 . While bet a-o xida tio n  \nsupports c arbon acqu isit ion, it a lso in cr eases reducti v e s tr es s by g ener a t in g NADH , which c an be  \nc ount er ac t ed thr ough sev er al  mechanisms. Fir s t, NADH is c onsumed thr o ugh r espir a t ion, an d  \nw e obser v e mut a tions in put ative nitrite ef flux pr o t eins narK3  and narU ,  which m a y f acilit ate  \nthe use of ni trate  a s  a  t erminal elec tron accept or in the cont e xt of micr oa er ophili c c ond itions\n33 . \nSec ond, NADPH equiv alen ts  ar e util ized f or b ios yn th esis of c ell  en v e lope componen ts and lipid  \ns t or ag e molecules by la r g e modular polyk etide s y n thase (p k s). The pk s en zymes ar e r esponsible  \nf or pr oducing k ey cel l w all c o mponen ts (e. g., dim y c ocer osa t e s, m y c ocer osic ac ids,  \nlipoar abinomannan, mannos yl-ß-1  phosphom y c ok et ides, sulf o lipid  and the s ider opho r e  \nm y c obactin)  using l ipid pr ecur s ors\n34 ;  and recent w o rk has  show n that p h thio cer ol  \ndim y c ocer osat e  pr oduct ion is r eq uir ed f o r optimal Mtb  gr owth o n pr opionat e, whic h  \npr esumably acts  t o  r e-balance  the r e ductiv e  s t r ess  fr om odd-chain l ipid d e gr ada tion 35 .  Her e, w e  \nfind signi fic a n t  enrichment o f i n tr ah os t mut ations—both in NHP and hu man da t asets—within  \npk s  g enes, which ma y be bene ficia l to Mtb in the human popula tion; f or ex ample, w e iden tifie d  \na fr ameshift in pk s7  tha t also appe ar s t o be independently mut at ed 7 times acr oss human \nclini c al str ains and has b een tr ansmi t t ed bet w een 24 indiv iduals (T able 1 ). In t e r es t ingly , while  \npk s7  kn ock out  str ains hav e be en re port ed  to be  a tt enu a t ed  in  aer oso l inf ect ion in C57BL/6 \nmice\n36 , ou r dat a would  sug g es t thi s en z yme c an  be  dispensabl e in h uman inf ecti on an d  \ntr ansmission, which w ould  be c ons i s t e n t with  r e cen t  w or k showing p ks7  insert ion mut an t s \nr et aining the abili ty t o i n tr a v enou sly i nf ect a g enetic a lly diver se panel of mice 37 . T og ether , these  \nmut a tional s igna tur es h ighligh t th e met abolic fle x ibili ty o f Mtb in balanc ing ener gy and r edo x  \nneeds under ho s t-imposed s tr e sses.  \n \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2026. ; https://doi.org/10.64898/2026.04.03.714442doi: bioRxiv preprint \n\n 13\nLimit at ions t o t he s tudy  \nSev er al limit a t ions t o this s tudy r e main. Fir s t, our da t aset li k ely unde res tima t e s true  \nin tr ahost mut a tiona l div e r sit y , as la te-emer ging  mut a tions (which will be  a t low er pr ev a lenc e  \nc ompar ed t o earl y arising mut a tions)  ar e unlik ely t o be sampled f or the sequencing depth used  \nin this study . Further , some v ariants  will also ha v e b een fi lt e r ed out by  our 10% pr ev alence  \nthr eshold, which w as imposed to reduce f alse posi ti v e c al ling of low-fr equency sequen cing  \nerr or s. Final ly , highly r epeti tive g enes  of th e PE/PP E f amilies ar e di f f icult  to uniquely  map using  \nshort r ead sequencing t echnol ogy , so w e ha v e mask ed these r egions in ou r r ef erence g enome\n20  \nand subsequen t anal y ses.  \nSec ond, our ana ly ses  ar e  r e s tri ct ed  to an ear ly in f ect ion time poi n t when  the bac t er ial  \npopula ti on siz e remains siz eable but  mut a tional d iv er s ity ma y be limit ed  c ompar ed t o human  \ninf ect ion with  Mtb, wher e  inf e ctions  of mon ths  t o  y ear s  enable  Mtb t o accumula t e in tr aho st  \nmut a tions. On the opposi t e end, human longitudinal s tudies ha v e s hown tha t an tibio ti c  \ntr ea tment (and  pr esumably immune a tt ack leading to l a t e n t inf ec tion)  will shr ink the in  vi v o  \nairw a y mut an t pool\n13–15 , which will l ik ely el imina t e subsets of mut ations  tha t w er e ben e fi cia l  \nearly—but not l a t e—in in f ect ion. Further , this NHP s tudy do es not  c aptur e popu la t ion  \nbot tlen eck s o ccurr ing during in t er-h os t tr ansmission, wher e mut a tions t ha t ma y be bene ficial  \nwithin a ho s t al so mus t suc cessful ly access  the a irw a y , sur v iv e e xpo sur e t o th e outs ide  \nen vir onmen t and tr ansmit inf ec tion to a new hos t. This high bar f or sp r ea d betw een hosts ma y \ne xplain wh y the v as t majority of mut a tions f ound acr oss ov er 55,000 Mtb clini c al s tr ains e xi s t at  \nt erminal br anches  within  single indiv iduals\n27  and tha t  81% of in tr a-NHP mut a tions f ound  here  \ndid not ha v e  a dir e ct c ount erpar t in t he human clinic a l s tr ain c o llect ion.  \nFinally , while g ene-lev el dat a sug g es ts ongoing Mtb mut a tion lipid and met abolic g enes ,  \nit is di f f icult t o inf er whether a gi v en missense or s ynon ymous mut ation is pos iti v ely or  \nneg a tiv ely impacting g ene function  and ther eby nomina ting pa thw a y s  as essen tia l or non-\nessen tia l f or  host adapta tion.  Additi onally , as  the e x act l ipid subs tr at es o f man y par alogues of  \ng enes in v olved in lipid met abolism and bios yn thesis r emain unknown, futur e w ork will b e  \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2026. ; https://doi.org/10.64898/2026.04.03.714442doi: bioRxiv preprint \n\n 14\nneeded t o d e fin e the nutr ient div ersi ty of Mtb host micr oen vir onments—especiall y a t r e la t iv e ly  \ninaccessib le anat omic si t es bey ond sp utum—tha t pr omot e and r estri ct suc cess fu l Mtb in f ect ion.  \n \n \n \n \n \nMETHODS \n \nSequencing o f Mtb  genomic po pul ati ons from in f ect ed NHP tissues \nW e cur a t ed a set o f Mtb  g enomic DNA samples tha t  w er e  pr evi ously  g ene rate d  fo r  \nbar c ode s equencing 7 . In or der  t o  c om par e Mtb e v olut ion across similar inf e ction time sc al es, w e  \nchose t o ana ly z e samples f r om the 20 NHP s fr om the original tha t sur vi v e d 10-12 w eek s of Mtb  \ninf ect ion, though w e  did  sequence  tissues fr om 3 addi tional  SIV c oin f e ct ed NHP s r each ed a  \nhumane endpoin t much earlier . Wh ole g enome sequencing w as perf or med on the Illumina \nNov aSeq S4 300 cy cle  platf orm acr oss  4 sequen cing runs  (see  Supplemen t a ry T able  2 f or  metric s  \nand sequencing a ccession  c odes).  R e ads w er e  f ilter ed  f or  good sequencin g quality  r eads  using  \nfa st p\n38  and  then  mapped t o  the  r ec en tly  g ener at ed ba r c od ed Mtb  Er dman r ef e r ence  g enome  \n(GenBank Access ion CP172229.1) 20 , which w as obt ained  fr om an al iquot of the  libr a ry used  to  \ninf ect  NHP s acr oss s t udies. Af t er alignmen t, w e applied  Mut e ct2 f rom the GA TK t oolki t  \n( h t tps:// g a tk.br oadins t itu t e.or g /hc/ e n-us\n) t o iden t ify al l mut a tiona l chang es (single nucleotid e  \npolymorphisms and insertion/de letio ns) a t an y fr eque ncy r e la ti v e t o the ref er en ce g enome. The \nfunctiona l eff ect  of each mut a t ion (e. g. in t er g en ic, s ynon ymous, missense, fr ameshift) w as a lso  \npr edic t ed using SnpE f f based on  our recen t Er dman annot ation 20 .  \nThe Mut ect2 ou tput y ielded  a pr e liminary l is t of o v er 100,000 det ect ed g enomi c  \nchang es acr oss al l samples, which wer e mainly f ound a t low pr ev a lence (<10% mut an t allel es  \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2026. ; https://doi.org/10.64898/2026.04.03.714442doi: bioRxiv preprint \n\n 15\nv er sus wi ldtype  sequenc es) and  dri ven primary  by a subset  of  samples. T o fi ler these  v aria n ts,  \nw e enacted 3 QC s teps:  firs t, w e us ed the i n ter quar tile  r ang e (IQR) method t o  de f ine ou tlie r  \nsamples with a number of Mu t ec t2 varian ts a t 1.5x gr eat er than the  IQR a cr oss all  the samples  \nfr om each sequen cing ba tch. This  yielde d 46 out lier  samples that w er e remov ed fr om  \nsubsequen t anal y ses  (Supplemen t al  Figur e 1A, ‘ QC1’). Not ably , the v ast majority mut a tion s in  \nthese outl ier s w er e enr iched f or C to A tr ans v er sions (Supplemen t al Figur e 1B), a mut a tional  \nchang e tha t has been pr ev iously ass ociat ed with DNA damag e artif acts arising during libr ar y  \npr epar a tion due t o eleva t ed he a t\n39 .   Sec ond, w e filt e r ed r emaining v ari an ts t o remov e low-\nc onfidenc e c a lls at si t es with subst a ndar d sequencing depth, which inc l uded only inc luded 1)  \nsequencing runs wi th an a ver ag e r ea d depth abov e  50x; 2) g enomic v aria n ts th a t s a t in r egion s  \nwher e ther e w as ov er a ll sequenc ing depth tha t e x ceeded 50% of the a v erag e sequencing depth  \nof the  sample; and 3) v ariants whos e pr ev alenc e w as abo v e 10% (r e la t ive t o  all  r eads  a t  that  \nposition ) t o  acc ou n t f o r r ar e sequen cing err or s (Supplement al Figur e 1A, ‘ QC2’).  Thir d, due to  \ndif f icult y in uniquely ass igning short r eads t o r epetit iv e r egions in t he Mtb g enome, w e  \nr easoned th a t the r e wil l be s ys t ema t ic mismapping ev en ts yield ing the sa me e x act ‘mut a tion’ \n(in both chr omosomal position and type of DNA base chang e) being f o und in samples ev en  \nacr oss diff eren t animals.  Filt ering o ut the e x act same v arian ts f ound in mor e than 1 animal,  \nyielded  a c andidat e  li s t  of  217 v ariants f ound  acr oss  125 tissu es (Suppl emen t al  Figur e 1A, ‘ QC3  \ns t ep’). Finall y , w e visuall y inspec t ed  the r ead pi leups g ener a ted by eac h mapping file using \nSam t ools and c onfi rmed tha t the d iff er en t r eads w ere mapping acr oss the sit e o f int eres t an d  \ntha t v aria n ts w ere not clu s t e ring a t  the 5’ and 3 ’ ends of r eads.  This  pr oduced our fina l  \nv alidat ed t able of 143 uniqu e hig h c onfid ence d e nov o  mut a t ions f or fur ther ana ly s is  \n(Supplemen t ary T able 1).  \n \nDefinin g pre-e xis ting a nd i n tra hos t-de rived Mtb mut a tions  \nAs our r ef er e nce Er dman g enome was deri v ed fr om a single barc oded  c lone with in a  \nlar g er barc oded l ibr ary , w e c annot e xclude that some mut a tions iden t ifi ed  by sequencing c ould  \nha v e arisen in vitr o is spec ific t o diff er en t barc oded s tr a in. T o c onser v a ti v ely separ at e in vivo  \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2026. ; https://doi.org/10.64898/2026.04.03.714442doi: bioRxiv preprint \n\n 16\nderiv ed  mut a ti ons v er sus those  that ma y ha v e been p r e-e xis ting i n the  lib r ary , w e c ompiled  th e  \nMtb bar c od ed s tr ain s pr ese n t i n eac h tissue sample from prior barc ode amplic on sequenc ing 7  \nand c ompar ed this  t o  the  Mutect2 det ected v ar ian ts  pr es en t  in  each tis sue. As diagr amed in  \nFigur e 1B , w e c onside r ed mut a t ions i n viv o-deri v ed i f they satisfied 1 o f th e 2 f ollowing criteria.  \nFir s t, w e fir s t lo ok ed f or tiss ues tha t c ont ained a g enomic v arian t and a si ngle bar c oded  \nMtb s tr ain, which  allow ed  us t o link a g enomic v ariant t o a barc oded  Mt b s tr ain g en ome. W e \nthen iden t ifi ed all tissues c ont a ining tha t bar c ode d Mtb s tr ain  and assessed the \npr esence/ absence of the g enomic v arian t of i n t e r est in those tissu es. If w e obser v ed a p atte r n  \nwher e some ti ssues had  only  the  wild t ype al lele  while  other s  had the g enomic v ariant, we  \nr easoned the det ec t ed mut a t ion lik e l y origina t ed dur ing inf ection. This sit ua tion is diagr ammed  \nas ‘BC2’ in Figur e 1B.  \nSec ond, when w e c annot link a g enomic v arian t t o a single bar c od ed Mt b s tr ain (often  \nthe c as e when a muta tion  is found in  a tissue  that w as in f ec t ed by mor e  th an one barc oded  Mtb  \ns tr ain), w e  look ed f or  the  g enomic v a rian t  of int er e s t  acr oss  all tissues  c ont aining ev e ry p ossibl e  \nbar c oded M tb s tr ain  it c ould b e link e d t o. If w e obser v ed bo th pr esen ce of wild typ e Mtb and  \nmut an t alle les in every  poten t ial ba r c ode  netw ork, w e also  conside r ed this v ar ian t  as l ik el y  \nemer ging in viv o. This situation i s dia gr a med as ‘BC3’ and ‘BC4’ in Fi gur e 1B. \nIf a v ariant does no t sa t isfy th e cri t e ria abov e, w e c annot ru le ou t the m ut a ti on could  \nha v e emer g ed in vitr o , so it is consid er ed ‘pr e-e xis ting ’ and e x cluded fr om further analy ses. An  \ne x ample of this is ‘BC1’ in Figur e 1 B , wher e a mut a tion w as pr esent i n all tissues th a t w as  \ninf ec t ed that barc oded str ain.  \n \nDefinin g mut a n t origin  tissues a nd t i ming of or igin  emergence  \nT o de fine mut a ti on ‘ origin’ sit es, w e fir s t id en t ifie d tissues c ont aining a si ngle bar c oded  \nMtb s tr ain.  If si t es c o n t ain  an unfix ed mut an t al lele  (i.e., <75% of a ll reads a t tha t  si t e are  \nmut an t), then we c onside r ed th ese  tissues  as harbor ing both wild typ e (i.e., par e n t al ) and  \nmut an t alle les. These ar e  lik ely the  origin si t es where the muta tion  or iginally ar ose. In the  \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2026. ; https://doi.org/10.64898/2026.04.03.714442doi: bioRxiv preprint \n\n 17\npar en t s tudy , animal lungs w er e imag ed using PET -C T on a mon thly basis at 4-, 8, and 1 2- w eek s \npos t inf ec tion t o  r egist er  the lo c a ti o n and pr es ence o f newly  det ec t ed  lu ng lesions 7 .  Her e, w e  \nc ompar ed the time of PET-C T det ecti on of the  lung-specific origin si t es (PET-C T r egis tr a tion w a s  \nnot done f or thor acic lymph node an d e xtr apulmonary si t es) a cr oss t r eat men t gr oups.  \n \nMut at ion ra t e qu antificat ion  \nSimilar t o p r evious w o rk us ing c ol on y based sequenc ing fr om inf ec t e d NHP s\n16 , w e  \nc alcul a t ed  a Mtb mut ation ra t e  f or e ach animal by dividing th e number of unique muta tiona l  \nev en ts (af t er normalizing f or number of tissues sampled) by the number of bact erial  \ng ener a tions  (per  unit  of inf ec tion ti me) occurring in  the  hos t.  Her e, su m med the number o f  \ndiff er e n t  mut a ti ons across al l tissues  in a NHP t o e s tima te t he mut ation  r at e o f the en t ir e  Mtb  \npopula ti on within one animal using t he f ollowing equa ti on: μ = m 4i354i35[ N4i35 ∗ 4i35( t4i354i354i35 g )] / D    \nIn this equation, μ\n r ep r esents the t o tal mut a tion ra t e a cr oss an animal, m  is the number  \nof uniqu e mut a t ional  ev ents de t ec t e d by sequenc ing acr oss  all  tiss ues in  the an imal, N  is  the  \nMtb g enome siz e (set a t 4,400,000  bp), t  is the t ot al inf ec tion t ime of each NHP (da y s of  \ninf ect ion multipl ied by 24 hour s), g  is  the Mtb in viv o  g ener a t ion time (arb itr arily used 18 hour s  \nf or a single  doubl ing) and D  is th e tot al number of tissues  that w e r e s e quenced per  animal. \nPlease not e that as the e x act Mtb div ision r a te within a giv en animal is unknown, the numeric al  \nmut a tion r at e is not  absolute; it is in s t ead the rela tive\n mut a ti on r at e per  fix ed un it o f inf ecti on  \ntime in this s tudy .  \n \nBact erial burden qua n ti ficatio n acros s animals  \nW e obt ained pr eviously published t is sue-lev el li v e bact e rial bur den (CFU) and t ot al ( liv e  \nand dead) bacterial  chr omosomal eq uiv alents (CE Q ) measur emen ts f or t he animals tha t  w er e  \nincluded  in this  s tudy 7 . T o t al CFU an d CE Qs w er e summed f or a ll sampl ed tissu es fr om each  \nanimal f or s t atisti c al comparisons. \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2026. ; https://doi.org/10.64898/2026.04.03.714442doi: bioRxiv preprint \n\n 18\n \nBact erial barc o ding network visual iz a tion  \nUsing pr eviously publ ished amplicon bar c oding dat a 7  fr om the tis sues seq uenced in th is  \ns tudy , w e  g ener at ed netw ork  plo ts by c on vert ing a t abl e o f g enomic varian ts  pr ese n t  in a ll  \nsequenced tissu es int o a netw ork plo t using the R pack ag e, igr aph. Specific all y , each tissue tha t  \nc ont ained a giv en barc oded Mtb str ain w as initiall y plott ed as nodes given the same c olor .  A  \nring w as added in  a di ff er e n t  c o lor to a ti ssue/node if it c ont ained  a uni que g enomic v aria nt  \naft er s equencing such  that d iff eren t  v arian ts  within  the  same bar coded d i ssemina tion ne tw ork  \nar e r epr es ent ed by diff er e n t c olo red rings.  Using the pr esence or absence of g enomic \nmut a tions shar ed ac r oss ti ssues, w e inf erred dissemina t ion subnetw o rk s with th e f ew e st  \ndissemina tion e v ents. In the c ase of netw ork s f o r which w e a r e unable to or der th e ev e n ts of  \ndissemina tion, we arb itr aril y repr es en t ed  dissemination  as a s ingle star-lik e netw or k. If  a  \ng enomic v arian t w as f ound in mor e than one tissu e with a netw or k, w e manually r e-or der e d  \nc onnec ting lines betw een th ese nod es t o f orm sub-netw ork s. W e finally  import ed the igr aph \nbar c ode netw or k s int o Adobe Illustr a t or t o visuall y impr ov e line de f init i on, upda t e c olo r s f or  \nvisualiza tion  and sc a le f or  siz e.  The e dg elis t r epresenting the c onnec tions betw een tis sues in  f o r  \nall visual iz ed barc oded netw o rk s is provided as an R objec t in Supplementary Dat a File 1. No t e:  \nsome tissues harb or ed mult iple g en o mic mut a tions bu t igr aph is limit ed  to adding a single r ing , \nso w e added these v ar ian ts and th e sit e o f origin si t es (as bo x e s) in Adobe Illus tr at or . The  \npr esence  of g en omic mut a ti ons and origin si t es wi thin the networ k s ar e  i ndic at ed by  met ada ta  \nc olumns in Supplemen t a ry Dat a File 1 . \n \nSTRING and In t erpro a nalyses  \nThe lis t o f all  g enes with  in v iv o d eri v ed mut a t ions from our NHP da t a, Li u et al.\n17  and  \nLiberman et al. 18  w ere anal y z ed o n the STRING da t abase ( h t tps:// s tri ng-db.or g )  to  d ef i n e  \nput a ti v e int er act ions. All int er act ing nodes w ere then  colored by th eir p r edic t ed 23  functi ona l  \nc at egories. Finally , pr o t ein domain en richmen t analysis (Int erPr o) w as performed on the STRING  \nda t abase f or a ll g enes.  \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2026. ; https://doi.org/10.64898/2026.04.03.714442doi: bioRxiv preprint \n\n 19\n \nIden tif ication  of i n tr a-NHP mut ations in human  Mtb cl inical strains  \nW e obt ained the lis t of g enomic mut a tions identif ied acr oss appr o xi ma t ely 55,000  \npublicly a v ai lable Mtb c lini c al iso late sequences fr om a r ece n t publi c at ion 27 , which mapped  \nmut a tions ag ains t a r e c onstruc t ed a nces tr al Mtb g enome and c on s truc t e d a ph ylog enetic tree  \nt o ide n ti fy independe n t mut a t ional ev en ts and sha ring of mut a ti ons ac r oss indiv iduals. W e \nsear ched f or  NHP deriv ed  intr ag eni c mut a tions  that also  show ed up with the same DNA  \nmut a tion (e. g. C>T) a t the same c odon position in the human Mtb cli nic al is olat e dat a. F or \nin t e r g enic sequen ces, w e used BLAST t o sear ch f or iden t ic a l s tr e t ches o f g enomic sequence  \nar ound the mut a tion si t e and then i den ti fied the r e lev an t g enomic posit i on of the mut a tion in  \nthe ancestr al Mtb g enome used t o map the human clini c al iso lat es.  \n \nDat a ava ilab ili ty \nThe g enomic mappin g , quality c on tr o l and v arian t det ec tion c od e and met hods ar e full y  \ndescribed at the f ol lowing Github li nk: h t tps:// github.c om/F ortune-Lab/ Mtb-NHP -g enotyping .  \nAll sequencing da t a has been deposited on the Sequencing R ead Ar chiv e, with accession c od es  \nand link s  f or ind ividual  samples pr ovi ded in Supplemen t a ry T able 1. All seq uencing da t a are als o  \na v ailable under B iopr ojec t Accession PRJNA1432 747. All met ada t a associat ed with the d a t aset s  \nin this  w or k ar e  depos ited on F ai r domhub a t th e f ollowing lin k:  \nh t tps://f air domhub.or g / s tudies/1401\n \n \nAcknowledgments \nThis pr oject has been funded in part  with f eder al funds f r om the Na tion al Ins titu t e o f  \nAller gy & I nf ectious Dis eases, Na tio nal Ins titu t es of Health under con tract 75N93019 C000 71 \nand NIH,  R01 AI134 195.  The fund er s had no r ole in s tudy design, da t a c olle ction and  \nin t e rpr eta ti on, or the de cision  t o sub mit the w ork f o r publi c ation.  \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2026. ; https://doi.org/10.64898/2026.04.03.714442doi: bioRxiv preprint \n\n 20\n \n \n \n \n \n \n \n \n \nREFERENCES \n1. 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Nat Comm un  12, 2716 \n(2021).  \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2026. ; https://doi.org/10.64898/2026.04.03.714442doi: bioRxiv preprint \n\n 23\n20.  Marin, M. G. et al.  Complet e g enome sequence of a virul en t ba r c od ed Mycobact er ium \ntuber culo sis str . Er dman c ommonly used f or non-human prima t e inf e ction s tudies. \nMicrobiol R esour An nounc  e0123224 (2025) doi:10.112 8/ mr a.01 232 -24.  \n21.  K r eutz er , D . A. & Essigmann, J . M. Oxi diz ed, deamina t ed c yt os ines are a so ur ce of C --> T \ntr ansitions in vi v o. Proc Natl Acad Sci U S A  95, 35 78–358 2 (1998).  \n22.  K urthk oti, K. et al.  A dis ti nct ph ysiolog ic al role o f MutY in mut a t ion pr e v en t i on in \nm y c obact er ia. Microbio logy (R ea ding )  156 , 88–93 (20 10).  \n23.  K apopoulou, A., Lew , J . M. & Cole, S. T . The My c oBr ow se r por t al: a c ompr eh ensiv e and \nmanually annot at ed r es our ce f o r m y cobact er ial g enomes. T uberculosis (E di nb)  91, 8–13 \n(2011).  \n24.  Szklar c z yk, D . et a l.  The STRING da t abase in 2023: pr ot ein-pr otein assoc ia t i on netw orks and \nfunctiona l enri chmen t anal y ses f or any sequenced g enome of int er e s t. Nucl eic Acids R es  51, \nD638–D646 (202 3).  \n25.  Culviner , P . H. et al.  E v olu tion o f Myc o bact erium tube r culos is tr anscr iption regula tion is \nassociat ed with incr eased  tr ansmissio n and drug r esist ance. Pr epri n t at \nh t tps://doi.or g /10.1101/2 025. 05.01 . 651750 (20 25).  \n26.  Martini, M. C. et al.  Loss of RNase J l e ads t o multi-drug t ol er ance and accu mula tion of h ighly \ns tructu r ed mRNA fr agmen ts in My c ob act erium tube r culos is. PLoS P athog  18 , e10107 05 \n(2022).  \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2026. ; https://doi.org/10.64898/2026.04.03.714442doi: bioRxiv preprint \n\n 24\n27.  Culviner , P . H. et al.  E v olu tion o f <em>My c obac t er ium tuber culos is</ em> tr anscription \nr egula tion is assoc ia ted with in cr ease d tr ansmission and drug r esi s t ance. b i oRxiv  \n2025.05 .01.6 5175 0 (2025) doi:10.1 10 1/2025 .05.0 1.651 750.  \n28.  Diedrich, C. R. et al.  SIV and My cobac t erium tuberculosi s s yner gy within th e gr anuloma \nacceler at es th e r eac tiv ation p a tt ern o f lat ent tuberculosis. PLoS P ath og  16, e1008413 \n(2020).  \n29.  Diedrich, C. R. et al.  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Ener g etics of R espir a ti on and \nOxida tiv e  Phosphoryl a ti on in Myc oba ct eria. M icrobiol  Spectr  2 , (2014). \n34.  Quadri, L. E. N. Bios yn thesis  of m y c ob act eria l lip ids by poly k etide s y n thases  and bey ond. Crit \nR ev Biochem Mol Biol  49, 179–211 (2014).  \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2026. ; https://doi.org/10.64898/2026.04.03.714442doi: bioRxiv preprint \n\n 25\n35.  Mulholland, C. V . et al.  Pr opionat e prev en ts loss of  the PDIM virul ence l ipi d in \nMy c obac t er ium tuber culos is. Nat M icrobiol  9 , 1607–1618 (2024).  \n36.  R ousseau, C. et al.  Virulence at t en ua t ion of two Mas-lik e po lyk etide  s yn tha se mut an ts of \nMy c obac t er ium tuber culos is. Microbi ology (R ead ing )  149 , 1837–18 47 (200 3). \n37.  Smith, C. M . et al.  Hos t-pa thog en g en etic int er act ions unde rlie tuber cu losis  susceptibi lity in \ng enetic al ly di v er se mice. Elif e  11, (2022). \n38.  Chen, S. Ultr af as t one-pass F A ST Q da t a pr epr ocess ing , quality c o n tr o l, and deduplica ti on \nusing f as tp. Imet a  2 , e107 (2023).  \n39.  Cos t ello , M. et al.  D isc o v ery  and charact eri z a t ion of a rtif ac tual mut a t ions i n deep c over ag e \nt ar g et ed c aptur e sequencing dat a du e t o o xidati v e DNA damag e during sample pr epar a tion. \nNucleic Acids R es  41, e67 (201 3).  \nFIGURE LEGENDS \nFigure 1. Whole genome sequencing identifies intrahost mutations.  (A) Study design in which \nnaïv e, SIV inf ected NHP s (with and without suppressive AR T init iat ed a t 3 d a y s pos t inf ec tion) \nw er e chal leng ed with a barc oded M t b libr ary . All bac t eria wer e isolat ed from inf ect ed tissues, \ng enomic DNA e xtr act ed f or who le g e nome sequencing and in tr ahost mut ations ide n ti fied. ( B) \nT op: crit er ia f or d e fin ing in vivo deri ved mut a tions, wher e e v er y r ow (Tissu es A-D) r epr esents \nsit es inf ec t ed by a barc oded M tb s tr ai n (c olumns BC1-4).  Tissues c ont ainin g a g enomic \nmut a tion ar e c o lor ed gr een whi le whi t e indi c at es p r esence  of wild type M t b.  Bot tom: \nMut a ti ons ar e gr ouped by their p r edi ct ed impact on p r ot e in coding. (C) T op: f or each animal \n(lines), the  g enomic posit ions with de t ec t ed mut a t ions in an y tissue a r e plo t t ed (d ots). B ott om: \nthe number s of mut a t ions (f r om all N HP s) obser v ed within 100 kb g enomic window s ar e plo t t ed \n(y ellow). Gr ey shad ing indica tes the 9 5% c onfidence le v el o f the number o f  e xpect ed mut ations \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2026. ; https://doi.org/10.64898/2026.04.03.714442doi: bioRxiv preprint \n\n 26\nin a giv en 100 kb window when samp ling 116 r andom sit es in the Mtb g en ome acr oss 1000 \nsimula tions.  \n \nFigure 2. Mutational rates and profiles across SIV coinfection and ART . (A ) R ela ti v e mut a t ional  \nr a t es w ere c alcu lat ed f or each animal per unit of inf ection time. (B) The t ot al ch r omosomal \nequiv alents (CE Qs) fr om all inf ect e d tissues in each NHP w as c ompar ed acr oss tr eatment  \ngr oups. (C) The t ot al c ol on y f orming units (CFU) in each NHP  a t necr ops y w as c ompar ed acr oss  \ngr oups. (B and C) St a ti s ti c al t esting w as perf ormed using a K rusk al W allis t est with Dunn’ s  \nc orrecti on. (D) The t o t al Mtb CFUs a t necr ops y f or origin s it es c o n t ain ing an o xida tive- (C>T /G>A  \nand C>A/G>T) or non-o xida tiv e da mag e mut a tion (all o ther SNP s) w e r e c ompar ed us ing a  \nMann-Whitney U t es t. (E) The pe r cent of a ll mut ations obser v ed in  a t r ea t men t gr oup a t t ribu t e d  \nt o a dis tinc t type of g enomic mut a tio n. P -v alue w as de fined by a Fisher ’ s E x act t es t of o xida t ive  \nmut a tions v e r sus non-o xida ti v e mut ations in the AR T gr oup c ompar ed t o t he TB only gr oup. (F)  \nThe per cent of  all muta tions  in each NHP (dot) separ at ed by type o f muta tion. ( G) Corr e la t ion  \nbetw een the  number of o xid a ti v e  damag e-associa t ed mut a t ions pe r  NHP (normaliz ed by \nnumber of tissues sampled) with eac h animal’ s Mtb r ela ti v e mut a t ion r a te (one-t ailed P ear so n  \nc orrela t ion).  \n \nFigure 3 . In vivo variants uncover discrete dissemination events within barcoded Mtb \ndissemination networks . Color ed no des denote ti ssues shar ing the same Mtb s tr ain  as defined  \nfr om prior bar c ode sequ encing in r ef\n7 . Rings indic a te Mtb mut a tions prese n t in tha t t issue, with  \neach c ol or repr ese n ting a di ff er e n t g enomic mut a tion. Tissues with  one  bar c oded M tb str ain  \nand a mixtur e  of wi ldtype  and mut ant alle les lik e ly ser v e as  ‘ origin’ sit es,  which ar e d enoted as  \nbo x es. Inf err ed dissemina t ion ev e n t s fr om origin si t es a r e ind ica t ed  by arr ow s (r ed  arr ows  \nindica t e  origin s it e  is a  lung tissue  dissemina ting t o o ther lung tissue s, while blue a rr ows  \nindica t e origin si t e is  a thor acic  lymph node dissemina t ing t o lung tis sues).  \n \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2026. ; https://doi.org/10.64898/2026.04.03.714442doi: bioRxiv preprint \n\n 27\nFigure 4. Intr ahost mutations are enriched in lipid metabolism genes and confer Mtb fitness . \n(A) Ov erlap o f g enes with in tr aho s t mut a tions (s ynon ymous and non-s yn on ymous) in NHP s (95  \ng enes) and tw o  human da t asets (257  and 403 g enes f or Liu e t al. 17  and L i eberman et al. 18 ). (B)  \nFr action of  i n tr ahos t  mut a t ions in g enes fr om each  Myc obr ow se r c ategory is p lott ed. The  \nenrichmen t o f i n tr ahost mut a ti ons in each fun ctiona l ca t egory w as c a lculat ed ag ains t the  \nnumber of g enes ann ot at ed  f or eac h  c at egory i n the  H37R v g enome using a Fisher ’ s e x act  t e s t.  \n(C) The pr oportion o f mut an t r eads ver sus all r eads w as c ompar ed a t ea ch  origin tissue. (D) The  \nper cent of tissues con t a ining a bar c oded Mtb s tr ain tha t also c a rried a  g enomic v arian t of  \nin t e r est w as c ompar ed. (C-D) St a tistical analy ses w e r e perf ormed using an unpair ed t-t e s t. (E) A  \nFisher ’ s Ex act t es t w as used to c omp ar e the pr opo rtion o f C-t o-T tr ansitions f or v arian t a llel es  \ntha t w e r e and w e r e not f ound  acr oss 55,000 hu man clinic al str ain g enomes. \n \nFigure 5. Model of intrahost mutations in metabolic and cell envelope pr ocesses . NHP -deriv ed  \nMtb mut a tions are gr ouped int o fun ctional pr o cesses using li t er atur e cura tion and conser v e d  \ndomain similarity  t o micr ob ial h omologs. Genes in bla ck r epresen t  al l i ntr ahos t deri v ed M tb  \nmut a tions, while g enes in r ed are mut a tions assigned t o t he ‘pr e-deri v ed’ mut a tion gr oup.  \nGenes mark ed with a s t e ris k s repr es en t fr ameshift mut a t ions; g enes  in par en thes is repr ese nt  \ns ynon ymous mut a tions and the r e maining ar e pr edict ed missens e mut a ti ons. Functiona l  \nc at egories ar e c ol or ed as f ollow s:  lipid met abolism and bios yn thesis  (r ed), cell en v e lope  \n(y ellow), nucl eotid e s yn th esis/r ep air (or ang e), met aboli t e tr anspo rt (gr een ), pr ot e i n  \nhomeos t asis/tr ansl a tion  (blue), r ed o x pr ocesses/r esp ir a ti on (gr ey), v ir ulence (pu rple). The  \nschema tic dep icts  lipas es breaking down c omple x host and ba ct e rial  li pids i n t o  f at ty a cids,  \nwhich c an then be act iv at ed f or degr ada tion (or in c orpor at ed i n to other molecules ) by  \nc onjug a tion t o CoA thr ough f a tty ac id  CoA lig ases (F A CL).  Dif f er ent acy l-CoA subs tr a t es a r e then  \ndegr aded thr ough it era ti v e c y cles  of  bet a-o xida tion, y ielding acet yl-CoA tha t c an be  used to  \ndriv e  the  T CA cy cle and  r esp ir a ti on as w e ll  as be ing incorpor at ed  int o  Mtb sec on dar y  \nmet abolit es. F a tty acid lig a t ion t o AMP via f a t ty acid AMP lig ases (F AAL) c an be dir ectly  \ni n co r p o rate d  b y  p ks  (and oth er b iosyn thesi s g enes)  int o  lar g e r l ipid-based s tru ctur es,  man y of  \nwhich ar e c omponen ts of th e cell envelope. Bet a-o xida tion o f lipids also y i elds NADH, leading t o  \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2026. ; https://doi.org/10.64898/2026.04.03.714442doi: bioRxiv preprint \n\n 28\nincr eased r educt iv e s tress that needs  t o be r e-balanced. This c an o ccur th rough c on v er sation o f  \nNADH t o NAD PH , which is utiliz ed f or sec ondary met aboli t e s yn th esi s as w ell as NADH  \nc onsumption during r espira tion, whic h c an bec ome impair ed due t o ho s t p r essur es lik e h ypo xia,  \nacidifi c ation and inflamma tion. This ma y lead t o leak ag e of electr ons from the r espir at or y chain, \ng ener a ting r eacti v e o xy g en species a nd macr omolecule dama g e in Mtb. I n the c ont e xt of AR T \ntr ea tment, w e  h ypothes iz e an  augmen t ed inf lamma t ory  en vir onment not seen  in  Mtb  only  \nanimals, which ma y yield gr ea t er M tb damag e and incr eased det ec tion  of o xida ti v e-damag e \nassociat ed mut a t ions. Imag e w as pr epar ed using BioR end er .  \n \nT able 1. NHP intrahost mutations are also observ ed in human Mtb clinical strains . Iden tic a l  \ng enomic mut a tions in the NHP da t aset w er e ma t ched t o mut a t ions obs er v ed in ov er 55,000  \nclini c al isol a t es\n27 .  Clini c al  s tr ain  muta tions  and th e number o f independ en t mut ations  ev e n ts  \nw er e pr ed ic t ed by comparing each clini c al s tr ain ag ains t a M t b ances tr al g enome  \nr ec on s truc tion. Nons yn = non-s ynon ymous, Syn = s ynon ymous, IG = in t er genic. Pk s7 mut a tion s  \nwith an ‘ A ’ and ‘B’ indic at e di f f er e n t mut a tions lead ing t o a fr ameshift in t he same c odon, while  \nw e find two di f f e r ent type s of i nt er g e nic whiB6 mut a ti ons a t the same posi tion.  \n \nSupplementary Figur e 1. (A) The number of g enomic v arian ts (par e n theses) de t ected by \nMut ect2 per sequ enced sample (dot s) be f or e and after quali ty c o n tr o l (QC) s t eps. (B) The time  \nof PET -CT  det ection (e ither 4-, 8- or 1 2-w eek s pos t in f ecti on) of a lung gr anuloma tha t ser v ed as  \nan origin si t e f or an in vi v o mut a t ion  is c ompar ed across gr oups. St a ti s ti cal t est compar ed the  \ndis tr ibution o f 8 and 12-w eek lesions using a K rusk al- W allis t es t with Dunn’ s c orr ec tion. (C)  \nSTRING analy ses showing the p r edic ted int er act ions bet w een a ll g enes  in  t he NHP da t aset. On ly  \nin t er ac ting nodes are shown and ar e c olo r ed by the ir Myc obr ow se r ca t egory .  \n \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2026. ; https://doi.org/10.64898/2026.04.03.714442doi: bioRxiv preprint \n\n 29\nSupplementary Figure 2. All genes with intrahost mutations (all mutations = synonymous and \nnon-synonymous mutations) for each dataset was analyzed for InterPro protein domain \nenrichment on the STRING database. \n \nSupplementary Table 1.  List of genomic accession numbers for all sequenced Mtb \npopulations from infected tissues in this study.  All samples are also available under the \nBioproject accession: PRJNA1432747.  \n \nSupplementary Table 2.  Final table of high confidence Mutect2-detected genomic mutations \noccurring in vivo.  \n \nSupplementary Data File 1. An R object that provides data frames containing the tissues sharing \ndisseminated barcoded Mtb strains. The name of each list item matches the following format: \n“ Animal ID_QtagBarcodeSequence” .  Each table (using only the Node1 and Node2 columns) can \nbe used as an edgelist by the igraph R package to generate the network structures shown in \nFigure 3.  However , additional metadata columns in each table—i.e., columns indicating the \ndiff erent variants associated with this barcoded strain (column names indicate genomic position \nassociated with a mutation) and a final column indicating which tissues ser ve as origins for each \nmutation—will need to be incorporated to fully reproduce Figure 3.  In the metadata columns, \n‘Node1’ and ‘Node2’ ref ers to the tissues in the same row . \n \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2026. ; https://doi.org/10.64898/2026.04.03.714442doi: bioRxiv preprint \n\nA\nB High Confidence Variants (143)\n31\n53\n19\n11\n2\nPre-existing? (27) In vivo defined (116)\n6\n17\n8\n2\nMissense\nSynonymous\nFrameshift\nIntergenic\nTruncation\nTissue A\nTissue B\nTissue C\nTissue D\nBC2 BC3 BC4\nOR\nTissue A\nTissue B\nTissue C\nTissue D\nBC1\nN = 7\nN = 4\nN = 9\nTBSIV + TBSIV + ART \n+ TB\nSIV \ninfection\nART (3 dpi) Barcoded Mtb \nInfection (bronchoscope)\nIsolation of Mtb and bacterial \nDNA from infected tissues\n1) Population-level whole \ngenome sequencing\n2) Alignment to Mtb Erdman \ngenome and SNP / indel \ndetection (Mutect2)\n3) QC analyses and in vivo \nvariant filtering\n5 months\n10-12 \nweeks\nGenomic Analyses\nC\nMtb genomic position (x 106 nt)\n# of mutations / \n100kb window\nTB\nSIV+TB\nSIV+ART+TB\n0.0\n5.0\n×10\n5\n1.0\n×10\n6\n1.5\n×10\n6\n2.0\n×10\n6\n2.5\n×10\n6\n3.0\n×10\n6\n3.5\n×10\n6\n4.0\n×10\n6\n4.5\n×10\n6\n0\n2\n4\n6\n8\n50\n100\n150\n200\nMtb genomic position (nt)\n# variants / 100Mb window\nMutation vs NHP and genomic window\n21818\n22218\n22318\n23218\n23518\n24018\n33519\n33919\n34319\n22417\n22717\n22817\n23017\n30018\n30118\n33419\n22117\n22317\n22517\n22617\nSIM\n0.0\n5.0\n×10\n5\n1.0\n×10\n6\n1.5\n×10\n6\n2.0\n×10\n6\n2.5\n×10\n6\n3.0\n×10\n6\n3.5\n×10\n6\n4.0\n×10\n6\n4.5\n×10\n6\n0\n2\n4\n6\n8\n50\n100\n150\n200\nMtb genomic position (nt)\n# variants / 100Mb window\nMutation vs NHP and genomic window\n21818\n22218\n22318\n23218\n23518\n24018\n33519\n33919\n34319\n22417\n22717\n22817\n23017\n30018\n30118\n33419\n22117\n22317\n22517\n22617\nSIM\nTB\n22417\n22717\n22817\n23017\n30018\n30118\n33419\nSIV + TB\n22117\n22317\n22517\n22617\nSIV + ART + TB\n21818\n22218\n22318\n23218\n23518\n14018\n33519\n33919\n34319\n0 0.5 1 1.5 2 2.5 3 3.5 4 4.5\n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2026. ; https://doi.org/10.64898/2026.04.03.714442doi: bioRxiv preprint \n\nNon-oxOxidative\n1\n10\n100\n1000\n10000\n100000\nFig 2D: Total origin CFU by mutation type\nTotal CFUs\n0.0036\nC>T_G>AC>T_G>AC>T_G>AC>A_G>TC>A_G>TC>A_G>TG>C_C>GG>C_C>GG>C_C>GT>C_A>GT>C_A>GT>C_A>GA>C_T>GA>C_T>GA>C_T>GA>T_T>AA>T_T>AA>T_T>ADeletionDeletionDeletionInsertionInsertionInsertion\n0.0\n0.2\n0.4\n0.6\n0.8\n1.0% of all mutations / NHP\nFig 2E: Fig 2E: Avg %mutation_by NHP\nB C D\nTB SIV ART\n0.0\n5.0×10-10\n1.0×10-9\n1.5×10-9\nMutation / bp / generation\nFig 2A: Fig 2A: MutationRate per time_hrs\n0.0221\n0.4613\nA\nMutations (x10-10) / bp / unit time\n5\nMtb\nSIV + \nMtb\nSIV + ART + \nMtb\n0\n15\n10\np = 0.46\n*p = 0.02\nMutations (x10-10) / bp / generation\n# Oxidative mutations / tissue\n105 150\n0.1\n0.2\n0.3\n0.4\n0.5\n0.0 5.0×10-10 1.0×10-9 1.5×10-9\n0.0\n0.1\n0.2\n0.3\n0.4\n0.5\nMutation Rate / unit time\nOxidative mutations / tissue \n#ox_mut vs. Mutation rate\nTB-ox\nSIV-on\nART-ox\nPearson r\nr\n95% confidence interval\nR squared\nP value\nP (one-tailed)\nP value summary\nSignificant? (alpha = 0.05)\nNumber of XY Pairs\nMutation Rate / unit time\nvs.\nTB-ox\n0.1334\n-0.6889 to 0.8055\n0.01779\n0.3878\nns\nNo\n7\nMutation Rate / unit time\nvs.\nSIV-on\n-0.4642\n-0.9856 to 0.8971\n0.2155\n0.2679\nns\nNo\n4\nMutation Rate / unit time\nvs.\nART-ox\n0.7048\n0.07652 to 0.9325\n0.4968\n0.0170\n*\nYes\n9\nMutation Rate / unit time\nvs.\nTB-CT\n0.1091\n-0.7016 to 0.7967\n0.01190\n0.4079\nns\nNo\n7\nMutation Rate / unit time\nvs.\nSIV-CT\n0.1014\n-0.9525 to 0.9681\n0.01029\n0.4493\nns\nNo\n4\nMutation Rate / unit time\nvs.\nART-CT\n0.7051\n0.07709 to 0.9325\n0.4972\n0.0169\n*\nYes\n9\nTB: r = 0.13; p = 0.39\nSIV+TB: r = -0.46; p = 0.27\nSIV+ART+TB: r = 0.70; p = 0.02\nF\nTB-CEQ SIV ART\n106\n107\nTotal CEQ/NHP\nFig 2B: Total NHP outcomes (all lesions): CEQ\n0.0945\n0.3197\nSIV + ART + \nMtb\nTotal CEQ / NHP\nSIV + \nMtbMtb\n107\n106\np = 0.32\np = 0.09\nTB-CFU SIV ART\n103\n104\n105\n106\n107\nTotal CFU/NHP\nFig 2C: Total NHP outcomes (all lesions): CFU\n0.0066\n0.5502\nSIV + \nMtb\nSIV + ART + \nMtbMtb\n105\n103\n104\n107\n106\nTotal CFU / NHP\n*p < 0.01\np = 0.55\n100\n80% of all mutations\n0\n20\n40\n60\nMtb\nSIV + \nMtb\nSIV + ART + \nMtbTB SIV ART\n0\n20\n40\n60\n80\n100% of all mutation events\n%type mutation\nC>T_G>A\nC>A_G>T\nG>C_C>G\nT>C_A>G\nA>C_T>G\nA>T_T>A\nDeletion\nInsertion\nG\nOxidative damage\n100\n0\n20\n40\n60\n80% of all mutations\nTB\nSIV + TB\nSIV + ART + TB\nC>T / G>AC>A / G>TG>C / C>GT>C / A>GA>C / T>GA>T / T>ADeletionInsertion\nE\n104\n102\n103\n105\n10\nOxidative \nmutationsNon\n-oxidative \nmutations\nTotal CFU at origin site\np = 0.09\n*p < 0.01\n1\n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2026. ; https://doi.org/10.64898/2026.04.03.714442doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2026. ; https://doi.org/10.64898/2026.04.03.714442doi: bioRxiv preprint \n\nSynonymous\nNon-synonymous\nIntergenic\n0.0\n0.2\n0.4\n0.6\n0.8\nFig 4C: %Prevalance at origin sites\n% Alternate Alleles at tissue\n0.0381\nIn vivo SynIn vivo NonIn vivo IG\n0.0\n0.2\n0.4\n0.6\n0.8\n1.0% of tissues within network\nFigure 4D: Fig 4D: Dissemination: % of network\n0.3458\nA\nC\nLieberman: 403 Liu: 257\nNHP: 95\n40\n1\n16 15\nNumber of genes \nwith mutations\ncell wall and cell processes\nconserved hypotheticals\ninformation pathways\nintermediary metabolism and respiration\nlipid metabolism\nvirulence, detoxification, adaptation\nregulatory proteins\nPE/PPEunknown\nstable RNAs\ninsertion seqs and phages\n0.0\n0.1\n0.2\n0.3Fraction of genes\nH37Rv\nNHP_all\nQL_all\nTL_all\nIntermediary Metabolism & \nRespiration\np < 0.001\nCell wall / Cell ProcessesConserved Hypotheticals\nInformation Pathways\nLipid Metabolism\nPE/PPE\nVirulence, detoxification & \nadaptation\nRegulatory Proteins\nFraction of genes in dataset\n0\n0.1\n0.2\n0.3\np = 0.05\np < 0.05\nNHP in vivo derived\nLieberman et al\nLiu et al\nH37RV\np < 0.05\np < 0.001\np < 0.05\np = 0.02\nB\nD E\nPercent variant alleles at \neach origin tissue\nSynonymous\nNon\n-synonymousIntergenic\n0\n20\n40\n60\n80\n*p < 0.04\nrv1925 (fadD31)\nrv1648\nrv0232\nCell wall process\nLipid metabolism\nIntermediatory metabolism\nInformation system\nTRUEFALSE TRUEFALSE\n0\n20\n40\n60\n80\n100\nClinical strain by % type of mutation\n% of all mutations\nC>T_G>A\nC>A_G>T\nG>C_C>G\nT>C_A>G\nA>C_T>G\nA>T_T>A\nDeletion\nInsertion\nTB\nSIV\nART\nIn clinical\nstrains?\n100\n80\n% of all mutations\n0\n20\n40\n60\nYes\n*p = 0.04\nNo\nA>C / T>G\nA>T / T>A\nDeletion\nInsertion\nC>T / G>A\nC>A / G>T\nG>C / C>G\nT>C / A>G\nSynonymous\nNon\n-synonymousIntergenic\n% barcode network with variant\n0\n20\n40\n60\n80\nrv1648\nrv0154c (fadE2)\n100\np = 0.35\nrv1384\nrv3665\n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2026. ; https://doi.org/10.64898/2026.04.03.714442doi: bioRxiv preprint \n\nLipases\nppe28*\nlipC \nlipJ\nrv2075 \nrv2565\n(rv1835)\n(rv3479)\nlipG\n(lucA)\nFACL\nfadD3\n1\nFAAL\n(fadD34)\nLipid / metabolite synthesis\npks7*, pks8, pks11, pks12 , rv0232, pta, entC, \ntesB1, grcC1, rv1520, ptbB\n(pks1), (pks2), (rv1524), (mbtD)\nubiA, (rv1520)\nMycolic Acids\nPDIM / TDM\nMPM\nTransport\nirtA\nkdpC\nctpI\nrv3728\nrv3273\nrv0987*\nrv0943\ndppA\ndppB\n(ceoB)\npitB\ncorA\n(ctpI)\n(rv2508c)\nCell Envelope\notsB1\nrv1648\nrv0225\nrv3594\nPE_PGRS36\nPE_PGRS43\n(rv2402)\n(glgE)\n(murE)\n(glfT2)\n(PE_PGRS9)\nPPE16\n(rv1230)\n(PPE42)\n(aftA)\nRv1433*\nPE_PGRS1\nrv1648\nRedox / \nRespiration\npntAa\nfbiA\nrv1812c \nrv2295\nrv0785\nrv0839* \nnarK3\ntcrA*\nrv3698\nmoaA2\n(narU)\n+\nLAM\nSulfolipids\nIsonitrile Lipopetides (Cu)\nMycobactins (Fe)\n• Rewired (lipid) \nmetabolism?\n• Biosynthetic \nelectron sinks?\n• Mtb virulence \n(lipid) synthesis?\n• Redox imbalance?\n• Metabolic toxicity?\n• Host immune damage?\nNucleotide \ndinP*\nruvB*\nuvrD1\nparE2 \nrnj\nupp\nparA\nparE2\n(rv3202)\nrecA*\nnrdF2\nercc3\nrv2559c\ndnaN\nrv2435c\nProtein / translation\npepC*, folC*, rv1002, rv0075, metE, rpsA, \n(ilvD), (rv0484), (carB), (rv2426), (rv3228)\nrv1112\nVirulence\nsapM\nIG_whiB6\nmycP2?\nBeta-\noxidation\nfadE2*\nechA11\n(fadE31)\n(echA9)\nOther metabolism\nrv0939.hpaG \n(catechol)\n(rv1026 (ppGpp?))\noxcA (oxalate)\n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2026. ; https://doi.org/10.64898/2026.04.03.714442doi: bioRxiv preprint \n\nGene DNA \nchange \nProtein \nChange Mutation # Events # \nStrains \nrv0785 C>CG NonSyn Ala561fs 2 2 \nrv0987 A>G NonSyn Ser710fs 3 3 \npks7 CG>C NonSyn Ala1174fs \nA: 5 \nB: 2 \nA: 21 \nB: 3 \nrv0075 G>A NonSyn Gly175Ser 1 1 \nrv0943 G>A NonSyn Ala54Val 1 1 \nrv1648 A>G NonSyn Ser26Gly 1 3 \npks8 C>T NonSyn Thr1274Met 1 1 \nlipJ G>A NonSyn Ala390Val 1 4 \nrv2075 C>A NonSyn Val415Phe 1 1 \nrv2565 C>T NonSyn Ser292Phe 1 1 \nrnj T>C NonSyn Arg361Gly 1 10 \nentC C>T NonSyn Ala270Val 1 8 \nrv3594 G>A NonSyn Arg215His 2 3 \nfadD34 C>T Syn Gly143Gly 1 1 \nRv1520 T>C Syn Arg81Arg 1 1 \nRv1835c C>T Syn Ala326Ala 2 2 \nRv2402 G>A Syn Ala324Ala 1 1 \nRv2426c T>G Syn Ala256Ala 3 12 \nRv3228 G>A Syn Leu102Leu 2 6 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2026. ; https://doi.org/10.64898/2026.04.03.714442doi: bioRxiv preprint \n\nfadE31 C>T Syn Asp190Asp 2 4 \naftA C>T Syn Arg5Arg 5 27 \nglfT2 G>A Syn Asn229Asn 1 3 \nPE-PGRS38-\npbpB C>A IG PGRS38 (-20) 1 1 \nRv3268-\nRv3269 A>C IG rv3269 (-110) 1 2 \nwhiB6-\nRv3863 C>T IG whiB6 (-25) \nC>T: 4 \nC>G:2 \nC>T:4 \nC>G:10 \n \nTable 1. NHP intrahost mutations are also observed in human Mtb clinical strains . Identical \ngenomic mutations in the NHP dataset were matched to mutations observed in over 55,000 \nclinical isolates 27 . Clinical strain mutations and the number of independent mutations events \nwere predicted by comparing each clinical strain against a Mtb ancestral genome \nreconstruction. Nonsyn = non-synonymous, Syn = synonymous, IG = intergenic. Pks7 mutations \nwith an ‘ A ’ and ‘B’ indicate different mutations leading to a frameshift in the same codon, while \nwe find two diff erent types of intergenic whiB6 mutations at the same position. \n \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted April 4, 2026. ; https://doi.org/10.64898/2026.04.03.714442doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}