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Isku-darka genome-ka ayaa dib u soo nooleeyay goobta cilmi-baarista cudurrada faafa, muujinta cudurrada faafa ee cudurrada, pathogenesis, isdhexgalka martida-pathogen, iyo habka korriinka ee lagu soo rogay cudurada. Dhismaha qaaxada ee Mycobacterium (MTBC) waxa ay u arka Mycobacterium bovis mid ka mid ah xubnaheeda la qabsiga xayawaanka ee keena qaaxada (TB) ee naasleyda dhulka, waana qaabka caadiga ah ee horumarka bakteeriyada. Sida xubnaha kale ee MTBC, Mycobacterium bovis waxa loo malaynayaa in uu yahay mid si adag u xidhan, si tartiib ah u kobcaya cudur-sidaha, mana jirto wax calaamad ah oo dib-u-habayn ah ama wareejinta hidde-sidaha tooska ah. Shaqadan, waxaan ku dabaqeynaa genomics isbarbardhigga xogta guud ee taxanaha genome (WGS) oo ka kooban 70 lo'da M. oo ka kala yimid faracyo kala duwan (Yurub iyo Afrika) si aan u helno fikrado ku saabsan kala duwanaanshaha hidde ee lo'da M. Awoodda korriinka. Saddex hab oo kala duwan ayaa loo isticmaalaa in lagu qiyaaso calaamadaha dib-u-habaynta. Caalamka, tiro yar oo ah dhacdooyinka dib-u-habaynta ayaa la aqoonsaday oo la xaqiijiyay laba hab oo madaxbannaan oo taageero adag leh. Si kastaba ha ahaatee, marka la barbar dhigo isbeddellada, dib-u-habaynta waxay saameyn daciif ah ku leedahay kala duwanaanshaha M. bovis (guud ahaan r/m = 0.037). Farqiga r/m celceliska ee laga helay isku-dhafka clonal ee Mycobacterium bovis ee xogtayada xogta waxay la socotaa fikradda guud ee ah in heerka dib-u-habaynta ay si weyn u kala duwanaan karto inta u dhaxaysa nalalka loo qoondeeyay isla noocyada taxonomic. Iyada oo ku saleysan shaqadan, dib-u-habaynta Mycobacterium bovis lama saari karo, markaa waa in ay noqotaa mawduuca dadaalka dheeraadka ah ee cilmi-baarista isbarbardhigga mustaqbalka, kaas oo WGS ee xogta ballaaran ee xaaladaha kala duwan ee cudurrada faafa ee adduunka oo dhan ay muhiim tahay. Falanqaynta dheeraadka ah ayaa markaa lagu sameeyay xogta Mycobacterium bovis-ka yar (n = 42) oo ka soo jeeda tirada badan ee TB-da, iyo in ka badan 1,800 loci ayaa la aqoonsaday, kuwaas oo ugu yaraan mid ka mid ah uu muujiyay hal nucleotide polymorphism (SNP). Inta badan (87.1%) waxay ku yaalaan gobolka codaynta, iyo saamiga caalamiga ah ee isbeddellada aan isku midka ahayn (dN/dS) ee isbeddellada isku midka ah ayaa ka sarreeya 1.5, taas oo muujinaysa in xulashada togan ay tahay awood horumarineed oo muhiim ah oo lagu sameeyay M. bovis. Qayb sare oo ka mid ah SNP-yada ayaa lagu ogaadey hiddo-wadaha hodanka ku ah qaybaha shaqada ee "shiid kiimikaad", "derbiga unugga iyo hababka gacanta", iyo "shiid dhexdhexaad ah iyo neefsasho", oo muujinaya kartidooda bayoolaji iyo horumarka Mycobacterium bovis muhiimadda. Fiirin dhow ee hidda-wadaha awoowayaasha MTBC ee u nugul wareejinta hidde-sidaha tooska ah oo lagu daray nidaamka 3R (dib-u-habaynta DNA, dib-u-celinta, iyo dib-u-habaynta) waxay muujineysaa celceliska qiimaha xun ee caalamiga ah ee tijaabada dhexdhexaadka ah ee Taijima D, taas oo muujinaysa iskaanka xulashada ee la soo dhaafay.
Mycobacterium tuberculosis complex (MTBC) waa mid ka mid ah canshuuraha ugu guulaha badan ee cudurada bakteeriyada iyo kiis caadi ah oo kobcinta bakteeriyada. Xubnaheedu waxay muujinayaan aqoonsiga nucleotide ee la yaabka leh ee heerka genomic (> 99%)1,2 . Noocyada kala duwan ee MTBC Ecotypes waxay keeni karaan tiibisho (TB), oo ah cudur granulomatous faafa, oo ka mid ah noocyada kala duwan ee martida loo yahay laga bilaabo naasleyda yaryar ilaa aadanaha3,4,5. Hadda, kakan waxa ku jira bini'aadamka [M. Qaaxada (Mtb), Mycobacterium africanum] iyo cudur-sidaha xoolaha la qabsada (Mycobacterium bovis, Mycobacterium capitum, Mycobacterium pinnipedum, Mycobacterium microtobacter, Mycobacterium mongee, Mycobacterium miysani, Mycobacterium surika, "Bacillus chimpanzee" iyo "dassie" M. canettii (oo sidoo kale loo yaqaan "Nodobacter glabrata") Celceliska aqoonsiga nucleotide ee mycobacteria aan kor ku soo sheegnay waa 98%, iyo shaqada genomics ee isbarbardhigga ayaa muujisay in M. canettii iyo inta kale ee MTBC ay dhawaan ka soo jeedaan awoowayaasha caadiga ah.
MTBC si nidaamsan ayaa loogu sifeeyaa inay tahay iskudhaf adag oo clonal ah, qaabdhismeedkeeda dadwaynahana waxa si cad u maamula kala duwanaanshiyaha la dhimay, dhalooyinka, sawirida xulashada, iyo qulqulka hiddaha9,10. Iyadoo loo malaynayo in koboca clonal adag oo adag, sida polymorphisms maqan laguma soo celin karo dib-u-habayn. Iyadoo lagu salaynayo fikradan, dhacdooyinka isdaba jooga ah ee tirtirka genomic ee gobolka kala duwan (RD) iyo TbD1 (Mtb gaar ah tirtirka gobolka 1) ayaa loo soo jeediyay inay yihiin calamadaha molecular ee MTBC evolution2,5,11. Genomics isbarbardhigga iyo isku-xiridda genome-ga oo dhan (WGS) waxay taageertaa kala qaybinta xubnaha bani-aadamka-qabsiga ee sagaal qaybood (Mycobacterium tuberculosis L1 ilaa L4, L7, iyo L8; iyo Mycobacterium africanum L5, L6, iyo L9), lineages L2 ilaa L4 la wadaago tirtirka TbD1 gobolka2,131.12 Intaa waxaa dheer, xubnaha xayawaanka la waafajiyay ayaa la soo jeediyay inay wadaagaan awoowe wadaag ah, kaas oo lagu qeexay tirtirid gaar ah oo ku jira RD7, RD8, RD9 iyo RD102, 5, iyo 14.
Gudbinta hidda-sidaha horizontal (HGT) iyo dhacdooyinka dib-u-habaynta ayaa loo arkaa naadir waxayna ku dhacaan awoowayaashii MTBC, halkii ay ka ahaan lahaayeen taariikhda kala duwan ee xubnaha MTBC oo dhan15,16,17. Laba warbixinood oo hore oo ay soo saareen Hughes iyo wada-shaqeeyayaasha (2002) iyo Gutacker and collaborators (2006) ayaa soo jeediyay in dhacdooyinka dib-u-habaynta ay gacan ka geysan karaan qaabaynta polymorphisms kuwaas oo calaamad u ah loci gaar ah oo ku jira noocyada M. qaaxada18,19. Sababaha muuqda ee dib-u-habayn la'aanta MTBC waa: (1) habka farsamada iyo luminta awoodda HGT; (2) naadirka dhacdooyinka HGT; (3) ma jirto wax fursad ah oo ah dhacdooyinka dib-u-habaynta ee MTBC niche14,17. Dhawaan, qaar ka mid ah daraasadaha isku xigxiga-genome-ga ah (WGS) ee lagu dabaqay cadaadiska MTBC 20 iyo Mycobacterium bovis 21 ayaa bixiyay caddaynta dib-u-habaynta, marka ugu horeysa ee muujisa in noocyada MTBC ay inta badan beddelaan jajabyada DNA-da yar, laakiin sababtoo ah kala duwanaanshaha isku xigxiga nucleotide xaddidan, dhacdooyinkan Wali lama ogaan.
Mycobacterium bovis waa xubinta MTBC ee inta badan laga soo kabsado xoolaha (inta badan lo'da), inkasta oo sidoo kale laga go'doomi karo xayawaanka duurjoogta ah ee xorta ah iyo deyr leh4,22,23,24. M. bovis waxa uu u xuub siibtay shan dhismo oo waaweyn oo clonal ah [European 1 (Eu1), European 2 (Eu2), European 3 (Eu3), African 1 (Af1) iyo Africa 2 (Af2)], marka loo eego profile spoligotyping, tirtirid gaar ah Iyo hal nucleotide polymorphisms (SNPs) Dhismayaashan clonal waxay muujinayaan qaabka kala duwan ee dadka Mycobacterium bovis iyo xiriirka ay la leeyihiin gobollada juqraafiyeed. Intaa waxaa dheer, shaqadii WGS ee dhawaan ay fulisay Zimpel iyo la-hawlgalayaasheeda (2020) waxay naqshadeeyeen phylogeny ku salaysan SNP ee Mycobacterium bovis, oo leh in ka badan 1,900 genomes, taas oo muujinaysa in ay jiraan ugu yaraan afar nooc oo kala duwan (oo lagu magacaabo Lb1 ilaa Lb1 ilaa Lb4), ma aha kuwo gebi ahaanba la jaan qaadaya kakanaanta hore ee 3. Qorayaashani waxay sameeyeen falanqayn kala duwan oo ku saabsan phylogeny iyo shukaansi molecular, laakiin ma aysan baran dib-u-habaynta30.
Shaqadii hore iyadoo la adeegsanayo farsamooyin molecular oo kala duwan, sida spoligotyping, MIRU-VNTR (mycobacterial interspersed reply unit-variable tandem repeat number), iyo SNP dhawaan ku qorid ayaa muujisay heer gaar ah oo kala duwanaansho hidde ah oo ka mid ah noocyada M. bovis 31,32,33, 34,35. Kala soocida kala duwanaanshiyaha hidde-sidaha ayaa noqday qalab muhiim u ah daraasadda cudurrada faafa, taas oo caawinaysa faham qoto-dheer oo ku saabsan cudur-sidaha, fayraska iyo gudbinta cudurrada. Soo ifbaxa habka WGS wuxuu bixiyaa suurtogalnimada in lagu muujiyo arrimaha wadista kobcinta ee ay ku soo rogeen Mycobacterium bovis genome ee habka la qabsiga iyo ku adkaysiga martida kala duwan iyo xaaladaha cudurrada faafa.
Shaqadan, waxaan u isticmaalnaa falanqaynta genomic isbarbardhigga ee kala duwan ee Mycoplasma bovis datasets (n=70), oo ay ku jiraan go'doominta dhismooyinka clonal ee kala duwan, si aan u helno fikrado ku saabsan geeddi-socodka kobcinta ee Mycoplasma bovis, gaar ahaan si loo xalliyo xiriirada phylogenetic iyo dhacdooyinka dib u habeynta. Sida lagu kabo falanqayntan, xog-hoosaadyada M. bovis go'doomin (n = 42) laga helay aagga qaaxada badan ee martida loo yahay oo si fiican loo yaqaan oo ku taal Portugal 31,36 ayaa la sii baadhay si loo ogaado aqoonsi la'aanta dheelitirka u dhexeeya saamiga qaraabo ee dareenka (dN) ee isku midka ah (dS) beddelka nucleotide, iyo sidoo kale horumarinta qayb ka mid ah suugaanta ayaa lagu sheegay. 37,38 helay awoowayaasha MTBC iyada oo loo marayo HGT, iyo encode 3R (dib-u-dayactirka DNA, ku-noqoshada, iyo dib-u-habaynta) qaybaha hidda-socodka Hidde-sidayaasha ku jira nidaamka 3R ayaa la doortay sababtoo ah shaqadii hore ee M. noocyada qaaxada waxay muujiyeen doorashooyin guud oo taban / nadiifin ah oo ka shaqeeya hiddo-wadahaas, waxaana laga yaabaa inay door muhiim ah ka ciyaaraan horumarinta 39. Hadafka kale ee shaqadani waa in la ogaado jiritaanka dhacdooyinka dib-u-habeynta. Sababtan awgeed, iyada oo la tixgelinayo in xogtayada Portugal oo kaliya ay ka kooban tahay genome-ka isku-dhafka ah ee Yurub ee clone 2 iyo noocyada aan loo qoondeeyn dhismaha clone, waxaan go'aansanay inaan ku darno xogta genome-ka ee dadweynaha ee la heli karo si aan ugu dambeyntii u helno wakiil ka mid ah dhammaan dhismayaasha clone, iyo hagaajinta xoojinta iyo baaxadda natiijooyinka.
42 cusub oo Mycoplasma bovis genomes ah oo laga soo qaaday goobta qaaxada ee martida loo yahay ee Bortuqiisku (faahfaahinta hoose), oo hore loogu sifeeyay aragtida epidemiological36, ayaa ah xarunta shaqadan. Iyadoo la tixgelinayo in xogta laga soo xigtay Portugal oo kaliya ay leedahay wakiilo ka mid ah dhismayaasha 2 clone ee Yurub iyo noocyada iyada oo aan lahayn dhismo loo qoondeeyey, si guud oo la heli karo xogta isku xigxiga-genome-ga ayaa lagu daray si loo ballaariyo xogta xogta oo ay ku jiraan dhammaan wakiilada M. bovis clone. Sidaa darteed, saddex ilo xogta isku xigxiga oo dhan-genome ah ayaa loo adeegsaday shaqadan: dhammaystiran/qabyo ururinta genome-ga, ilaa 10 xabo oo lagu kaydiyay NCBI (Xarunta Qaranka ee Macluumaadka Biyotechnoolajiyada) (n = 15 go'doomin); lagu kaydiyay SRA (Faylka Illumina fastq ee kaydka akhriska ee taxanaha ah) wuxuu u taagan yahay kala duwanaanshaha kakan ee M. bovis clones (n = 12 go'doomin)30; iyo 42 genome oo dhowaan la soo dejiyey oo ka yimid Portugal. Mycobacterium bovis BCG (Bacille Calmette-Guerin) ayaa laga saaray raadinta NCBI. M. bovis AF2122/97 waxaa badanaa loo isticmaalaa genome tixraac ahaan si loogu daro xogta. Sababtoo ah helitaan la'aanta dadwaynaha ee dhammaan taxanaha genome-ga ee ay matalaan iskudhafka 1 ee Afrika, iyo tirada yar ee genome ee noocyada matalayaasha Af2 iyo Eu1, xogta isku xigxiga asalka ah ee ay bixisay SRA ayaa loo adeegsaday kiisaskan. Shaqada Zimpel iyo la-hawlgalayaasheeda (2020) waxay gacan ka geysteen in la aqoonsado genome-ka ka soo baxa isku-dhafka cloning ee aan soo sheegnay waxayna gacan ka geysteen xulashada Mycobacterium bovis si loogu daro xogta. Eu3, kaliya hal nooc oo genome ah ayaa lagu sifeeyay (Branger et al., 2020), marka genome-ka aan ku darno waa wakiil gooni ah oo ka kooban Eu3.
Caalamka, xogta this set ka mid ah 70 lo'da M. bovis ka go'doomin 8 noocyada martida loo yahay, qaybiyey 12 dal laga bilaabo 1985 ilaa 2016. 36 noocyada waxaa loo qoondeeyey sida Eu2, 7 noocyada waa Eu1, 1 nooc waa Eu3, 3 nooc waa Af1, 4 noocyada waa Af2 iyo 19 aan loo saarin wax kakan. Macluumaadka faahfaahsan (oo ay ku jiraan lambarka gelitaanka) ee Mycobacterium bovis ee loo adeegsaday daraasaddan waxa lagu muujiyey Shaxda 1 iyo Kaabista Shaxda 1.
42 cusub oo isku xigxigay oo dhan genome of Mycobacterium bovis oo ka yimid meelaha qaaxada xoolaha ee Portugal oo la qaybiyey in ka badan 12 sano ayaa udub dhexaad u ah daraasaddan, maadaama nidaamyada cudurada duurjoogta-xoolaha si joogto ah loola socdo 31,36 (Jaantuska Dheeraadka ah 1). Marka loo eego habraacyada xiga, noocyadan ayaa laga soocay lo'da (n = 14), deerada cas (n = 16) iyo doofaarka duurjoogta ah (n = 12) laga bilaabo 2003 ilaa 2015: ururinta iyo maaraynta xoolaha iyadoo la raacayo habraaca habraaca lagu taliyey. Shaybaarada unugyadu waxay ku jiraan OIE Terrestrial Animal Handbook waxayna ku jiraan Stein-Jeen iyo Löculated oo ku yaal Buug-gacmeedka Xayawaanka ee OIE Terrestrial. dareere dhexdhexaad ah. Dhaqamada waxaa lagu beeray 37 °C waxaana korriinka la eegaa todobaadkii hal mar ugu yaraan 12 toddobaad. Deegaannada waxaa si toos ah loogu kaydiyaa xal glycerol ah -80ºC. Dhexdhexaadinta xulashada Mycobacterium (Middlebrook 7H9, BD Diagnostics), muunada asalka ah ee kaydsan ayaa la dhex maray hal mar oo vitro in vitro si loo helo DNA-da barnaamijka WGS. Taas awgeed, xalka kaydka dhaqanka ee barafaysan waxaa lagu hodmay 5% sodium pyruvate iyo 10% ADS (50 g albumin, 20 g glucose, 8.5 g sodium chloride in 1 L water) on Middlebrook 7H9 at 37°C Retrain. Kadib 4 toddobaad oo korriin ah, dhexdhexaadinta ayaa dib loo cusboonaysiiyay waxaana si joogto ah loola socon jiray dhaqanka ilaa koritaanka la arkay. Unugyada waxaa lagu soo guray centrifugation, pellet-ka waxaa dib loogu celiyay 500 µL phosphate saline buffered (PBS), kulaylka 99 °C 30 daqiiqo, sentifuga, iyo supernatant waxaa lagu kaydiyay -20 °C ilaa WGS. Nidaamyada oo dhan waxaa lagu fuliyaa heerka 3 ee xarumaha badbaadada noolaha.
Maktabadda genome-ga ee WGS-dhamaadka-dhamaadka ah waxaa loo diyaariyey iyadoo la adeegsanayo tusmada gaarka ah ee muunad kasta oo DNA ah, waxayna isticmaashaa Illumina MiSeq (2 × 250 pb) (40 muunado) iyo HiSeq (2 × 150 pb) (laba go'doomin) tignoolajiyada (Eurofins Genomics, Jarmalka) isku xigxiga. Marka loo eego tilmaamaha soo-saaraha, isticmaal Illumina Genome Analyzer oo leh lifaaqa moduleka laba-dhammaadka ah si aad isugu xigto DNA-da genomiga ah, oo isticmaal Qalabka Diyaarinta Maktabadda Nextera XT DNA ee Illumina si aad u dhisto maktabadda.
Iyadoo la tixgelinayo xogta laga helay SRA (n = 12), aqoonsiga dhismaha clone waxaa loo isticmaali karaa sida metadata daabacaadda u dhiganta 30, 41, 43. Marka la tixgeliyo genome dhamaystiran, marka laga reebo Mycobacterium bovis AF2122/97 iyo Mycobacterium bovis 3601, kuwaas oo la aqoonsan yahay 19 iyo xubnaha E5 ee E5. siday u kala horreeyaan, waxay la mid tahay genome-ka dhamaystiran ee qaaxada Mycobacterium H37Rv (lambarka gelitaanka NCBI NC_000962.3). alignment genome waxaa lagu sameeyaa iyadoo la isticmaalayo MAFFT (barnaamijka isku toosinta badan ee amino acid ama taxanaha nucleotide, nooca 7.458) iyo parameter -addfragments48. Kadib, raadi maqnaanshaha dhismooyinka clonal ee kala duwan iyo/ama joogitaanka astaamaha SNP.
Mycobacterium bovis (n = 42) iyo akhrinta asalka ah ee qabyada genome-ga la soo ururiyay (n = 3) waxay la jaanqaadaan kakanaanta tixraaca genome-ga Mycobacterium tuberculosis H37Rv iyada oo loo sii marayo dhuumaha vSNP iyo joogitaanka tirtirka iyo/ama SNP sifooyinka kala duwan ee clones Baadhitaan ayaa la sameeyay.
Ka soo ururi macluumaadka la'aanta sifooyin iyo/ama joogitaanka/maqnaanshaha SNP iyo profiles-ka-soo-saarista si loogu meeleeyo xogta genomiga isku-dhafka clonal ee u dhigma. Afarta gole ee qabyo qoraalka ah, astaanta qalafsanaantu lama qiyaasi karo, sidaas darteed waxay ku jiraan kooxda "wax kakan".
Socod-socodka bioinformatics-ka ee ay raacdo shaqadani waxay ka bilaabataa de novo kulanka iyo khariidaynta istaraatiijiyadda tixraaca, iyada oo ujeedadu tahay sahaminta dhacdooyinka dib-u-habaynta iyo polymorphisms-ka genome-ga gaarka ah. Jaantuska 1 waxa uu bixiyaa jaantuska socodka tallaabooyinka la raacay. Falanqaynta dib-u-habaynta, dhammaan genome-yada ayaa loo isticmaalaa si loo kordhiyo adkaanta fikradaha iyo tilmaamayaasha la xidhiidha.
Si loo yareeyo khaladaadka jiilka isku xigxiga genome-ga, waxaan marka hore helnay de novo shirka, ka dibna waxaan helnay alignments badan oo asaasiga ah. Dhuumaha Unicycler ayaa hadda laga heli karaa https://github.com/rrwick/Unicycler49 waxaana loo isticmaalaa in lagu sameeyo kulanka de novo ee 54 genomes taxane ah (42 cusub oo taxane ah iyo 12 faylal fastq ah oo laga helay SRA). Marka la soo koobo, ka hor inta aan la soo ururin xoqin, falanqaynta tayada akhrinta waxaa lagu sameeyay nooca FastQC 0.11.7 (https://github.com/s-andrews/FastQC), iyo nooca Trimmomatic 0.36 (ikhtiraacida "gooynta Adapters iyo taxane kale oo iftiin gaar ah oo laga soo bilaabo akhrinta" iyo "gooy saldhigyada tayada hoose ee 2) (http://www.usadellab.org/cms/?page= trimmomatic) 50. Ka dib, SPAdes optimiser49 waxa loo isticmaalay isu imaatinka genome, iyo nooca Pilon 1.1851 ayaa loo isticmaalay hagaajinta golaha kadib. Habka isku xidhka muxaafidka ah ayaa la doortay si looga fogaado isu imaatinka khaldan, iyo cabbirka k-mer waa la baadhay oo la doortay inta u dhaxaysa 20% iyo 95% dhererka akhrinta. Raac tilmaamaha SPAdes oo tixgeli cabbirka akhrinta, tirtir wareegyada ka yar 300 bp, oo samee 20 akhrinta qoto dheer ee daboolida daboolida 52. Istaraatijiyadda de novo shirka, gobollada genomic sida proline-glutamate (PE) iyo proline-proline glutamate (PPE) paralogs lama saarin.
Tayada shirka de novo waxaa lagu qiimeeyaa dhuumaha QUAST (http://quast.sourceforge.net/quast.html), taas oo sahlaysa cusboonaysiinta contig iyo genome tixraaca M. bovis AF2122/97 (NCBI accession number LT708304.1) Mapping (eeg Jadwalka Dheeraadka 1 ee cabbirada tayada).
Iyada oo la kaashanayo dhuumaha vSNP (https://github.com/USDA-VS/vSNP), faylka FASTQ ee taxanaha cusub ee M. bovis ee taxanaha Illumina ayaa la barbardhigay M. bovis AF2122/97 genome tixraaca (LT708304.1)) . Marka loo eego talooyinka ku-dhaqanka ugu wanaagsan ee Qalabka Falanqaynta Genome (GATK) 53, 54, 55 waxay dabaqaan cabirrada shaandhada caadiga ah ama buundooyinka tirada badan ee kala duwan ee dib u habeynta. Natiijooyinka waxaa lagu sifeeyaa iyadoo la isticmaalayo dhibcaha guud ee SAMtools ugu hooseeya ee 150 iyo AC = 2. Sidoo kale isticmaal Kraken (http://ccb.jhu.edu/software/kraken/) si aad u hubiso akhrinta si aad meesha uga saarto wasakhda. Dhuumaha vSNP ee loo isticmaalo in lagu khariidado istiraatijiyado isku xigxiga shaqadeena waxa ay baadhaan taxane ah SNPs iyo bartilmaameedyo, waxa kale oo ay meesha ka saaraysaa xaaladaha caabuqa isku dhafan. Daboolida genome-ka akhrida ayaa ka wanaagsan 99% (Shaxda Dheeraadka ah 1).
Si looga fogaado khaladaadka khariidadaha iyo SNP-yada khaldan, sifee kala duwanaanshiyaha xaaladahan soo socda: (1) waxaa taageeraya wax ka yar 20 akhrin, (2) waxaa lagu helaa inta jeer ee ka yar 0.9, (3) waxay ku jirtaa ugu yaraan hal Strains, laakiin ugu yaraan waxaa jira nusqaamo dhinaca kale ah. Nooca isku-dhafan ee genomics-ka (IGV) 2.4.19 (http://software.broadinstitute.org/software/igv/)56 ayaa loo adeegsaday si muuqaal ahaan loo xaqiijiyo SNPs iyo jagooyinka leh dhibaatooyinka khariidaynta ama toosinta. Tan iyo markii hiddo-wadaha proline-glutamate (PE) iyo proline-proline glutamate (PPE) ay aad u nuqulan yihiin oo ay qayb ka yihiin qoyska hiddo-wadaha badan, waxay si fudud u fahmi karaan taxanaha Illumina iyo khariidad khaldan, sidaas darteed waxay doorbidaan shaqada bioinformatics mycobacterial ee laga saaray xubnaha tuberculosis waxay xaqiijinayaan isku-dhafka marka la isticmaalayo istaraatiijiyada khariidaynta si isdaba-joog ah. Sidaa darteed, waxaanu falanqaynta ka soo saarnay hiddo-wadaha PE/PPE iyo indels-ka.
Sida laga soo xigtay Bovilist (http://genolist.pasteur.fr/BoviList/), dhammaan SNP-yada waxaa loo qaybiyaa qaybo shaqaynaya. Dhuumaha SnpEff (https://pcingola.github.io/SnpEff/) waxaa loo isticmaalaa in lagu ogaado cawaaqibka SNP (isbeddellada la midka ah ama kuwa aan isku midka ahayn). Waxay abuurtay xog cusub oo ah Mycobacterium bovis AF2122/97 genom (LT708304.1).
Isku-habaynta genome-ka xudunta u ah waxaa lagu sameeyay Parsnp v1.2, oo hadda laga heli karo https://github.com/marbl/parsnp57, iyadoo la adeegsanayo 69 genomes / qabyo ah oo dhammaystiran (oo leh ikhtiyaar -c) iyo M. bovis AF2122/97 (LT708304.1) ayaa loo isticmaalay tixraac ahaan. Afar qaybood oo kala duwan ayaa la sameeyay: kaliya xubnaha Eu2 cloning complex (n = 37), oo ay ku jiraan dhammaan xubnaha isku xidhka isku xidhka Yurub (n = 44), oo ay ku jiraan barta isku xidhka ee isku xidhka Yurub iyo Afrika cloning complex (n = 51), oo ay ku jiraan dhammaan Mycobacterium bovis daraasaddan (n = 70).
Isku toosinta xudunta u ah Parsnp waxaa loo isticmaalaa in lagu qiyaaso suurtogalnimada ugu badan (ML) geedka phylogenetic iyadoo la isticmaalayo CIPRES Science Gateway v3.3
Saddex algorithms oo kala duwan iyo qalab bioinformatics ayaa loo isticmaalaa si loo hubiyo joogitaanka dhacdooyinka dib-u-habaynta isbarbardhigga: SplitsTree4 software, Gubbins (lineage aan eex lahayn iyada oo loo marayo dib-u-habaynta taxanaha nucleotide) dhuumaha, iyo RDP4 (barnaamijka dib u soo celinta, nooca beta 4.101) software.
Habka kala qaybsanaanta ee lagu hirgaliyay SplitsTree4 v4.15.1 (http://www.splitstree.org/)59 waxa loo isticmaalaa in lagu xisaabiyo shabakada phylogenetic ee xidid la'aanta ah, iyada oo la adeegsanayo tijaabada Phi si loo xaqiijiyo tirakoobka, iyo muhiimada xadku waa p = 0.05. Falanqaynta is-waafajinta badan ee asaasiga ah ee Parsnp ayaa loo istcimaalaa gelinta ahaan, iyo kala qaybsanaanta kala-goynta sida halbeegga shabakad ahaan waa la xaqiiqsaday.
Dhuumaha Gubbins v2.3.1 LT708304.1.
Ugu dambeyntii, si loo xaqiijiyo dhacdada dib-u-habeynta ee ay soo jeedisay dhuumaha Gubbins, lixda algorithms ee lagu hirgeliyay RDP467 (RDP61, GENECONV62, Bootscan63, Maxchi64, Chimaera65 iyo SiScan66) ayaa lagu dabaqaa is-waafajinta badan ee Parsnp ee hoos yimaada goobaha caadiga ah. Waxaan go'aansanay in ugu yaraan saddex ka mid ah algorithms-yada lagu hirgeliyay RDP4 ay tahay inay si joogto ah u muujiyaan calaamad muhiim ah si loo xaqiijiyo dhacdo kasta oo dib-u-habayn.
Iyadoo la tixgelinayo in labadaba Gubbins iyo software RDP ay raadiyaan calaamadaha dib-u-habeynta iyagoo hubinaya isku-dhafka kala duwan ee asaasiga ah ee daaqada ilaa 500 bp, iyo xaqiijinta in ku darida hiddo-wadaha PE/PPE inta lagu guda jiro shirka de novo aysan faragelin doonin calaamadaha dib-u-habaynta ee la helay, falanqayn dheeraad ah ayaa lagu sameeyaa homolinearity Hubi agagaarka hidda-socodka aqoonsiga dhacdada dib-u-dhiska. Khariidadda isku-dhafka ah ee la adeegsanayo genome-ga dhammaystiran ayaa la dhisay iyadoo la adeegsanayo isku-habaynta MAUVE-multi-genome (http://darlinglab.org/mauve/mauve.html) si looga saaro rogrogmada genome-ga maxalliga ah ama leexinta. Intaa waxaa dheer, dhammaan genome-ka waxaa loo isticmaalay in lagu sameeyo falanqaynta homolinearity ee taxanaha amino acid iyada oo loo marayo server-ka SyntTax (https://archaea.i2bc.paris-saclay.fr/SyntTax/).
Falanqaynta qoto dheer ee xogta genome-ka ee laga helay nidaamka qaaxada ee martida badan ee Boortaqiiska waa in la hubiyo polymorphism-ka hiddo-wadaha lagu sheegay suugaanta. Hidde-sidayaashani waa 37,38 iyo hidda-sidaha 3R ee ay heleen awoowayaasha MTBC iyada oo loo marayo HGT ( hagaajinta DNA, dib u habeynta iyo dib u habeynta) qaybaha nidaamka 39. Isticmaal ClustalX v2.1 (http://www.clustal.org/clustal2/) oo isticmaal DnaSP v6.12.03 (http://wwwsp.ub. (π) iyo Tajima's D gelidda cabbirka tijaabada dhexdhexaadka ah.
Suurtagalnimada ugu badan (ML) geed phylogenetic ah oo ku salaysan 69 Mycoplasma bovis go'doomin iyo genome tixraac ah ayaa la helay (Jaantuska 2A). Marka la barbardhigo geedaha hal-abuurka ah ama geedo badan oo ku salaysan, istaraatiijiyadani waxay u oggolaanaysaa abuurista geedo awood badan oo aan qabsanayn isbeddelka genome-ga oo dhan sidaas darteedna waxay muujinayaan awood hoose oo lagu takooro noocyada 68,69. Qaab dhismeedka sare ee geedka ML wuxuu caadi ahaan la socdaa kala soocida adag ee clones. Genome-ka Eu2 waxa uu ku urursan yahay laan, genome-ka Af1 sidoo kale waa la isku keenay (Jaantuska 2A). Natiijadu waxay sidoo kale la socotaa xiriirka horumarinta ee la yaqaan ee Mycobacterium bovis, taas oo ah, waxaa jira farqi weyn oo u dhexeeya xubinta Eu1 iyo kooxda ka kooban dhammaan noocyada kale ee clonal iyo genome, laakiin isku-dhafka clonal 30 lama cayimin. Isku dheelitir la'aanta yar ee udhaxeysa isku-dhafka clonal iyo xiriirka lagu arkay geedka phylogenetic waxaa lagu sharxi karaa xaqiiqda ah in isku-dhafka clonal lagu qeexay iyadoo lagu saleynayo gobollo gaar ah oo genomic ah, halka geedka phylogenetic uu ku salaysan yahay isku-xiryo badan oo ah genome-ga asaasiga ah ee matalaya genome-ga oo dhan.
Suurtagalnimada ugu badan ee geedka phylogenetic (GTR) ayaa la dhisay iyadoo lagu salaynayo isku toosinta genome-ga xudunta u ah Mycobacterium bovis genome ka hor (A) iyo ka dib (B) ka saarida goobta dib-u-habaynta. Midabada laanta waxay u taagan yihiin dhismaha Mycobacterium bovis clone: Yurub 1 waa guduud, Yurub 2 waa casaan, Yurub 3 waa buluug, Afrika 1 waa orange, iyo Afrika 2 waa cagaar. Geedku waa la rujiyey oo loo sawiray miisaan, dhererka laantana waxaa lagu qiyaasaa beddelka goob kasta.
Kakanaanta qaaxada Mycobacterium waxaa lagu sifeeyaa sida clonally horumarsan, iyo inta badan cadaymaha la ururiyey sannadihii la soo dhaafay waxay taageertaa fikradda ah in HGT socda iyo dhacdooyinka dib-u-habaynta aysan ku dhici doonin heerka la ogaan karo ee MTBC15,17,18.
Shaqadii hore waxay muujisay in ay jiri karto dib-u-eegis xaddidan oo u dhexeeya MTBC strains20,21, halka kuwa kale ay ku guuldaraysteen inay aqoonsadaan dhacdooyinka dib-u-habaynta la qiyaasi karo70,71. Dib ugala hadal arrintan adigoo diiradda saaraya Mycobacterium bovis, taas oo ka duwan shaqadii hore ee kaliya ee loo tixgeliyey qaaxada Mycobacterium 70,71; ama tixgeli MTBC guud ahaan, iyada oo ku dhawaad uusan jirin M. bovis oo matalaya 20; ama ka fiirso oo kaliya jajabyada lo'da xaddidan. Xogta loo yaqaan 'mycobacteria dataset', shaqadan, waxaa jira wadar dhan 70 nooc, oo ka dhigan dhammaan isku-dhafka 'clonal complexes', oo loo isticmaalo baaritaanka dib-u-habaynta. Qaabka xogta waxaa loo miisaamay iyadoo loo eegayo afar heerar oo isugeyn ah: (1) xubnaha Eu2, (2) dhammaan xubnaha kakooban Yurub ee clone (ie Europe), (3) Yurub iyo Afrika clone complex (Eu + Af) iyo (4) Xog ururinta oo dhan (oo ay ku jiraan genomes oo aan lagu darin dhismooyinka cloning ee horay loo sharraxay).
Si loo sii daraaseeyo mala-awaalkan, shabakad kala-bax ayaa la sameeyay si loo qiimeeyo maqnaanshaha dhacdooyinka dib-u-habaynta ee u dhexeeya genomes, sababtoo ah habkani wuxuu sawiri karaa xiriirka awoowayaasha ee shakhsiyaadka iyo muujinta calaamadaha phylogenetic ee iska soo horjeeda. Dhammaan afarta xog ee falanqaynta ayaa xaqiijiyay jiritaanka wareegyada shabakadda (taas oo ah, meelaha aan ku dhicin hal geed), laakiin imtixaanka Phi ma laha taageero xisaabeed (Eu2, p = 0.0956; Europe, p = 0.1637; Eu + Af p = 0.2774; xogta oo dhan p = 1), taas oo muujinaysa jiritaanka saboolka ah ee dhacdooyinka. 3A-D).
Yurub 2 genomes (n = 37) (A), genomes Yurub (n = 44) (B), genome Yurub iyo Afrika (n = 51) (C) iyo xogta oo dhan (n = 70) (D).
Falanqayntan ka dib, iyada oo la tixgelinayo indho-indheynta wareegtada ee dhammaan shabakadaha, algorithm-ka dib-u-dhiska ee lagu hirgeliyay dhuumaha Gubbins ayaa lagu dabaqay dib-u-dhiska lineage clonal iyo in lagu kordhiyo qiyaasta saameynta dib-u-dhiska ee genome-ka M. bovis. Sheeg tirada isku-darka ah ee dhacdooyinka dib-u-habaynta, kuwaas oo intooda badan ka dhacay laamaha terminal (taas oo ah, hal genome) (Shaxda 2). Tilmaamayaashani waxay muujinayaan joogtaynta xogta xogta oo dhan waxayna tilmaamayaan in inta jeer ee dhacdooyinka dib-u-habaynta ay tahay 200 ilaa 300 jeer ka beddelka. Marka qiyaasta rho/theta ee ka dhigan qiimaha qaraabada ee dib-u-habaynta iyo isbeddelka dhibcaha ee laanta waxay u muuqataa inay u dhexeyso 0.0037 iyo 0.0056 (Shaxda 3). Dhawaan, shaqada la daabacay ee cadaadiska 38 M. bovis waxay muujisay qiimo sare oo rho / theta ah (rho / theta = 0.1) marka loo eego tan laga helay xogtan, laakiin shaqada Patane iyo asxaabtiisu waxay adeegsadeen golaha tixraaca ku salaysan si ay u muujiyaan cabbirada dib-u-habaynta , Faahfaahinta habraaca, iyada oo ay ugu wacan tahay habka golaha, ayaa lala xiriiriyay dhacdooyinka dib-u-kicinta badan ee laanta.
Marka xigta, cabbirka r/m wuxuu u taagan yahay saamiga kala duwanaanshaha dib-u-habaynta iyo is-beddelka is-beddelka, iyo celcelis ahaan qiimihiisu wuxuu u dhexeeyaa 0.025 iyo 0.037, taas oo muujinaysa in marka la barbardhigo isbeddellada, dib-u-dhiska ay saameyn hoose ku leedahay kala duwanaanshaha hidde-sideyaasha M. bovis (Shaxda 3)). Isbarbardhigga ballaaran, hab la mid ah ayaa loo adeegsaday in lagu qiyaaso cabbirka r/m ee xogta MTBC oo ka kooban 23 genomes, taasoo muujinaysa celceliska qiimaha 0.48620, halka Patane iyo asxaabtiisa '38 M. bovis dataset, ay caddeeyeen celceliska qiimaha waa 0.98. Daraasaddii ugu horreysay, laba kaliya oo ka mid ah genome-yada 23 ee ku jira shaqada M. bovis (M. bovis BCG iyo cadaadiska tixraaca), sidaas darteed qiimaha la helay ayaa laga yaabaa inuu u xagliyo sababtoo ah xad-dhaafka ah ee genome M. qaaxada. Warbixinta labaad, dadka Mycobacterium bovis ee la lafa-guray ayaa inta badan laga soo kabsaday Maraykanka iyo xoolaha. Taas bedelkeeda, xogtayada, meelo badan oo juqraafi ah iyo noocyada martida loo yahay ayaa lagu soo bandhigay, iyo genome-ga oo loo kala qaybiyay dhismayaal clonal oo kala duwan oo leh astaamo hidde-siyeedyo kala duwan ayaa sidoo kale la adeegsadaa, sidaas darteedna lagu gaaro aqoon qoto dheer oo ballaaran oo dadweynaha ah. Farqiga r / m ee qiimaha celceliska ee la helay xogtayada xogta waxay la socotaa fikradda ah in heerka dib-u-soo-celinta ay si weyn u kala duwan tahay inta u dhaxaysa farriimaha loo qoondeeyey noocyada taxonomic isku mid ah, sidaas darteed natiijooyinkani waxay muujinayaan in isku-dhafka M. bovis clone laga yaabo inuu muujiyo kala duwanaansho dib-u-habeyneed Saamaynta sidoo kale waa sida ay soo jeediyeen Didelot & Maiden72. Si kastaba ha ahaatee, in si weyn loo balaadhiyo xogtan la dejiyay iyadoo lagu darayo tiro badan oo ah genome-ga M. bovis waxay oggolaanaysaa in si dheeraad ah loo caddeeyo qodobkan. Halbeegyada r/m iyo rho/theta labaduba waxay muujinayaan kala duwanaanshiyaha laamaha, natiijaduna waxay la socotaa warbixinnada noocyada kale ee bakteeriyada72,73.
Ugu dambeyntii, si loo xaqiijiyo dhacdooyinka dib-u-habaynta ee lagu aqoonsaday dhuumaha Gubbins, lix algorithms oo kala duwan ayaa loo adeegsaday software-ka RDP4 si ay si madaxbannaan u tijaabiyaan isbarbardhigga kala duwan ee asaasiga ah. Caalamka, in ka yar kala badh dhacdooyinka lagu aqoonsaday Gubbins waxaa xaqiijiyay RDP4 (Shaxda 4 iyo 5). Iyadoo la tixgelinayo dhammaan xogta xogta, saddex dhacdo oo dib-u-habeyn ah ayaa la xaqiijiyay, laba ku lug leh qanjidhada gudaha iyo kan kale oo ku lug leh hal genome oo ku yaal laan terminal, kaas oo dhismayaasha clonal aan loo qoondeyn karin (Shaxda 4 iyo 5). Aqoonsiga dhacdooyinka laamaha terminalku waxa laga yaabaa inay tilmaanto in dib-u-habaynta ay wali ku socoto noocyada M. bovis ee casriga ah ama natiijadu ay meel khaldan tahay70. Gobolkan dib-u-habaynta mala-awaalka ah, qiyaastii 20% boosasku waxay leeyihiin nucleotides (N), taas oo saameynaysa calaamadda dib-u-habaynta (Jaantuska Dheeraadka ah 2). Intaa waxaa dheer, gobolkani wuxuu saameeyaa hidda-wadaha rrs, isagoo ku dhejinaya 16S ribosomal RNA oo la filayo in si heer sare ah loo ilaaliyo, markaa calaamaddan dib-u-habaynta dib-u-celinta waxay noqon kartaa natiijada khaladaadka isku xigxiga ama is-waafajinta. Kadibna dhammaan isku-dhafka genome ee u dhexeeya Mb0003 iyo Mycobacterium bovis AF2122/97 ayaa la sameeyay, iyo jiritaanka nucleotides-ka aan la cayimin iyo SNP-yada ayaa la xaqiijiyay, sidaas darteed dhibaatooyinka suurtagalka ah ee la xidhiidha isku-dheellitirka khaldan ma ahayn sababtoo ah macluumaadka bayoolojiga ee lagu fuliyay shaqadan ayaa soo muuqday ka dib barashada barnaamijka.
Wax nusqaan ah ama nukleotide aan la qeexin lagama helin gobollada dib-u-habaynta qanjidhada gudaha (Jaantus 4 iyo 5). Marka laga hadlayo dhacdooyinkan, mid ka mid ah wuxuu ka kooban yahay kaliya Eu2 genome wuxuuna saameeyaa hidda-wadaha pks12, kaas oo qeexaya suurtagalnimada polyketide synthase; halka kan kale uu ka diiwaan gashan yahay Eu1 genome oo uu saameeyo hidda-wadaha narX kaas oo qeexaya reductase nitrate suurtagal ah (Shaxda 4). Guud ahaan, falanqaynta dib-u-habaynta waxay muujinaysaa inay jiraan tiro xaddidan oo jajab ah oo dib-u-habeyn ah oo leh taageero xisaabeed, iyo tilmaamayaasha la qiyaaso waxay muujinayaan in dib-u-habeynta ay saameyn hoose ku leedahay M. bovis lineage. Digniinta dib-u-habeynta ayaa la filayaa inay hooseyso, laakiin waxaa muhiim ah in la kala saaro calaamadda kobaca dhabta ah ee dhawaaqa asalka ah, taas oo ah hawl adag. Si loo yareeyo dhawaaqa dhawaaqa ee lagu soo bandhigo isu-ururinta ku salaysan tixraaca iyo dhibaatooyinka is-waafajinta 70, 71, dhammaan inta soo hartay marka laga reebo genome-ga dhamaystiran ayaa laga soo ururiyay xoq, tayada shirka ayaa la hubiyay oo la hubiyay iyadoo la eegayo falanqaynta dhuumaha QUAST (Shaxda Dheeraadka ah 1) . Intaa waxaa dheer, taxana ah falanqayn dheeri ah ayaa la sameeyay si loo bixiyo adkaanta iyo saxnaanta sahanka guud. Sidaa darteed, tayada isku xigxiga ee hiddo-wadaha narX iyo pks12 ayaa lagu qiimeeyay akhrinta khariidad ka dhan ah Mycobacterium bovis AF2122/97. Booska SNP ee lagu taliyey ee gobolka dib-u-habaynta ayaa la xaqiijiyay iyadoo la adeegsanayo shuruudaha lagu xusay qaybta habka (ugu yaraan 20 akhrin iyo 0.9 isbeddelka isbeddelka). Polymorphism-ka hiddo-wadaha narX ayaa si buuxda loogu xaqiijiyay labada genome (Mb1792361 iyo Mb7240415; 2.3%) iyo hiddaha pks12 genome: Mb0891, Mb1711, Mb1789, Mb1870, Mb17046, Mb1756, iyo Mb1756. Si kastaba ha ahaatee, genome Mb2043, lix ka mid ah siddeedda boos kama buuxin shuruudaha qoto dheer ee akhrinta sababtoo ah SNP waxaa taageera ugu badnaan 17 akhrinta, taas oo ka hooseysa qiimaha goynta la aasaasay ee 20. Sidaa darteed, dib-u-habaynta lix genomes (8.6%) goobtan genome waa la xaqiijin karaa (Jaantus 4 iyo 5).
Aragtida faahfaahsan ee isku-habaynta gobolka ee isku-habboonaanta Mycobacterium bovis dataset waxay saamaysaa hidda-wadaha narX kaas oo qeexaya reductase nitrate oo suurtogal ah. Wax nukleotide ah oo aan la qeexin lagama helin gobolka dib-u-habaynta qanjidhada gudaha. Dhacdadan gaarka ah waxay ka diiwaan gashan tahay Eu1 genome. Tayada isku xigxiga ee hiddo-wadaha narX ayaa lagu qiimeeyay iyadoo la naqshadeeyay akhrinta Mycobacterium bovis AF2122/97. Xaqiiji goobta lagu taliyey ee SNP ee aagga dib-u-habaynta adoo adeegsanaya shuruudaha lagu sheegay qaybta habka (ugu yaraan 20 akhrin iyo 0.9 isbeddelka soo noqnoqda). Polymorphism-ka hiddo-wadaha narX ayaa si buuxda loogu xaqiijiyay genome-yada Mb1792361 iyo Mb7240415 (2.3%).
Aragti faahfaahsan oo ku saabsan isku toosinta gobolka dib u habeynta ee Mycoplasma bovis dataset ee saameeya hidda-wadaha pks12. Wax nukleotide ah oo aan la qeexin lagama helin gobolka dib-u-habaynta qanjidhada gudaha. Marka laga hadlayo dhacdada saamaynaysa hidda-wadaha pks12 ee dhigaya suurtogalnimada polyketide synthase, waxa ay ka kooban tahay oo kaliya genome Eu2. Tayada isku xigxiga ee pks12 waxaa lagu qiimeeyay akhrinta khariidadda Mycobacterium bovis AF2122/97. Xaqiiji goobta lagu taliyey ee SNP ee aagga dib-u-habaynta adoo adeegsanaya shuruudaha lagu sheegay qaybta habka (ugu yaraan 20 akhrin iyo 0.9 isbeddelka soo noqnoqda). Polymorphisms-ka genome-ga Mb0891, Mb1711, Mb1789, Mb1870, Mb1758, Mb2043, iyo Mb1960 ayaa si buuxda loo xaqiijiyay.
Hidde-sidayaasha PE iyo PPE waxay leeyihiin gobollo soo noqnoqda kuwaas oo si sahlan loo akhrin karo isku xigxiga Illumina iyo khariidaynta khaldan, sidaa darteed waxaa badanaa laga tirtiraa socodka shaqada bioinformatics ee xubnaha qaaxada ee M. kaliya marka la isticmaalayo istaraatiijiyadda isku-xigta. Fikradda dhacdooyinka dib-u-habaynta ee lagu dabaqay shaqadan waxay ku salaysan tahay golaha de novo iyada oo aan la shaandhayn PE/PPE. Waxaan aaminsanahay in hirgelinta saddex hab oo dhameystiran oo kala duwan iyo algorithms iyada oo loo marayo SplitsTree, Gubbins pipeline iyo software RDP4, xeeladaha lagu dabaqay ay ku adag yihiin habaynta iyo shaandhaynta gobollada dib loo habeeyey ee ay sababtay calaamadaha khaladku. Si kastaba ha ahaatee, si looga saaro faragelinta hidda-wadaha PE / PPE ee Gubbins iyo software RDP4 si loo aqoonsado kooxaha SNP, sidaas darteed aqoonsiga gobollada dib-u-dhiska ayaa la soo jeediyay inay saameeyaan hiddo-wadaha narX iyo pks12, xaafadda gen-gan ayaa la baaray (Sawir Dheeraad ah 3-5). M. bovis AF2122/97, hiddo-wadaha narX waxaa kala soocay narK2 iyo Mb1764c, halka pks12 ay ku wareegsan tahay Mb2075c e Mb2073c (Jaantuska Dheeraadka ah 3-5). Khariidadda la sameeyay iyadoo la adeegsanayo khariidadda isku-dhafka MAUVE ee genome-ka dhammaystiran waxay ku siinaysaa macluumaadka ku saabsan ilaalinta iyo dib u habeynta isku xigxiga hidda-wadaha, oo muujinaya afar baloog oo kollinear ah, oo aan lahayn calaamado beddelka genome-ga ama rogidda. Intaa waxaa dheer, falanqaynta dhamaystirka ee isku xigxiga amino acid waxay caddeeyeen homology-ga dhammaan genomeyada dhamaystiran, mana jirto PE/PPE laga helay gobollada ku dhow ee narX ama pks12. Marka loo eego narX, hal genome (Mb0030) ayaa leh dhibco isku mid ah oo hoose sababtoo ah hiddo-wadaha narX waxaa loo aqoonsaday inay yihiin laba jajab (jarjar 1891 iyo 1890). pks12, isku ekaanshaha dartii, Mb0030 iyo Mb003 waxay muujiyeen buundooyinka isku midka ah ee hoose, halka pks12 lagu aqoonsaday laba iyo saddex qaybood, siday u kala horreeyaan, oo matalaya qaybaha kala duwan ee borotiinka (Jaantuska Dheeraadka 3-5). Iyadoo la tixgelinayo macluumaadkan, iyo Gubbins iyo software RDP4 labaduba waxay sameeyaan falanqeyn, hubi isku-dhafka kala duwan ee asaasiga ah ee ugu badnaan 500 bp ee daaqada, waxaan xaqiijinay in hidda-wadaha PE/PPE uusan faragelin doonin calaamadda dib-u-habeynta ee saameeya narX iyo pks12.
Inkasta oo calaamadaha dib-u-habaynta ee laga helay xogtan xogta loo tixgelin karo inay hadh yihiin, waa run in dib-u-habaynta M. bovis aan meesha laga saari karin, sidaas darteed waa in ay sii ahaato mawduuca falanqayn dheeraad ah, taas oo genomes oo dhan oo ka soo jeeda xaalado kala duwan oo epidemiological ah ayaa loo taxaabay si Muhiim ah.
Isbarbardhigga dhirta phylogenetic ee ML ee la helay ka hor iyo ka dib hagaajinta dib-u-habaynta (Jaantus 2A, B) ma keenin isbeddel weyn oo ku yimaada xiriirka phylogenetic ee la qiyaaso, iyo noocyada M. bovis ayaa lagu ururiyay isla koox isku mid ah.
Ka dib markii la sawiray 42 taxane cusub oo M. bovis ah oo ay la socoto genome-ka tixraaca ee M. bovis AF2122/97, isku toosin SNP ah oo ka kooban 1816 boosaska polymorphic ayaa la helay. Inta badan SNP-yada (87.1%) waxay ku yaalaan gobolka codaynta, hiddo-wadaha saameeyana waxaa lagu gartaa iyadoo loo eegayo qaybaha shaqada ee lagu muujiyay Bovilist (Jaantus 6A, B). Iyadoo la tixgelinayo tirada guud ee gen ee qayb kasta oo shaqeynaya, gen-yada ku jira qaybta "shiidka lipid" waxay muujiyeen SNPs badan, oo ay ku xigto "darbiga unugyada iyo hababka unugyada" iyo "shiid dhexdhexaad ah iyo neefsasho", oo muujinaya inay ku jiraan horumarinta M. bovis.
Falanqaynta kala sareynta ee xogta M. bovis ee Portugal (n = 42). Tirada guud ee SNP-yada diiwaangashan iyo hiddo-wadaha saameeya qayb kasta oo shaqaynaysa (A). Tirada guud ee isbeddellada isku midka ah iyo kuwa aan isku midka ahayn ee ka diiwaangashan qaybta shaqada (B).
Miisaanka caalamiga ah, celceliska saamiga dN/dS wuu ka fiican yahay 1.5, taas oo muujinaysa in cadaadiska korriinka caalamiga ah uu yahay in laga takhaluso dawladda awoowayaasha oo uu ka dhigan yahay xaalad togan (kala duwan ama la hago) iyo/ama nasteexo doorasho doorasho. Qaybaha "virulence, sun-saarid, la-qabsi", "isku-darka gelitaanka iyo phages", iyo "protein-ka nidaamsan", in ka badan saddex-meelood laba meelood SNP-yada waa kuwo aan isku mid ahayn (Jaantuska 6B).
Dhammaan qaybaha, waxaa jira hidde-sideyaal badan oo SNPs ah, taasoo keentay celceliska isbeddelka (taas oo ah, celceliska SNP halkii hidde) oo ka weyn 1 (Jaantus 6A). Pks12 (Mb2074c) oo leh 15 SNPs iyo fas (Mb2553c) oo leh 8 SNPs ayaa leh qiimayaal isbeddel sare leh. Labadan hidde-side waxay ku lug leeyihiin dheef-shiid kiimikaadka aashitada dufanka leh. Hidde-sidaha pks wuxuu qeexayaa polyketide synthase (PKS), kaas oo ah enzyme multifunctional oo ku lug leh biosynthesis dufanka unugyada mycobacterial 74,75. Hidde-sidahani waxa uu dhigayaa polypeptide multifunctional kaas oo ku lug leh soo saarista mycoketides74,76. Hidda-wadaha fasku waxa uu ku lug leeyahay isku-xidhka mycolic acid. Labadan hidde-sideyaashani waxay door muhiim ah ka ciyaaraan biosynthesis ee gidaarka unugga marka ay la xiriiraan martida loo yahay.
Si loo sii daraaseeyo horumarka Mycobacterium bovis, laba qaybood oo hidde-sideyaal gaar ah ayaa la falanqeeyay. Shaqooyinka hore loo daabacay iyadoo la adeegsanayo isku dhafka isku xigxiga iyo hababka phylogenetic ayaa lagu aqoonsaday hiddo-wadaha ay awoowayaasha MTBC ka heleen HGT ka hor kala-duwanaanshaha37,38. Hidde-sidayaashan waxay ku taxan yihiin Shaxda Dheeraadka ah 2. Qaybinta SNP ee wadarta 77 hiddo-wadaha laga yaabo inay la xiriiraan HGT ayaa la falanqeeyay, iyo 26 goobood oo polymorphic ah ayaa la aqoonsaday, taas oo inta badan (78%) ay keentay isbeddel aan isku mid ahayn (NS) (Shaxda Dheeraadka ah 2). Shaqadii hore ee genome-ka MTBC waxay muujisay in gobolka HGT ee la saaray uu muujinayo saamiga NS SNP sare marka loo eego inta kale ee genome-ga. Haddii qofku u maleynayo in gobolladan dib-u-habaynta ay heleen awoowayaasha MTBC, sidaas darteed, waxay si xad dhaaf ah u muujinayaan polymorphisms qadiimiga ah, markaa saamiga isbeddellada isku midka ah ayaa la filayaa inay ka sarreeyaan, sababtoo ah beddelka NS ayaa la filayaa in lagu baabi'iyo xulashada xun sababtoo ah isbeddellada amino acid waxay bedeli kartaa shaqada borotiinka. Sidaa darteed, natiijooyinkayagu waxay muujinayaan in cawaaqibta shaqeyntu ay ka iman karto beddelka hiddo-wadaha-HGT-ka, taas oo ka tarjumaysa muhiimadda ay u leeyihiin kala duwanaanshaha hidde-qabsiga ee qiimaha leh.
Marka la barbar dhigo falanqayntan, hiddo-wadaha soo koobaya qaybaha nidaamka 3R (dib-u-habaynta DNA, dib-u-celinta, iyo dib-u-habaynta) ayaa si fiican loo baadhay iyada oo la raacayo liiska ay hore u daabacday dos Vultos iyo la-hawlgalayaashu (2008) 39. Isweydaarsiga jajabyada DNA-da ee isku midka ah si toos ah looma arki karo, inkasta oo laga yaabo inay noqoto nidaam soo noqnoqda marka bakteeriyadu si dhow ula xiriiraan, sida kiiska xogtan; Intaa waxaa dheer, habkani wuxuu noqon karaa furaha hababka dayactirka DNA72, markaa door ka ciyaar dib-u-habayn isku mid ah. Wadarta 26 boosas polymorphic ah oo ay u qaybiyeen hiddo-wadaha 54 ayaa la aqoonsaday (Shaxda Dheeraadka ah 3). Qaybtan hidde-sideyaasha ah, isbeddellada NS waxay ku xisaabtameen qiyaastii 65% cawaaqibta, taas oo la socota warbixinadii hore ee noocyada qaaxada Mycobacterium.
Waqtiga boostada: Oct-21-2021