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Ukulandelana kwe-genome kuye kwavuselela umkhakha wocwaningo lwezifo ezithathelwanayo, kwembula i-epidemiology yezifo, i-pathogenesis, ukusebenzisana kwe-host-pathogen, kanye nenqubo yokuziphendukela kwemvelo ebekwe kumagciwane. I-Mycobacterium tuberculosis complex (MTBC) ithatha i-Mycobacterium bovis njengelinye lamalungu ayo aguquguqukayo ezilwane abangela isifo sofuba (TB) ezilwaneni ezincelisayo zasemhlabeni, futhi iyimodeli evamile yokuguquguquka kwamagciwane. Njengamanye amalungu e-MTBC, i-Mycobacterium bovis ithathwa njengeyigciwane elihlanganiswe ngokuqinile, elivela kancane kancane, futhi ngokusobala alukho uphawu lokuhlangana kabusha noma ukudluliswa kofuzo okuvundlile. Kulo msebenzi, sisebenzisa i-genomics yokuqhathanisa kudathasethi yedatha ye-genome sequence (WGS) eyakhiwe izinkomo ezingu-70 M. ezivela ezizukulwaneni ezahlukene (i-Europe ne-Afrika) ukuze sithole ulwazi ngokuhlukahluka kofuzo lwezinkomo M. Amandla Okuziphendukela kwemvelo. Kusetshenziswa izindlela ezintathu ezahlukene ukulinganisa izimpawu zokuhlela kabusha. Emhlabeni jikelele, inani elincane lezehlakalo zokuhlanganiswa kabusha ziye zabonakala futhi zaqinisekiswa izindlela ezimbili ezizimele ezinokusekelwa okuqinile. Noma kunjalo, uma kuqhathaniswa nokuguqulwa kwezakhi zofuzo, ukuhlanganisa kabusha kunomphumela obuthaka ekwahlukeni kwe-M. bovis (sekukonke r/m = 0.037). Umehluko ongu-r/m omaphakathi otholwe kunhlanganisela ye-clonal ye-Mycobacterium bovis kusethi yethu yedatha uhambisana nomqondo ovamile wokuthi izinga lokuhlanganisa kabusha lingahluka kakhulu phakathi kwemigqa eyabelwe uhlobo olufanayo lwe-taxonomic. Ngokusekelwe kulo msebenzi, ukuhlanganiswa kabusha kwe-Mycobacterium bovis akunakukhishwa, ngakho kufanele kube isihloko semizamo eyengeziwe ocwaningweni oluzayo lwe-genomics oluqhathaniswayo, lapho i-WGS yamasethi amakhulu wedatha evela ezimeni ezihlukene ze-epidemiological emhlabeni jikelele ibalulekile. Ukuhlaziywa okwengeziwe kwabe sekwenziwa kusethi yedatha ye-Mycobacterium bovis encane (n = 42) kusukela ekusakazekeni kwe-TB enabantu abaningi, futhi kwahlonzwa ama-loci angaphezu kuka-1,800, lapho okungenani uhlobo olulodwa lwabonisa i-nucleotide polymorphism (SNP) eyodwa. Iningi (87.1%) litholakala endaweni yokubhala amakhodi, futhi isilinganiso somhlaba wonke sezinguquko ezingezona izinto ezifanayo (dN/dS) zezinguquko ezifanayo zidlula u-1.5, okubonisa ukuthi ukukhetha okuhle kungamandla abalulekile okuziphendukela kwemvelo asetshenziswa ku-M. bovis. Ingxenye ephakeme yama-SNP itholwe ezakhini zofuzo ezinothile ezigabeni zokusebenza ze-"lipid metabolism", "udonga lwamaseli nezinqubo zamangqamuzana", kanye "nometabolism omaphakathi nokuphefumula", okuveza amandla awo kubhayoloji nasekuguqukeni kokubaluleka kwe-Mycobacterium bovis. Ukubhekisisa izakhi zofuzo kumadlozi e-MTBC athambekele ekudluliselweni kofuzo okuvundlile futhi afakwe ohlelweni lwe-3R (ukulungisa i-DNA, ukuphindaphinda, nokuhlanganisa kabusha) kuveza isilinganiso somhlaba wonke esingesihle sokuhlolwa kokungathathi hlangothi kwe-Taijima, okubonisa ukuskena okukhethiwe kwesikhathi esidlule.
I-Mycobacterium tuberculosis complex (MTBC) ingenye yezindlela eziphumelela kakhulu ze-bacterial pathogens kanye nesimo esijwayelekile sokuvela kwebhaktheriya. Amalungu ayo abonisa ubunikazi be-nucleotide obuphezulu ngokumangalisayo ezingeni le-genomic (> 99%)1,2. Izinhlobonhlobo ze-MTBC ecotypes zingabangela isifo sofuba (i-TB), okuyisifo esithathelwanayo se-granulomatous, ezinhlobonhlobo zezinhlobo eziningi ezivela ezilwaneni ezincelisayo kuya kubantu3,4,5. Njengamanje, inkimbinkimbi ihlanganisa abantu [M. Isifo Sofuba (Mtb), Mycobacterium africanum] kanye namagciwane aguqulelwe ezilwaneni (Mycobacterium bovis, Mycobacterium capitum, Mycobacterium pinnipedum, Mycobacterium microtobacter , Mycobacterium mongee, Mycobacterium miysani, Mycobacterium surika, "Bacillus chimpanzee") kanye "ne-Bacillus chimpanzee" 5,6. M. canettii (owaziwa nangokuthi "i-Nodobacter glabrata") Isilinganiso sobunikazi be-nucleotide ne-mycobacteria eshiwo ngenhla singama-98%, futhi umsebenzi wokuqhathanisa we-genomics ubonise ukuthi uM. canettii kanye nayo yonke i-MTBC isanda kuhlukana nokhokho ovamile.7 Ngokucabangela lo mqondo, abanye ababhali be-MT canetti 8 M.
I-MTBC ichazwa ngokuhlelekile njengenkimbinkimbi eqinile ye-clonal, futhi isakhiwo sayo sabantu sibuswa ngokucacile ukuhlukahluka okuncishisiwe, izingqinamba, ukuskena okukhethiwe, kanye ne-genetic drift9,10. Kucatshangwa ukuthi i-clonal evolution eqinile eqinile, efana ne-polymorphisms engekho ayikwazi ukubuyiselwa ngokuhlanganisa kabusha. Ngokusekelwe kulesi sisekelo, izehlakalo ezilandelanayo zokususwa kwe-genomic yesifunda esihlukanisayo (RD) kanye ne-TbD1 (isifunda esiqondile se-Mtb sokususa 1) kuhlongozwe njengezimpawu zamangqamuzana ze-MTBC evolution2,5,11. Ukuqhathanisa i-genomics nomsebenzi wokulandelanisa i-genome (WGS) wonke usekela ukuhlukaniswa kwamalungu aguqulwe abantu abe yimigqa eyisishiyagalolunye (Mycobacterium tuberculosis L1 kuya ku-L4, L7, kanye ne-L8; kanye ne-Mycobacterium africanum L5, L6, kanye ne-L9), imigqa engu-L2 kuya ku-L4 eyabiwe susa i-TbD1,132,1. Ukwengeza, amalungu aguqulelwe ezilwaneni ahlongozwa ukuthi abelane ngedlozi elilodwa, elichazwa ngokususwa okuqondile kwe-clade ku-RD7, RD8, RD9 kanye ne-RD102, 5, kanye ne-14.
Ukudluliswa kofuzo okuvundlile (HGT) kanye nemicimbi yokuhlanganisa kabusha kuthathwa njengeyivelakancane futhi kwenzeka kumadlozi e-MTBC, kunomlando ohlukile walo lonke ilungu le-MTBC15,16,17. Imibiko emibili yokuqala ka-Hughes nabahlanganyeli (2002) kanye no-Gutacker nabahlanganyeli (2006) baphakamise ukuthi izehlakalo zokuhlanganisa kabusha zingasiza ekulolongeni ama-polymorphisms amaka i-loci ethile ku-M. tuberculosis strains18,19. Izizathu zokushoda okusobala kokuhlanganiswa kabusha ku-MTBC yilezi: (1) inqubo yemishini kanye nokulahlekelwa ikhono le-HGT; (2) ukungavamile kwemicimbi ye-HGT; (3) alikho ithuba lemicimbi yokuhlanganisa kabusha ku-MTBC niche14,17. Muva nje, ezinye izifundo ze-whole-genome sequencing (WGS) ezisetshenziswe ku-MTBC strain 20 kanye ne-Mycobacterium bovis 21 zinikeze ubufakazi bokuhlanganiswa kabusha, ezokuqala ezibonisa ukuthi izinhlobo ze-MTBC zivame ukushintshanisa izingcezu ezincane ze-DNA, kodwa ngenxa yokuhluka okulinganiselwe kokulandelana kwe-nucleotide, lezi zehlakalo azikabonwa.
I-Mycobacterium bovis iyilungu le-MTBC elivame ukutholwa emfuyweni (ikakhulukazi izinkomo), nakuba ingase futhi ihlukaniswe nezilwane zasendle ezihlala mahhala nezibiyelwe4,22,23,24. I-M. bovis iguquke yaba ama-clonal complexes amahlanu amakhulu [European 1 (Eu1), European 2 (Eu2), European 3 (Eu3), African 1 (Af1) kanye ne-Africa 2 (Af2)], ngokuya ngephrofayela ye-spoligotyping, ukususwa okuqondile Kanye nama-nucleotide polymorphisms (SNPs) 25, 27, 26, 26, 26, 92 ku-genes ethize. Lezi zakhiwo ze-clonal zibonisa ukwakheka okuhlukahlukene kwenani labantu be-Mycobacterium bovis kanye nokuhlotshaniswa kwayo nezifunda zendawo. Ngaphezu kwalokho, umsebenzi wakamuva we-WGS owenziwe yi-Zimpel nabahlanganyeli (2020) uklame i-phylogeny esekelwe ku-SNP ye-Mycobacterium bovis, enama-genomes angaphezu kwe-1,900, okubonisa ukuthi kukhona okungenani imigqa emine ehlukene (eqanjwe ngo-Lb1 kuye ku-Lb1 kuye ku-Lb4), ayicaciswanga ngokuphelele i-clographic3 yinkimbinkimbi. Laba babhali benza ukuhlaziywa okuhlukile kwe-phylogeny nokuthandana kwamangqamuzana, kodwa abazange bafunde kabusha i-recombination30.
Umsebenzi wangaphambili usebenzisa amasu ahlukene wamangqamuzana, njenge-spoligotyping, i-MIRU-VNTR (inombolo yokuphinda yeyunithi ye-mycobacterial interspersed repeat unit-variable tandem), futhi ukuthayipha kwakamuva kwe-SNP kwembule izinga elithile lokuhlukahluka kofuzo phakathi kwezinhlobo ze-M. bovis 31,32,33, 34,35. Ukuhlukaniswa kwezakhi zofuzo sekuphenduke ithuluzi elibalulekile ekutadisheni i-epidemiology yezifo, okuwusizo ekuqondeni okujulile kwe-pathogenesis, i-virulence kanye nokudluliselwa kwezifo. Ukuvela kwendlela ye-WGS kunikeza ithuba lokuveza izici zokushayela zokuziphendukela kwemvelo ezibekwe i-Mycobacterium bovis genome enqubweni yokujwayela nokuphikelela kubabungazi abahlukene kanye nezimo ze-epidemiological.
Kulo msebenzi, sisebenzisa ukuhlaziya okuqhathanisayo kwe-genomic kumadathasethi edatha e-Mycoplasma bovis ahlukahlukene (n=70), okuhlanganisa nokuhlukaniswa okuvela kuma-clonal complexes ahlukene, ukuze sithole imininingwane ngenqubo yokuvela kwe-Mycoplasma bovis, ikakhulukazi ukuxazulula ubudlelwano be-phylogenetic Kanye nemicimbi yokuhlanganisa kabusha. Njengesengezo salokhu kuhlaziywa, isethi yedatha engaphansi ye-M. bovis ihlukanisa (n = 42) etholwe endaweni enezimpawu ezinhle eziphethwe abantu abaningi abaphethwe isifo sofuba e-Portugal 31,36 yabuye yacutshungulwa ukuze kuqondwe ukungewona ubunikazi Ibhalansi phakathi kwesilinganiso esihlobene somqondo (dN) kuya kumqondo ofanayo (dS) we-nucleotide esikhundleni se-nucleotide, okukhulunywa ngakho njengengxenye ye-genome yezincwadi. 37,38 etholwe amadlozi e-MTBC nge-HGT, futhi encode 3R (DNA repair, replication, and recombination) izingxenye zofuzo zesistimu 39. Khetha izakhi zofuzo ezitholwe nge-HGT ngoba zingamelela ama-polymorphisms asendulo, ngakho kulindelekile ukuthi zingaqukatha ingxenye ephezulu yezinguquko ezifanayo. Izakhi zofuzo ezifakwe ohlelweni lwe-3R zikhethiwe ngoba umsebenzi wangaphambilini we-M. isifo sofuba ubonise ukukhetha okungalungile / ukuhlanzwa okujwayelekile okusebenza kulezi zakhi zofuzo, futhi zingase zibe nendima ebalulekile ekuziphendukeleni kwemvelo 39. Omunye umgomo walo msebenzi uwukucabangela ukuba khona kwezenzakalo zokuhlelwa kabusha. Ngenxa yalesi sizathu, uma sicabangela ukuthi isethi yethu yedatha evela e-Portugal iqukethe kuphela ama-genome e-European clone complex 2 kanye nezinhlobo ezingazange zinikeze i-clone complex, sinqume ukufaka idatha yofuzo etholakala esidlangalaleni ukuze ekugcineni sithole ummeleli wazo zonke izakhiwo ze-clone, futhi Sithuthukise ukuqina nobubanzi bemiphumela.
42 amagenome e-Mycoplasma bovis asanda kuhlelwa avela endaweni yesiPutukezi egcwele abantu abaningi besifo sofuba (imininingwane ngezansi), ngaphambilini ebonakala ngokombono we-epidemiological36, ayisizinda salo msebenzi. Uma kucatshangelwa ukuthi isethi yedatha evela e-Portugal inabameleli be-European 2 clone complexes kuphela kanye nezinhlobo ezingenazo izakhiwo ezikhethiwe, idatha etholakala esidlangalaleni yokulandelanisa i-whole-genome yengezwe ukuze kunwetshwe isethi yedatha ehlanganisa bonke abameleli bezakhiwo ze-M. bovis clone. Ngakho-ke, imithombo emithathu yedatha yokulandelana kwe-genome yonke isetshenziswe kulo msebenzi: ukuhlanganiswa okuphelele / okusalungiswa kwe-genome, kuze kufike kuma-scaffolds angu-10 agcinwe ku-NCBI (Isikhungo Sikazwelonke Solwazi Lwe-Biotechnology) (n = 15 ihlukanisa); egcinwe ku-SRA ( Ifayela le-Illumina fastq lengobo yomlando efundwayo ngokulandelana) limelela ukuhlukahluka okuyinkimbinkimbi kwama-clones we-M. bovis (n = 12 isolate)30; kanye nama-genomes asanda kulandelana angama-42 avela e-Portugal. I-Mycobacterium bovis BCG (Bacille Calmette-Guerin) ayifakwanga ekusesheni kwe-NCBI. I-M. bovis AF2122/97 ivamise ukusetshenziswa njengereferensi yofuzo okufanele ifakwe kudathasethi. Ngenxa yokungatholakali komphakathi kwalo lonke uhlelo lokulandelana kofuzo olumelelwe i-African 1 cloning complex, kanye nenani elincane lama-genome avela kuzinhlobo ezimele i-Af2 ne-Eu1, idatha yokulandelana yasekuqaleni enikezwe i-SRA yasetshenziswa kulezi zimo. Umsebenzi ka-Zimpel nabahlanganyeli bakhe (2020) usize ekuhlonzeni i-genome kwi-cloning complex eshiwo ngenhla futhi wasiza ekukhetheni i-Mycobacterium bovis ukuze ifakwe kusethi yedatha. Ku-Eu3, uhlobo olulodwa kuphela lwe-genome oluchazwayo (Branger et al., 2020), ngakho-ke i-genome esiyihlanganisayo ingummeleli ohlukile we-Eu3 complex.
Emhlabeni jikelele, le sethi yedatha ihlanganisa izinkomo ezingu-70 ze-M. bovis ezihlukanisiwe ezinhlotsheni ezingu-8 ezitholakalayo, ezisatshalaliswe emazweni angu-12 kusukela ngo-1985 kuya ku-2016. Izinhlobo ezingu-36 ziqokwa njenge-Eu2, izinhlobo ezingu-7 ziyi-Eu1, uhlobo olu-1 luyi-Eu3, izinhlobo ezi-3 ziyi-Af1, izinhlobo ezi-4 ziyi-Af2 kanye nezinhlobo ezingu-19 azikho ngaphansi kwe-clonal). Ulwazi oluningiliziwe (kuhlanganise nenombolo yokungena) ye-Mycobacterium bovis esetshenziswe kulolu cwaningo ikhonjiswe kuThebula 1 kanye neThebula Lokwengeza 1.
Ama-genomes aphelele angama-42 e-Mycobacterium bovis avela ezindaweni ezithandwa kakhulu yizilwane e-Portugal futhi asatshalaliswe iminyaka engaphezu kwe-12 ayisizinda salolu cwaningo, njengoba izinhlelo zezifo zezilwane zasendle ezingenzeka ziqashwe njalo 31,36 (Fig. 1). Ngokwezinqubo ezalandela, lezi zinhlobo zahlukaniswa izinkomo (n = 14), izinyamazane ezibomvu (n = 16) kanye nezingulube zasendle (n = 12) kusukela ngo-2003 kuya ku-2015: ukuqoqa futhi ukuphatha izilwane ngokuhambisana neziqondiso zephrothokholi ezinconyiwe Amasampula ezicubu aku-OIE Terrestrial Animal Handbook futhi afakwe i-solid-Jean liquid e-Stonewepynöpy kanye ne-Lyruvatesteine okuphakathi. Amasiko afukanyelwa ku-37 °C futhi ukukhula kuhlolwa kanye ngesonto okungenani amasonto ayi-12. Amakholoni agcinwa ngqo kwisisombululo se-glycerol ku -80ºC. Endleleni ekhethiwe ye-Mycobacterium (Middlebrook 7H9, BD Diagnostics), amasampula oqobo afakwe kungobo yomlando adluliswe endimeni eyodwa ye-in vitro in vitro ukuze kutholwe i-DNA yohlelo lwe-WGS. Ukwenzela lokhu, isixazululo sesitoko esiqandisiwe sesitokwe sanothiswa nge-sodium pyruvate engu-5% kanye no-10% we-ADS (50 g albumin, 20 g glucose, 8.5 g sodium chloride emanzini angu-1 L) eMiddlebrook 7H9 ngo-37°C Retrain. Ngemuva kwamaviki angu-4 okukhula, okuphakathi kwavuselelwa futhi isiko lalibhekwa njalo kuze kube yilapho ukukhula kubonakala. Amaseli avunwa nge-centrifugation, i-pellet yaphinde yamiswa ku-500 µL phosphate buffered saline (PBS), yashisisa ngo-99 °C imizuzu engu-30, i-centrifuged, futhi i-supernatant yagcinwa ku -20 °C kuze kube yi-WGS. Zonke izinqubo zenziwa ezingeni 3 izikhungo ze-biosafety.
Umtapo wolwazi we-WGS wokuphela kwegenome ulungiswa kusetshenziswa inkomba eyingqayizivele yesampula ngayinye ye-DNA, futhi isebenzisa i-Illumina MiSeq (2 × 250 pb) (amasampuli angu-40) kanye nobuchwepheshe be-HiSeq (2 × 150 pb) (i-isolates) (i-Eurofins Genomics, eJalimane) ukuze ilandelelane. Ngokusho kwemiyalo yomkhiqizi, sebenzisa i-Illumina Genome Analyzer enokunamathiselwe kwemojula eneziphetho ezimbili ukuze ulandele i-genomic DNA, futhi usebenzise Ikhithi Yokulungiselela Yelabhulali Ye-Nextera XT DNA esuka ku-Illumina ukuze wakhe umtapo wolwazi.
Uma kucatshangelwa idatha etholwe ku-SRA (n = 12), ukuhlonza inkimbinkimbi ye-clone kungasetshenziswa njengemethadatha yokushicilelwa okuhambisanayo 30, 41, 43. Uma kucutshungulwa i-genome ephelele, ngaphandle kwe-Mycobacterium bovis AF2122/97 kanye ne-Mycobacterium bovis 3601, amalungu e-Eu3 eu2 kanye ne-Eunal1 complex, abonwayo kanye ne-Eu31 29, ngokulandelana, iyafana ne-genome ephelele ye-Mycobacterium tuberculosis H37Rv (inombolo yokungena ye-NCBI NC_000962.3). Ukuqondanisa i-genome kwenziwa kusetshenziswa i-MAFFT (uhlelo lokuqondanisa okuningi lwe-amino acid noma ukulandelana kwe-nucleotide, inguqulo 7.458) kanye nepharamitha -addfragments48. Bese, sesha ukungabikho kwama-clonal complexes ahlukene kanye/noma ukuba khona kwezici ze-SNP.
I-Mycobacterium bovis esanda kuhlelwa (n = 42) nokufunda kwangempela kohlaka lwe-genome oluhlanganisiwe (n = 3) kuqondanisa inkimbi ne-reference genome Mycobacterium tuberculosis H37Rv ngepayipi le-vSNP kanye nokuba khona kokususwa kanye/noma izici ze-SNP zama-clones ahlukene Ukusesha kwenziwe.
Qoqa ulwazi kusuka ekuntulekeni kwezici kanye/noma ukuba khona/ukungabikho kwe-SNP namaphrofayela we-spoligotyping ukuze unikeze idatha ye-genomic ku-clonal complex ehambisanayo. Emihlanganweni emine esalungiswa, iphrofayela ye-spoligotyping ayikwazi ukuchazwa, ngakho-ke ifakwe eqenjini "elingekho inkimbinkimbi".
Ukugeleza komsebenzi we-bioinformatics okulandelwa yilo msebenzi kuqala kusukela ekuhlanganiseni kwe-de novo kanye nokwenza imephu kuye kwisu eliyireferensi, ngenhloso yokuhlola izehlakalo zokuhlanganisa kabusha kanye ne-genome polymorphisms ethile. Umfanekiso 1 unikeza ishadi lokugeleza lezinyathelo ezilandelwayo. Ukuze kuhlaziywe kabusha, wonke ama-genomes asetshenziselwa ukukhulisa ukuqina kwezinkomba kanye nezinkomba ezihlobene.
Ukuze kuncishiswe amaphutha ekwenziweni kokulandelana kokuvumelana kwe-genome, siqale sathola ukuhlanganiswa kwe-de novo, sabe sesithola ukuqondanisa okuningi okuyisisekelo. Ipayipi le-Unicycler liyatholakala ku-https://github.com/rrwick/Unicycler49 futhi lisetshenziselwa ukwenza ukuhlanganiswa kwe-de novo kwama-genomes alandelanayo angu-54 (amafayela angu-42 asanda kulandelwa kanye namafayela angu-12 fastq atholwe ku-SRA). Ngamafuphi, ngaphambi kokuhlanganisa kusukela ekuqaleni, ukuhlaziywa kwekhwalithi efundwayo kwenziwa ngenguqulo ye-FastQC engu-0.11.7 (https://github.com/s-andrews/FastQC), kanye nenguqulo ye-Trimmomatic 0.36 (inketho "yokusika ama-Adapter nokunye ukulandelana okuqondene nokukhanyisa okuvela ekufundeni" kanye "Sika izisekelo, ukubamba oku-2" kungaphansi kwekhwalithi yokufundwayo kusetshenzisiwe) (http://www.usadellab.org/cms/?page= trimmomatic) 50. Bese, i-SPAdes optimiser49 isetshenziselwe ukuhlanganisa i-genome, futhi inguqulo ye-Pilon 1.1851 yasetshenziselwa ukwenziwa kahle kwangemva komhlangano. Imodi yebhuloho elandelanayo ikhethiwe ukuze kugwenywe ukuhlanganisa okungalungile, futhi usayizi we-k-mer waseshwa futhi wakhethwa phakathi kuka-20% no-95% wobude obufundiwe. Landela imihlahlandlela ye-SPAdes futhi ucabangele usayizi wokufundwayo, susa ama-contigs amancane kuno-300 bp, bese usungula ukujula kokuhlanganisa okungu-20 kokufunda okungu-52. Kusu lokuhlanganisa i-de novo, izifunda ze-genomic ezifana ne-proline-glutamate (PE) ephindaphinda kakhulu i-proline-proline glutamate (PPE) azizange zisuswe.
Ikhwalithi yokuhlanganisa i-de novo ihlolwa ngepayipi le-QUAST (http://quast.sourceforge.net/quast.html), elenza ukuvuselelwa kwe-contig kanye ne-M. bovis AF2122/97 reference genome (inombolo yokungena ye-NCBI LT708304.1) Mapping (bona Ipharamitha Yethebula Lesengezo engu-1).
Ngosizo lwepayipi le-vSNP (https://github.com/USDA-VS/vSNP), ifayela le-FASTQ le-M. bovis esanda kulandelwa ukulandelana evela ku-Illumina liqhathaniswa ne-M. bovis AF2122/97 reference genome (LT708304.1)). Ngokwezincomo zokuzijwayeza ezihamba phambili ze-Genome Analysis Toolkit (GATK) 53, 54, 55 sebenzisa amapharamitha okuhlunga ajwayelekile noma izikolo ezihlukile zesisindo zokulinganisa kabusha. Imiphumela ihlungwa kusetshenziswa amaphuzu aphansi kakhulu we-SAMtools angu-150 kanye ne-AC = 2. Futhi sebenzisa i-Kraken (http://ccb.jhu.edu/software/kraken/) ukuze uhlole ukufundwa ukuze ukhiphe ukungcola. Ipayipi le-vSNP elisetshenziswa ukwenza imephu ukuhlela amasu emsebenzini wethu lihlola uchungechunge lwama-SNP achaziwe kanye nemigomo, futhi alifaki izimo ezixubile zokutheleleka. Ukufakwa kwe-genome efundiwe kungcono kuno-99% (Ithebula Lokwengeza 1).
Ukuze ugweme amaphutha emephu nama-SNP angalungile, hlunga okuhlukile ezimweni ezilandelayo: (1) isekelwa ukufundwa okungaphansi kuka-20, (2) itholakala nemvamisa engaphansi kuka-0.9, (3) okungenani i-Strains eyodwa, kodwa okungenani kukhona izikhala kolunye uhlobo. Inguqulo ehlanganisiwe ye-genomics viewer (IGV) 2.4.19 (http://software.broadinstitute.org/software/igv/)56 yasetshenziswa ukuze kuqinisekiswe ngokubonakalayo ama-SNP nezikhundla ezinezinkinga zemephu noma zokuqondanisa. Njengoba izakhi zofuzo ze-proline-glutamate (PE) kanye ne-proline-proline glutamate (PPE) ziyimpinda kakhulu futhi ziyingxenye yomndeni wezakhi eziningi zofuzo, ziqondwa kalula ngokulandelana kwe-Illumina nokungahambi kahle, ngakho zikhethwa I-mycobacterial bioinformatics workflow isuse amalungu e-tuberculosis complex uma usebenzisa isu lokulandelana kwemephu ye-SNP. Ngakho-ke, sihlunge izakhi zofuzo ze-PE/PPE nama-indel ekuhlaziyeni.
Ngokusho kwe-Bovilist (http://genolist.pasteur.fr/BoviList/), wonke ama-SNP ahlukaniswe ngezigaba zokusebenza. Ipayipi le-SnpEff (https://pcingola.github.io/SnpEff/) lisetshenziselwa ukukhomba imiphumela ye-SNP (izinguquko ezifanayo noma ezingezona ezifanayo). Idale isizindalwazi esisha se-Mycobacterium bovis AF2122/97 genome (LT708304.1).
Ukuqondanisa okuningi kwe-genome okuyinhloko kwenziwa kusetshenziswa i-Parsnp v1.2, okwamanje etholakala kokuthi https://github.com/marbl/parsnp57, kusetshenziswa ama-genomes/draft assemblies angu-69 (ngenketho -c) kanye ne-M. bovis AF2122/97 (LT708304.1) isetshenziswa njengereferensi. Ukuqondanisa okuningi okune okubalulekile kwenziwa: amalungu kuphela e-Eu2 cloning complex (n = 37), okuhlanganisa wonke amalungu e-European cloning complex (n = 44), okuhlanganisa nendawo yokuhlangana ye-European and African cloning complex (n = 51), futhi afaka yonke i-Mycobacterium bovis kulolu cwaningo (n = 70).
Ukuqondanisa okuyisisekelo okukhiqizwa yi-Parsnp kusetshenziselwa ukulinganisa amathuba aphezulu (ML) wesihlahla se-phylogenetic kusetshenziswa i-CIPRES Science Gateway v3.3 (http://www.phylo.org/)58 kusetshenziswa i-RAxML, futhi yenze iziphindaphindo eziqondisiwe eziyi-1000.
Ama-algorithms amathathu ahlukene kanye namathuluzi e-bioinformatics asetshenziselwa ukuhlola ukuba khona kwemicimbi yokuhlanganiswa kabusha ngokuhambisana: isofthiwe ye-SplitsTree4, ama-Gubbins (uhlu lozalo olungachemile ngokuhlanganiswa kabusha kokulandelana kwe-nucleotide), kanye nesofthiwe ye-RDP4 (uhlelo lokutholwa kabusha, inguqulo ye-beta 4.101).
Indlela yokuhlukanisa ukuhlukanisa isetshenziswe ku-SplitsTree4 v4.15.1 (http://www.splitstree.org/)59 isetshenziselwa ukubala inethiwekhi ye-phylogenetic engenazimpande, kusetshenziswa ukuhlolwa kwe-Phi ukuze kuqinisekiswe izibalo, futhi umkhawulo wokubaluleka ngu-p = 0.05. Ukuhlaziywa kokuqondanisa okuningi okuyisisekelo kwe-Parsnp kusetshenziswa njengokufakwayo, futhi ukubola okuhlukanisayo njengezinga lenethiwekhi kuyenziwa.
Ipayipi le-Gubbins v2.3.1 (https://github.com/sanger-pathogens/gubbins60 isebenza namapharamitha amisiwe njengenye indlela yokuhlola umthelela wokuhlanganisa kabusha ku-Mycobacterium bovis. I-algorithm esetshenziswa epayipini yakha kabusha uhlu olufanele lwe-clone Iqoqo eliphelele le-genome/okusalungiswa kwesethi yethu yedatha (AF1vine ireferensi ye-2bovid) LT708304.1) zihambisana; futhi skena indawo ye-SNP egatsheni ngalinye lesihlahla ukuze kutholwe iqoqo le-SNP elimele umcimbi wokuhlanganisa kabusha Uziro wegatsha Cabanga ukuthi awukho umcimbi wokuhlanganisa, okusho ukuthi ama-SNP avela egatsheni kufanele asatshalaliswe ngokulinganayo Amafayela aphakathi kwe-ParRA asetshenziswa ukusuka esihlahleni sokuhlanganisa okuningi.
Okokugcina, ukuze kuqinisekiswe umcimbi wokuhlela kabusha ophakanyiswe ipayipi le-Gubbins, ama-algorithms ayisithupha asetshenziswe ku-RDP467 (RDP61, GENECONV62, Bootscan63, Maxchi64, Chimaera65 kanye ne-SiScan66) asetshenziswa ekuqondaneni okuningi okuyisisekelo kwe-Parsnp ngaphansi kwezilungiselelo ezizenzakalelayo. Sinqume ukuthi okungenani ama-algorithms amathathu asetshenziswe ku-RDP4 kufanele abonise isignali ebalulekile ukuze kuqinisekiswe umcimbi ngamunye wokuphinda uhlanganiswe.
Uma kucatshangelwa ukuthi kokubili ama-Gubbins kanye nesofthiwe ye-RDP ibheka amasignali okuhlanganiswa kabusha ngokuhlola ukuqondanisa okuyisisekelo okuningi efasiteleni elifinyelela ku-500 bp, futhi kuqinisekiswe ukuthi ukufakwa kwezakhi zofuzo ze-PE/PPE ngesikhathi somhlangano we-de novo ngeke kuphazamise izimpawu zokuhlanganiswa kabusha ezitholiwe, ukuhlaziya okwengeziwe kwenziwa nge-homolinearity Hlola indawo ehlonza isizukulwane sokuhlanganiswa kabusha. Imephu ye-sylinear esebenzisa i-genome ephelele yakhiwe kusetshenziswa ukuqondanisa kwe-MAUVE-multi-genome (http://darlinglab.org/mauve/mauve.html) ukuze kukhishwe ukudluliselwa kwegenome yendawo noma ukuguqulwa. Ngaphezu kwalokho, lonke uhlobo lofuzo lwasetshenziswa ukwenza ukuhlaziya kwe-homolinearity ekulandelaneni kwe-amino acid ngeseva yewebhu ye-SyntTax (https://archaea.i2bc.paris-saclay.fr/SyntTax/).
Ukuhlaziywa okujulile kwesethi yedatha ye-genome etholwe ohlelweni lwesiPutukezi lwe-tuberculosis ye-multi-host ukuhlola i-polymorphism yezakhi zofuzo ezishiwo ezincwadini. Lezi zakhi zofuzo ziyi-37,38 kanye nofuzo olufaka ikhodi engu-3R olutholwe amadlozi e-MTBC ngokusebenzisa i-HGT (ukulungisa i-DNA, ukuphindaphinda nokuhlanganisa kabusha) izingxenye zesistimu 39. Sebenzisa i-ClustalX v2.1 (http://www.clustal.org/clustal2/) futhi usebenzise i-DnaSP v6.12.03 (http://u/dub divers generic and recombination). ukuhlukahluka kwe-nucleotide (π) kanye nokokufaka kwepharamitha yepharamitha ye-Tajima's D neutral.
Amathuba aphezulu (ML) wesihlahla se-phylogenetic esisekelwe ku-69 Mycoplasma bovis isolate kanye nama-genome ereferensi atholakele (Umfanekiso 2A). Uma kuqhathaniswa nezihlahla ezisekelwe kufuzo olulodwa noma izihlahla ezisekelwe ezindaweni eziningi, lelisu livumela ukukhiqizwa kwezihlahla ezinamandla kakhulu ezingathathi ukuhlukahluka kwe-genome yonke futhi ngenxa yalokho kubonisa amandla aphansi okubandlulula phakathi kwezinhlobo ezingu-68,69. Isakhiwo se-topological yesihlahla se-ML ngokuvamile sihambisana nokuhlukaniswa okuyinkimbinkimbi kwama-clones. I-genome ye-Eu2 ihlanganiswe egatsheni, futhi i-genome ye-Af1 nayo ihlanganiswe ndawonye (Umfanekiso 2A). Umphumela ubuye uhambisane nobudlelwano obaziwayo bokuzivelela kwe-Mycobacterium bovis, okungukuthi, kunomehluko omkhulu phakathi kwelungu le-Eu1 neqembu elihlanganisa zonke ezinye izakhiwo ze-clonal kanye nama-genome, kodwa i-clonal complex 30 ayicaciswanga. Ukungqubuzana okuncane phakathi kwenkimbinkimbi ye-clonal kanye nobuhlobo obubonwe esihlahleni se-phylogenetic kungachazwa ukuthi inkimbinkimbi ye-clonal ichazwa ngokusekelwe ezifundeni ezithile ze-genomic, kuyilapho isihlahla se-phylogenetic sisekelwe ekuqondaneni okuningi kwama-core genomes amele i-genome yonke.
Isihlahla esinamandla kakhulu se-phylogenetic (GTR) sakhiwe ngokusekelwe ekuqondaneni kwegenome okuwumongo we-Mycobacterium bovis genome ngaphambi (A) nangemuva (B) kokususwa kwesayithi lokuhlanganisa kabusha. Imibala yegatsha imele i-Mycobacterium bovis clone complex: I-Europe 1 inobubende, i-Europe 2 ibomvu, i-Europe 3 iluhlaza okwesibhakabhaka, i-Afrika 1 inombala osawolintshi, kanti i-Afrika 2 iluhlaza. Isihlahla sigxilile futhi sidonselwa esikalini, futhi ubude begatsha bulinganiswa njengokuthatha indawo yesiza ngasinye.
Inkimbinkimbi yesifo sofuba i-Mycobacterium ichazwa ngokuthi iguquguquke ngokuhlanganyela, futhi ubufakazi obuningi obuqoqwe phakathi neminyaka busekela umbono wokuthi i-HGT eqhubekayo nemicimbi yokuhlanganisa kabusha ngeke yenzeke ezingeni elibonakalayo le-MTBC15,17,18.
Umsebenzi wangaphambilini ubonise ukuthi kungase kube nokuhlanganisa kabusha okulinganiselwe phakathi kwe-MTBC strains20,21, kuyilapho abanye behlulekile ukuhlonza izehlakalo zokuhlanganiswa kabusha ezilinganisekayo70,71. Phinda uxoxe ngalolu daba ngokugxila ku-Mycobacterium bovis, ehlukile kunomsebenzi wangaphambili owawubheka kuphela isifo sofuba i-Mycobacterium 70,71; noma ucabangele i-MTBC iyonke, cishe kungekho M. bovis omele 20; noma ucabangele kuphela izingxenyana zezinkomo ezinemikhawulo. Isethi yedatha ye-mycobacteria, kulo msebenzi, kukhona isamba sezinhlobo ezingu-70, ezimele zonke izakhiwo ze-clonal, ezisetshenziselwa ukuhlolela ukuhlanganiswa kabusha. Isethi yedatha ikalwa ngokuya ngamaleveli amane anqwabelene: (1) Amalungu e-Eu2, (2) wonke amalungu ayinkimbinkimbi yaseYurophu (okungukuthi i-Europe), (3) I-European and African clone complex (Eu + Af) kanye (4) nawo wonke amaqoqo wedatha (okuhlanganisa nama-genomes angafakiwe kunoma yiziphi izakhiwo ze-cloning esezichaziwe).
Ukuze kuqhutshekwe nokufunda le nkolelo-mbono, inethiwekhi yokuhlukanisa-ukuhlukana yenziwa ukuze kuhlolwe ukungabikho kwezenzakalo zokuhlanganiswa kabusha phakathi kwama-genomes, ngoba le ndlela ingakwazi ukubona ngeso lengqondo ubuhlobo bokhokho phakathi kwabantu ngabanye futhi ibonise izimpawu ze-phylogenetic eziphikisanayo. Wonke amasethi edatha amane ekuhlaziyweni aqinisekisa ukuba khona kwezihibe kunethiwekhi (okungukuthi, izindawo ezingaguquki zibe isihlahla esisodwa), kodwa ukuhlolwa kwe-Phi akunakho ukusekelwa kwezibalo (Eu2, p = 0.0956; Europe, p = 0.1637; Eu + Af p = 0.2774; yonke idatha isethwe pganiza1 = 0, i-Figure 5 noma i-0.2 ihlinzeka ngobufakazi obumpofu = 0.2. 3A-D).
EYurophu 2 ama-genomes (n = 37) (A), ama-genomes aseYurophu (n = 44) (B), ama-genomes aseYurophu nawase-Afrika (n = 51) (C) kanye nesethi yonke yedatha (n = 70) (D).
Ngemva kwalokhu kuhlaziya, futhi kucatshangelwa ukubonwa komjikelezo kuwo wonke amanethiwekhi, i-algorithm yokwakha kabusha esetshenziswe epayipini le-Gubbins yasetshenziswa ukuze kwakhiwe kabusha uhlu lozalo lwe-clonal kanye nokwengezelela isilinganiso somphumela wokuhlanganisa kabusha ku-genome ye-M. bovis. Cabangela inombolo eqoqiwe yezenzakalo zokuhlanganisa kabusha, eziningi zazo ezenzeke emagatsheni agcinayo (okungukuthi, ku-genome eyodwa) (Ithebula 2). Lezi zinkomba zibonisa ukuvumelana kwayo yonke isethi yedatha futhi zibonisa ukuthi imvamisa yezenzakalo zokuhlanganiswa kabusha izikhathi ezingu-200 kuya kwezingu-300 zokuguqulwa kwezakhi zofuzo. Uma ipharamitha ye-rho/theta emele izilinganiso ezihambisanayo zokuhlanganisa kabusha nokuguqulwa kwamaphoyinti egatsheni ibonakala iphakathi kuka-0.0037 no-0.0056 (Ithebula 3). Muva nje, umsebenzi oshicilelwe we-38 M. bovis strain ubonise inani eliphezulu le-rho/theta (rho/theta = 0.1) kunalelo elitholwe kule dathasethi, kodwa umsebenzi kaPatané nozakwabo basebenzise ukuhlanganiswa okusekelwe kunkomba ukuze kufinyelelwe imingcele yokuhlanganisa kabusha , Imininingwane yenqubo, ngenxa yenqubo yokuhlanganisa, ihlotshaniswe nenqwaba yezenzakalo zegatsha.
Okulandelayo, ipharamitha ye-r/m imelela isilinganiso sokuhlukahluka sokuhlanganiswa kabusha nokwethulwa kokuguqulwa, futhi inani layo elimaphakathi liphakathi kuka-0.025 no-0.037, okubonisa ukuthi uma kuqhathaniswa nokuguqulwa kwezakhi zofuzo, ukuhlanganisa kabusha kunomthelela ophansi jikelele ezinhlobonhlobo zofuzo ze-M. bovis (Ithebula 3)). Ukuze kuqhathaniswe okubanzi, indlela efanayo yasetshenziswa ukulinganisa ipharamitha engu-r/m kudathasethi ye-MTBC eyakhiwe ama-genomes angu-23, okubonisa inani eliyisilinganiso elingu-0.48620, kuyilapho kudathasethi yedatha ka-Patané nozakwabo engu-38 M. bovis, ibonise inani eliyisilinganiso lingu-0.98. Ocwaningweni lokuqala, ama-genomes amabili kuphela kwangu-23 afakwe emsebenzini we-M. bovis (M. bovis BCG kanye ne-reference strain), ngakho-ke inani elitholiwe lingase libe nokuchema ngenxa yokuvezwa ngokweqile kwe-M. tuberculosis genome. Embikweni wesibili, inani labantu abahlaziywe be-Mycobacterium bovis latholwa ikakhulukazi e-United States kanye nakubasingathi bemfuyo. Ngokuphambene, kusethi yethu yedatha, izindawo eziningi zendawo kanye nezinhlobo zezilwane zimelwe, futhi ama-genomes aqoqwe abe ama-clonal complexes anezici ezihlukahlukene zofuzo zabantu nawo ayasetshenziswa, ngaleyo ndlela kuzuzwe ulwazi olujulile nolubanzi Lwenani labantu. Umehluko r/m isilinganiso senani elitholwe nesethi yethu yedatha lihambisana nomqondo wokuthi izinga lokuhlanganisa lihluka kakhulu phakathi kwemigqa eyabelwe uhlobo olufanayo lwe-taxonomic, ngakho le miphumela ibonisa ukuthi inkimbinkimbi ye-M. bovis clone ingase ibonise umehluko wokuhlanganisa kabusha Umthelela futhi njengoba uphakanyiswe yi-Didelot & Maiden72. Noma kunjalo, ukwandisa ngokuphawulekayo le datha esethwe ngokufaka inombolo enkulu ye-M. bovis genomes kuzovumela ukucaciswa okwengeziwe kwaleli phuzu. Kokubili amapharamitha we-r/m kanye ne-rho/theta abonisa ukuhlukahluka phakathi kwamagatsha, futhi lo mphumela uhambisana nemibiko yezinye izinhlobo zebhaktheriya72,73.
Okokugcina, ukuze kuqinisekiswe izenzakalo zokuhlela kabusha ezikhonjwe ipayipi le-Gubbins, ama-algorithms ayisithupha ahlukene asetshenziswa kusofthiwe ye-RDP4 ukuze kuhlolwe ngokuzimela ukuqhathanisa okuningi okuyisisekelo okuhlukile. Emhlabeni jikelele, ngaphansi kwengxenye yezehlakalo ezihlonzwe uGubbins zaqinisekiswa yi-RDP4 (Amathebula 4 no-5). Uma kucatshangelwa yonke isethi yedatha, izehlakalo ezintathu zokuhlanganisa kabusha zaqinisekiswa, ezimbili ezibandakanya ama-node angaphakathi kanti enye ihilela i-genome eyodwa egatsheni le-terminal, lapho ama-clonal complexes ayengakwazi ukunikezwa (Amathebula 4 no-5). Ukuhlonzwa kwezehlakalo emagatsheni esitheminali kungase kubonise ukuthi ukuhlanganisa kabusha kusaqhubeka kuhlobo lwamanje lwe-M. bovis noma umphumela ubekwe endaweni engafanele70. Kulesi sifunda sokuhlanganiswa kabusha okucatshangelwayo, cishe i-20% yezikhundla zine-nucleotides engachazwanga (N), ngaleyo ndlela ithinte isignali yokuhlanganisa kabusha (Umfanekiso Ongeziwe 2). Ngaphezu kwalokho, lesi sifunda sithinta isakhi sofuzo se-rrs, efaka ikhodi ye-16S ribosomal RNA okulindeleke ukuthi ilondolozwe kakhulu, ngakho-ke lesi siginali yokuhlanganisa kabusha ebekayo ingaba umphumela wokulandelana kwamaphutha noma ukungahambi kahle. Khona-ke ukuqondanisa konke kwe-genome phakathi kwe-Mb0003 ne-Mycobacterium bovis AF2122/97 kwenziwa, futhi ukuba khona kwama-nucleotide angachazwanga kanye nama-SNP kwaqinisekiswa, ngakho-ke izinkinga ezingenzeka ezihlobene nokuqondanisa okungalungile azizange zibe ngenxa yolwazi lwebhayoloji oluqaliswe kulo msebenzi Kuvele ngemva kokufunda uhlelo.
Azikho izikhala noma ama-nucleotide angachazwanga atholakala ezindaweni zokuhlanganisa kabusha ama-node angaphakathi (Izibalo 4 kanye ne-5). Ngokuphathelene nalezi zenzakalo, eyodwa iqukethe kuphela i-Eu2 genome futhi ithinta isakhi sofuzo se-pks12, esifaka i-polyketide synthase engenzeka; kuyilapho enye ibhaliswe ku-Eu1 genome futhi ithinta isakhi sofuzo se-narX esifaka i-nitrate reductase engase ibe khona (Ithebula 4). Ngokuvamile, ukuhlaziywa kokuhlanganiswa kabusha kubonisa ukuthi kunenombolo elinganiselwe yezingcezu zokuhlanganisa futhi ezinokusekelwa kwezibalo, futhi izinkomba ezibonisiwe zibonisa ukuthi ukuhlanganisa kabusha kunomthelela ophansi ohlwini lozalo lwe-M. bovis. Isignali yokuhlanganisa ilindeleke ukuba ibe phansi, kodwa kubalulekile ukuhlukanisa isignali yeqiniso yokuziphendukela kwemvelo emsindweni wangemuva, okuwumsebenzi oyinselele. Ukuze kuncishiswe isignali yomsindo eyethulwa ukuhlanganisa okusekelwe kunkomba kanye nezinkinga zokungahambisani 70, 71, konke okunye ngaphandle kwe-genome ephelele kwahlanganiswa kusukela ekuqaleni, futhi ikhwalithi yokuhlanganisa yahlolwa futhi yaqinisekiswa ngokuhlaziywa kwepayipi le-QUAST (Ithebula Lokwengeza 1) . Ukwengeza, uchungechunge lokuhlaziya olwengeziwe lwenziwa ukuze kuhlinzekwe ukuqina nokunemba kocwaningo lulonke. Ngakho-ke, ikhwalithi yokulandelana yezakhi zofuzo ze-narX ne-pks12 yahlolwa ngokufunda imephu ngokumelene ne-Mycobacterium bovis AF2122/97. Isikhundla esinconyiwe se-SNP endaweni yokuhlanganisa kabusha siqinisekiswe ngokusebenzisa imibandela eshiwo esigabeni sendlela (okungenani ukufundwa okungu-20 kanye no-0.9 wokushintsha imvamisa). I-polymorphism yofuzo lwe-narX yaqinisekiswa ngokugcwele kuma-genomes amabili (Mb1792361 kanye ne-Mb7240415; 2.3%) kanye nama-genomes we-pks12 genome: Mb0891, Mb1711, Mb1789, Mb1870, Mb, Mb1704 ne-M1704 Kodwa-ke, ku-genome Mb2043, izikhundla eziyisithupha kweziyisishiyagalombili azihlangabezani nesimiso sokujula sokufunda ngoba i-SNP isekelwa ukufundwa okungaphezulu kwe-17, okungaphansi kwenani elimisiwe le-cutoff elingu-20. Ngakho-ke, ukuhlanganiswa kabusha kwama-genome ayisithupha (8.6%) kule sayithi ye-genome kungaqinisekiswa (Izibalo 4 no-5).
Ukubonakala okunemininingwane yokuqondanisa kwesifunda sokuhlanganiswa kabusha kwedathasethi ye-Mycobacterium bovis kuthinta isakhi sofuzo se-narX esifaka i-nitrate reductase engase ibe khona. Awekho amagebe noma ama-nucleotide angachazwanga atholakala endaweni yokuhlanganiswa kabusha kwamanodi angaphakathi. Lo mcimbi othile ubhaliswe ku-Eu1 genome. Ikhwalithi yokulandelana yofuzo lwe-narX yahlolwa ngokuhlela ukufundwa kwe-Mycobacterium bovis AF2122/97. Qinisekisa indawo enconyiwe ye-SNP endaweni yokuhlanganisa kabusha ngokusebenzisa imibandela eshiwo esigabeni sendlela (okungenani ukufundwa okungu-20 kanye no-0.9 wokushintsha imvamisa). I-polymorphism ye-narX gene yaqinisekiswa ngokugcwele kuma-genomes we-Mb1792361 kanye ne-Mb7240415 (2.3%).
Ukubona ngeso okunemininingwane yokuqondanisa kwesifunda sokuhlanganiswa kabusha kwedathasethi ye-Mycoplasma bovis ethinta isakhi sofuzo se-pks12. Awekho amagebe noma ama-nucleotide angachazwanga atholakala endaweni yokuhlanganiswa kabusha kwamanodi angaphakathi. Mayelana nomcimbi othinta i-pks12 gene encoding polyketide synthase, iqukethe kuphela i-Eu2 genome. Ikhwalithi yokulandelana kwe-pks12 yahlolwa ngokufundwa kwemephu kwe-Mycobacterium bovis AF2122/97. Qinisekisa indawo enconyiwe ye-SNP endaweni yokuhlanganisa kabusha ngokusebenzisa imibandela eshiwo esigabeni sendlela (okungenani ukufundwa okungu-20 kanye no-0.9 wokushintsha imvamisa). Ama-polymorphisms we-genomes Mb0891, Mb1711, Mb1789, Mb1870, Mb1758, Mb2043, kanye ne-Mb1960 aqinisekiswe ngokugcwele.
Izakhi zofuzo ze-PE ne-PPE zinezifunda eziphindaphindayo ezifundeka kabi kalula ngokulandelana kwe-Illumina nokungalungile, ngakho-ke ngokuvamile ziyasuswa ekuhambeni komsebenzi kwe-bioinformatics yamalungu e-M. tuberculosis kuphela lapho kusetshenziswa isu lokuhlela-kuya-kulandelana. Ukucatshangwa kwemicimbi yokuhlanganiswa kabusha esetshenziswe kulo msebenzi kusekelwe ekuhlanganiseni kwe-de novo ngaphandle kokuhlunga i-PE/PPE. Sikholelwa ukuthi ngokusebenzisa izindlela ezihambisanayo ezintathu ezihlukene kanye ne-algorithms ngokusebenzisa i-SplitsTree, ipayipi le-Gubbins nesofthiwe ye-RDP4, amasu asetshenzisiwe aqinile ekucubunguleni nasekuhlungeni izifunda ezihlelwe kabusha ezibangelwa amasignali amaphutha. Kodwa-ke, ukuze kukhishwe ukuphazanyiswa kwesakhi sofuzo se-PE / PPE ku-Gubbins nesofthiwe ye-RDP4 ukukhomba amaqoqo e-SNP, ngakho-ke ukuhlonza izifunda zokuhlanganiswa kabusha okuhlongozwayo ukuthi kuthinte izakhi zofuzo ze-narX kanye ne-pks12, umakhelwane walezi zakhi zofuzo zahlolwa (I-Supplementary Fig. 3-5). Ku-M. bovis AF2122/97, isakhi sofuzo se-narX sihlukaniswa yi-narK2 ne-Mb1764c, kuyilapho i-pks12 izungezwe i-Mb2075c e Mb2073c (Umfanekiso Owengeziwe 3-5). Imephu ekhiqizwe kusetshenziswa imephu ye-syline ye-MAUVE ye-genome ephelele inikeza ulwazi mayelana nokugcinwa kokulandelana kwezakhi zofuzo nokuhlela kabusha, ebonisa amabhulokhi ama-collinear amane, futhi azikho izimpawu zokuguqulwa kofuzo noma ukuguqulwa. Ngaphezu kwalokho, ukuhlaziya okuhambisanayo nokulandelana kwe-amino acid kufakazele i-homology kuwo wonke ama-genomes aphelele, futhi ayikho i-PE/PPE etholakele ezindaweni eziseduze ze-narX noma i-pks12. Ku-narX, i-genome eyodwa (Mb0030) inesilinganiso esiphansi se-synonymy ngoba isakhi sofuzo se-narX sihlonzwe njengezingcezu ezimbili (izingcezu 1891 kanye no-1890). Ku-pks12, ngenxa yokufana, i-Mb0030 ne-Mb003 ibonise amaphuzu aphansi e-sylinearity, kuyilapho i-pks12 ikhonjwe ngezingcezu ezimbili nezintathu, ngokulandelana, ezimele izizinda ezihlukene zamaprotheni (Umfanekiso Owengeziwe 3-5). Ngokucabangela lolu lwazi, kanye nama-Gubbins kanye nesofthiwe ye-RDP4 bobabili benza ukuhlaziya, hlola ukuqondanisa okuningi okuyisisekelo kobukhulu obungu-500 bp efasiteleni, siqinisekisile ukuthi isakhi sofuzo se-PE/PPE ngeke siphazamise isignali yokuhlanganiswa kabusha ethinta i-narX ne-pks12.
Nakuba amasiginali okuhlanganiswa kabusha atholwe kule sethi yedatha angase abhekwe njengensalela, kuyiqiniso ukuthi ukuhlanganiswa kabusha ku-M. bovis akukwazi ukukhishwa, ngakho kufanele kuqhubeke kube isihloko sokuhlaziya okwengeziwe, lapho ama-genomes aphelele asuka kuzimo ze-epidemiological ezehlukene alandelana ukuze athi Okubalulekile.
Ukuqhathanisa izihlahla ze-ML phylogenetic ezitholwe ngaphambi nangemva kokulungiswa kokuhlanganiswa kabusha (Umfanekiso 2A, B) akuzange kuholele ekushintsheni okuphawulekayo ebuhlotsheni be-phylogenetic obuhlosiwe, futhi izinhlobo ze-M. bovis zahlanganiswa eqenjini elifanayo.
Ngemva kokwenza imephu 42 esanda kuhlelwa ngokulandelana kwe-M. bovis ifundeka ne-reference genome ye-M. bovis AF2122/97, ukuqondanisa kwe-SNP okuqukethe izikhundla ze-polymorphic ezingu-1816 kwatholakala. Ama-SNP amaningi (87.1%) atholakala endaweni yokufaka ikhodi, futhi izakhi zofuzo ezithintekile zibonakala ngokwezigaba zokusebenza eziboniswe ku-Bovilist (Umfanekiso 6A, B). Uma kucatshangelwa inani eliphelele lezakhi zofuzo esigabeni ngasinye sokusebenza, izakhi zofuzo ezisesigabeni "se-lipid metabolism" zibonise ama-SNP amaningi, alandelwa "izinqubo ze-cell wall and cell" kanye "ne-metabolism emaphakathi nokuphefumula", okuveza ukuthi zise-M. bovis evolution.
Ukuhlaziywa kwe-Hierarchical kwedathasethi ye-M. bovis evela e-Portugal (n = 42). Isamba senani lama-SNP abhalisiwe kanye nezakhi zofuzo ezithintekile esigabeni ngasinye sokusebenza (A). Isamba senani lezinguquko ezifanayo nezingezona ezifanayo ezibhaliswe ngokwesigaba somsebenzi (B).
Emhlabeni jikelele, isilinganiso se-dN/dS singcono kuno-1.5, okubonisa ukuthi ingcindezi yomhlaba wonke yokuguquguquka iwukuqeda isimo sokhokho futhi imele isimo esihle (esihlukahlukene noma esiqondisiwe) kanye/noma esikhululekile sokukhetha ukuhlanzwa. Ezigabeni "ze-virulence, detoxification, adaptation", "ukufakwa ngokulandelana kanye nama-phages", kanye "namaprotheni okulawula", ngaphezu kwezingxenye ezimbili kwezintathu ze-SNP azifanisi (Umfanekiso 6B).
Kuzo zonke izigaba, kukhona izakhi zofuzo ezinama-SNP amaningi, okuholela esilinganisweni sezinga lokuguqulwa (okungukuthi, isilinganiso se-SNP yisakhi sofuzo) esikhulu kuno-1 (Umfanekiso 6A). I-Pks12 (Mb2074c) enama-SNP angu-15 kanye ne-fas (Mb2553c) enama-SNP angu-8 anamanani aphezulu okuguqula. Zombili lezi zakhi zofuzo zibandakanyeka ku-fatty acid metabolism. I-pks gene ifaka i-polyketide synthase (PKS), okuyi-enzyme esebenzayo ehilelekile ku-mycobacterial cell wall lipid biosynthesis74,75. Lolu hlobo lofuzo luhlanganisa i-polypeptide esebenzayo ebandakanyeka ekuhlanganiseni kwe-mycoketides74,76. I-fas gene ibandakanyeka ekuhlanganiseni kwe-mycolic acid. Zombili lezi zakhi zofuzo zidlala indima ebalulekile ku-biosynthesis yodonga lwamaseli lapho luthintana nomsingathi.
Ukuze kuqhutshekwe nokufunda ngokuvela kwe-Mycobacterium bovis, kwahlaziywa amasethi amabili ezakhi zofuzo ezithile. Imisebenzi eshicilelwe ngaphambilini isebenzisa ukwakheka kokulandelana nezindlela ze-phylogenetic ezihlonze izakhi zofuzo ezatholwa okhokho be-MTBC nge-HGT ngaphambi kokuhlukahluka37,38. Lezi zakhi zofuzo zifakwe kuhlu lwe-Supplementary Table 2. Ukusatshalaliswa kwe-SNP kwengqikithi yezakhi zofuzo ezingama-77 ezingase zihlobane ne-HGT kwahlaziywa, futhi kwahlonzwa amasayithi e-polymorphic angama-26, okuvame kakhulu (78%) okubangele izinguquko ezingezona ezifanayo (NS) (Ithebula Lokwengeza 2). Umsebenzi wangaphambilini wofuzo lwe-MTBC ubonise ukuthi isifunda se-HGT esibekayo sibonisa isilinganiso esiphezulu se-NS SNP uma siqhathaniswa nayo yonke i-genome. Uma umuntu ecabanga ukuthi lezi zifunda zokuhlanganiswa kabusha zitholwe okhokho be-MTBC, ngakho-ke, zimelela ngokweqile ama-polymorphisms asendulo, khona-ke ingxenye yezinguquko ezifanayo kulindeleke ukuba ibe phezulu, ngoba ukufakwa esikhundleni kwe-NS kulindeleke ukuthi kuqedwe ngokukhethwa okungalungile ngenxa yezinguquko ze-amino acid Ingase ishintshe umsebenzi weprotheni. Ngakho-ke, imiphumela yethu ibonisa ukuthi imiphumela yokusebenza ingase ibangelwe esikhundleni sezakhi zofuzo ezifana ne-HGT, okubonisa ukubaluleka kwazo ezinhlobonhlobo zofuzo eziguqukayo ezibalulekile.
Ngokuhambisana nalokhu kuhlaziya, izakhi zofuzo ezifaka amakhodi ezingxenyeni ze-3R (ukulungisa i-DNA, ukuphindaphinda, nokuhlanganisa kabusha) uhlelo zahlolwa ngokucophelela ngokuhambisana nohlu olwanyatheliswa ngaphambilini ngu-dos Vultos nabahlanganyeli (2008)39. Ukushintshaniswa kwezingcezu ezifanayo ze-DNA akukwazi ukubonwa ngokuqondile, nakuba kungase kube inqubo evamile lapho amabhaktheriya ahlobene eduze ehilelekile, njengalapho kwenzeka le sethi yedatha; ngaphezu kwalokho, le nqubo ingaba yisihluthulelo sezindlela zokulungisa i-DNA72, ngakho-ke Dlala indima ekuhlanganiseni kabusha kwe-homologous. Ingqikithi yezikhundla ze-polymorphic ezingama-26 ezisatshalaliswe yizakhi zofuzo ezingama-54 zahlonzwa (Ithebula Lokwengeza 3). Kuleli sethi yezakhi zofuzo, izinguquko ze-NS zibalele cishe u-65% wemiphumela, ehambisana nemibiko yangaphambilini yezinhlobo ze-Mycobacterium tuberculosis.
Isikhathi sokuthumela: Oct-21-2021