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Ukulandelelana kwe-Genome kuye kwavuselela intsimi yophando lwezifo ezosulelayo, ukutyhila i-epidemiology yesifo, i-pathogenesis, i-host-pathogen interactions, kunye nenkqubo ye-evolution ebekwe kwii-pathogens. I-Mycobacterium tuberculosis complex (MTBC) ithatha i-Mycobacterium bovis njengelinye lamalungu ayo aqhelana nezilwanyana abangela isifo sephepha (TB) kwizilwanyana ezanyisayo ezisemhlabeni, kwaye ngumzekelo oqhelekileyo wokuzivelela kwebhaktiriya. Njengamanye amalungu e-MTBC, iMycobacterium bovis ithathwa njengeyintsholongwane eqinileyo, ephuma kancinane, kwaye akukho phawu lokuphinda ludityaniswe okanye lugqithise imfuza ethe tye. Kulo msebenzi, sisebenzisa i-genomics ethelekisayo kwi-genome sequence (WGS) yonke idathasethi eyenziwe ngeenkomo ezingama-70 M. ezivela kwiimvelaphi ezahlukeneyo (iYurophu kunye ne-Afrika) ukuze sifumane ulwazi malunga nokwahlukana kwemfuza yeenkomo M. Amandla e-Evolutionary. Kusetyenziswa iindlela ezintathu ezahlukeneyo ukuqikelela imiqondiso yohlengahlengiso. Ehlabathini lonke, inani elincinci leziganeko zokudibanisa kwakhona zichongiwe kwaye ziqinisekiswa ngeendlela ezimbini ezizimeleyo kunye nenkxaso eqinile. Nangona kunjalo, xa kuthelekiswa neenguqu, ukuhlanganiswa kwakhona kunempembelelo ebuthakathaka kwiintlobo ezahlukeneyo ze-M. bovis (i-r / m = 0.037 iyonke). Umahluko we-r/m ophakathi ofunyenwe kwi-clonal complex ye-Mycobacterium bovis kwiseti yethu yedatha ihambelana nengcamango ngokubanzi yokuba iqondo lokuphinda liphinde liphinde lihluke kakhulu phakathi kwemigca eyabelwe uhlobo olufanayo lwe-taxonomic. Ngokusekelwe kulo msebenzi, ukuhlanganiswa kwakhona kwi-Mycobacterium bovis akunakugwenywa, ngoko ke kufuneka ibe ngumxholo wemizamo eyongezelelweyo kuphando lwe-genomics oluthelekisayo lwexesha elizayo, apho i-WGS yeeseti ezinkulu zedatha ezivela kwiimeko ezahlukeneyo ze-epidemiological emhlabeni jikelele ibalulekile. Uhlalutyo olongezelelweyo lwenziwa emva koko kwiseti yedatha encinci ye-Mycobacterium bovis (n = 42) ukusuka kwi-TB ye-TB ye-multi-host, kwaye ngaphezu kwe-1,800 loci ichongiwe, apho ubuncinane i-strain enye ibonisa i-nucleotide polymorphism (SNP) . Uninzi (i-87.1%) lufumaneka kwingingqi yekhowudi, kwaye umlinganiselo wehlabathi jikelele weenguqu ezingabonakaliyo (dN / dS) zeenguqu ezifanayo zidlula i-1.5, ebonisa ukuba ukhetho oluhle luyinto ebalulekileyo yokuguquguquka kwemvelo eyenziwa kwi-M. bovis. Umlinganiselo ophezulu we-SNP ufunyenwe kwiintlobo zofuzo ezizityebi kwiindidi ezisebenzayo "ze-lipid metabolism", "udonga lweseli kunye neenkqubo zeselula", kunye "nemetabolism ephakathi kunye nokuphefumla", ebonisa amandla abo kwi-biology kunye nokuziphendukela kwe-Mycobacterium bovis ukubaluleka. Ukujongwa ngokucokisekileyo kwiijini kwi-MTBC izinyanya ezithambekele ekudluliselweni kofuzo oluthe tye kwaye zifakwe kwinkqubo ye-3R (ukulungiswa kwe-DNA, ukuphindaphinda, kunye nokudibanisa kwakhona) inkqubo ibonisa umyinge wehlabathi jikelele wexabiso elibi le-Taijima yovavanyo olungathathi hlangothi lwe-D, olubonisa ukuskena okukhethiweyo okudlulileyo
I-Mycobacterium tuberculosis complex (MTBC) yenye yeyona tata iphumeleleyo ye-bacterial pathogens kunye nemeko eqhelekileyo yokuzivelela kwebhaktiriya. Amalungu ayo abonisa ngokumangalisayo i-nucleotide identity ephezulu kwinqanaba le-genomic (> 99%)1,2. I-MTBC ecotypes eyahlukeneyo inokubangela isifo sofuba (i-TB), esisifo esosulelayo se-granulomatous, kwiintlobo ezininzi zezilwanyana ezivela kwizilwanyana ezincinci ukuya kubantu3,4,5. Sithetha nje, le complex iquka abantu [M. Isifo sephepha (Mtb), Mycobacterium africanum] kunye nezilwanyana ezilungelelanisiweyo (Mycobacterium bovis, Mycobacterium capitum, Mycobacterium pinnipedum, Mycobacterium microtobacter , Mycobacterium mongee, Mycobacterium miysani, Mycobacterium surika, "Bacillus chimpanzee") kunye ne "Bacillus chimpanzee" 5,6. M. canettii (eyaziwa nangokuthi "i-Nodobacter glabrata") I-avareji ye-nucleotide identity kunye ne-mycobacteria ekhankanywe ngasentla yi-98%, kunye nomsebenzi wokulinganisa we-genomics ubonise ukuba i-M. canettii kunye nayo yonke i-MTBC isandul 'ukuhlukana kwi-ancestor eqhelekileyo.7 Ukuqwalasela le ngcamango, abanye ababhali bangakwazi ukubiza i-MTBC iLungu le-8 M.
I-MTBC ichazwa ngokucwangcisiweyo njenge-clonal complex, kwaye ubume bayo babemi ngokucacileyo bulawulwa yiyantlukwano encitshisiweyo, imiqobo, ukuskena okukhethiweyo, kunye nemfuzo ye-drift9,10. Ukucinga ngendaleko engqongqo engqongqo yeclonal, enje ngepolymorphisms engekhoyo ayinakubuyiselwa ngokudibana kwakhona. Ngokusekelwe kwesi siseko, iziganeko ezilandelelanayo zokususwa kwe-genomic yommandla ohlukeneyo (RD) kunye ne-TbD1 (i-Mtb ecacileyo yokucima i-1 yengingqi) zicetywayo njengeziphawuli ze-molecular ze-MTBC evolution2,5,11. Ukuthelekisa i-genomics kunye nomsebenzi opheleleyo we-genome sequencing (WGS) uxhasa ulwahlulo lwamalungu alungelelanisiweyo kwimizila elithoba (Mycobacterium tuberculosis L1 to L4, L7, and L8; and Mycobacterium africanum L5, L6, and L9), lineages L2 to L4 shared delete TbD1 region22,1. Ukongezelela, amalungu alungelelanisiweyo ezilwanyana acetywayo ukuba abelane ngokhokho oqhelekileyo, ochazwa ngokucinywa kwe-clade-specific deletions kwi-RD7, i-RD8, i-RD9 kunye ne-RD102, i-5, kunye ne-14.
Ukudluliswa kwemfuza ethe tyaba (HGT) kunye neziganeko zokudibanisa zibhekwa njengezinqabileyo kwaye zenzeke kwizinyanya ze-MTBC, kunokuba imbali eyahlukileyo yelungu lonke le-MTBC15,16,17. Iingxelo ezimbini zangaphambili zikaHughes kunye nabasebenzisana nabo (2002) kunye noGutacker kunye nabasebenzisana nabo (2006) bacebise ukuba iziganeko zokuhlaziya zinokunceda ukubumba iipolymorphisms eziphawula i-loci ethile kwi-M. tuberculosis strains18,19. Izizathu zokungabikho okucacileyo kokuhlanganiswa kwakhona kwi-MTBC zezi: (1) inkqubo yomatshini kunye nokulahlekelwa kwamandla e-HGT; (2) ukunqaba kweziganeko zeHGT; (3) akukho thuba leziganeko zokudibanisa kwakhona kwi-MTBC niche14,17. Kutshanje, ezinye izifundo ze-whole-genome sequencing (WGS) ezifakwe kwiMTBC strain 20 kunye neMycobacterium bovis 21 zibonelele ngobungqina bokudibana kwakhona, eyokuqala ukubonisa ukuba iintlobo zeMTBC zihlala zitshintshiselana ngamaqhekeza amancinci eDNA, kodwa ngenxa yokwahluka kolandelelwano lwenucleotide olulinganiselweyo, ezi ziganeko azikaqatshelwa.
I-Mycobacterium bovis lelona lungu lixhaphake ukufunyanwa kwi-MTBC kwimfuyo (ingakumbi iinkomo), nangona inokuthi ibekwe yodwa kwizilwanyana zasendle ezikhululekileyo nezibiyelweyo4,22,23,24. I-M. bovis yavela kwii-clonal complexes ezintlanu ezinkulu [i-European 1 (Eu1), i-European 2 (Eu2), i-European 3 (Eu3), i-Afrika 1 (Af1) kunye ne-Afrika 2 (Af2)], ngokweprofayili ye-spoligotyping, ukucima okuthe ngqo kunye ne-polymorphisms eyodwa ye-nucleotide (SNPs) 25, 27, 27, 26, 26, 92, 25, 26, 92, Ezi zakhiwo ze-clonal zibonisa ubume obahlukeneyo boluntu lwe-Mycobacterium bovis kunye nokunxulumana nemimandla yejografi. Ukongeza, umsebenzi wamva nje we-WGS owenziwe nguZimpel kunye nabasebenzisana nabo (ngo-2020) wayila i-phylogeny esekwe kwi-SNP yeMycobacterium bovis, engaphezulu kwe-1,900 genomes, ebonisa ukuba kukho ubuncinci imigca emine eyahlukeneyo (ebizwa ngokuba yi-Lb1 ukuya ku-Lb1 ukuya ku-Lb4), ayichazwanga ngokupheleleyo, nangona i-clographic inokuthi ingqinelane. Aba babhali benza uhlalutyo oluhlukeneyo lwe-phylogeny kunye ne-molecular dating, kodwa abazange bafunde ukuphindaphinda30.
Umsebenzi wangaphambili usebenzisa iindlela ezahlukeneyo ze-molecular, ezifana ne-spoligotyping, i-MIRU-VNTR (i-mycobacterial interspersed repeat unit-variable tandem repeat number), kunye nokuchwetheza kwe-SNP yakutshanje kubonakalise inqanaba elithile lokwahluka kofuzo phakathi kwe-M. bovis strains 31,32,33, 34,35. Ukwahlula ukuhlukahluka kofuzo kuye kwaba sisixhobo esibalulekileyo kwisifundo se-epidemiology yesifo, esiluncedo ekuqondeni ngokunzulu i-pathogenesis, i-virulence kunye nokudluliselwa kwezifo. Ukuvela kwendlela ye-WGS kunika ithuba lokubonisa izinto zokuqhuba eziguquguqukayo ezibekwe yi-Mycobacterium bovis genome kwinkqubo yokulungelelaniswa kunye nokunyamezela kwimikhosi eyahlukeneyo kunye neemeko ze-epidemiological.
Kulo msebenzi, sisebenzisa uhlalutyo oluthelekisayo lwe-genomic kwiinkcukacha ezahlukeneyo ze-Mycoplasma bovis (n = 70), kubandakanywa i-isolates ukusuka kwii-clonal complexes ezahlukeneyo, ukufumana ulwazi malunga nenkqubo ye-evolution ye-Mycoplasma bovis, ngakumbi ukusombulula ubudlelwane be-phylogenetic kunye neziganeko zokubuyisela kwakhona. Njengesongezelelo kolu hlalutyo, iseti engaphantsi kwedatha ye-M. bovis yodwa (n = 42) efunyenwe kwindawo yesifo sephepha esaziwa kakuhle e-multi-host e-Portugal 31,36 yaphononongwa ngakumbi ukuqikelela ukungazazisi Ibhalansi phakathi komlinganiselo ohambelanayo wengqondo (dN) kwi-synonymous (dS) nucleotide ekhankanyiweyo kwi-evolution, njengoko kuchaziwe kwi-evolution I-37,38 efunyenwe yi-MTBC izinyanya nge-HGT, kunye ne-encode 3R (ukulungiswa kwe-DNA, ukuphindaphinda, kunye nokudibanisa) izakhi zofuzo zenkqubo 39. Khetha izakhi zofuzo ezifunyenwe nge-HGT kuba zingamela iipolymorphisms zamandulo, ngoko ke kulindeleke ukuba ziqulethe umlinganiselo ophezulu weenguqu ezifanayo. Izakhi zofuzo ezibandakanyiweyo kwinkqubo ye-3R zikhethwe ngenxa yokuba umsebenzi wangaphambili kwiintlobo zesifo sofuba ze-M. ubonise ukhetho olubi / oluhlambulukileyo oluqhelekileyo olusebenza kule mizi , kwaye banokudlala indima ebalulekileyo ekuziphendukeleni kwemvelo 39. Enye injongo yalo msebenzi kukuphazamisa ubukho beziganeko zohlengahlengiso. Ngesi sizathu, sithathela ingqalelo ukuba iseti yethu yedatha evela ePortugal iqulethe kuphela iigenomes ze-European clone complex 2 kunye neentlobo ezingakhange zinike i-clone complex, siye sagqiba ekubeni sibandakanye idatha ye-genome ekhoyo esidlangalaleni ukuze ekugqibeleni sifumane ummeli wazo zonke izakhiwo ze-clone, kunye nokuphucula ukomelela kunye nobubanzi beziphumo.
42 iMycoplasma bovis genomes ezisandula ulandelelwano ezisuka kwindawo yesiPhuthukezi yesifo sephepha esixhaphakileyo (iinkcukacha ezingezantsi), eziphawulwe ngaphambili ngokwembono ye-epidemiological36, zingundoqo walo msebenzi. Ukuqwalasela ukuba isethi yedatha evela ePortugal inabameli kuphela baseYurophu 2 i-clone complexes kunye neentlobo ngaphandle kwezakhiwo ezikhethiweyo, idatha efumaneka esidlangalaleni yokulandelelanisa i-genome yongezwa ukwandisa isethi yedatha equka bonke abameli be-M. bovis clone complexes. Ngoko ke, imithombo emithathu yedatha yokulandelelanisa i-genome yonke isetyenziswe kulo msebenzi: indibano epheleleyo / eyidrafti ye-genome, ukuya kwii-scaffolds ze-10 ezigcinwe kwi-NCBI (iZiko leSizwe loLwazi lwe-Biotechnology) (n = 15 i-isolate); igcinwe kwi-SRA (Ifayile ye-Illumina fastq yolandelelwano lokufunda i-archive) imele ukuhlukana okuyinkimbinkimbi kwe-M. bovis clones (n = 12 isolate)30; kunye nama-42 amatsha ohlobo lwe-genomes asuka ePortugal. I-Mycobacterium bovis BCG (i-Bacille Calmette-Guerin) yayingabandakanywanga kwi-NCBI yokukhangela. I-M. bovis AF2122/97 idla ngokusetyenziswa njenge-reference genome ukuba ibandakanywe kwidathasethi. Ngenxa yokungafumaneki koluntu lonke ulandelelwano lwe-genome olumelwe yi-African 1 cloning complex, kunye nenani elincinci le-genomes ukusuka kwiintlobo ezimele ze-Af2 kunye ne-Eu1, idatha yolandelelwano lwangaphambili olunikezelwe yi-SRA lusetyenziswe kwezi meko. Umsebenzi kaZimpel kunye namaqabane akhe (2020) uncede ekuchongeni igenome kwi-cloning complex ekhankanywe ngasentla kwaye wanceda ekukhetheni iMycobacterium bovis ukuze ifakwe kwiseti yedatha. Kwi-Eu3, lunye kuphela uhlobo lwe-genome oluchaziweyo (Branger et al., 2020), ngoko ke i-genome esiyibandakanyayo ngummeli owahlukileyo we-Eu3 complex.
Ehlabathini lonke, le seti yedatha iquka i-70 yeenkomo ze-M. bovis ezihlukanisiwe kwiintlobo ze-8 ze-host, ezisasazwe kumazwe angama-12 ukusuka kwi-1985 ukuya ku-2016. Iintlobo ze-36 zichongwa njenge-Eu2, iintlobo ezi-7 ze-Eu1, iintlobo ze-1 ziyi-Eu3, iintlobo ze-3 ze-Af1, iintlobo ezi-4 ze-Af2 kunye ne-19 azikho kwi-clonal complex. Iinkcukacha ezithe vetshe (kubandakanywa nenombolo yokungena) yeMycobacterium bovis esetyenziswe kolu phononongo iboniswe kwiTheyibhile 1 kunye neTheyibhile eyoNgezelelweyo 1.
Iigenomes ezingama-42 ezisandula ukulandelelana ezipheleleyo zeMycobacterium bovis ezivela kwiindawo zesifo sephepha sezilwanyana zasePortugal kwaye zasasazwa ngaphezu kweminyaka eyi-12 zingundoqo wolu phononongo, njengoko iinkqubo ezinokubakho zezifo zezilwanyana zasendle ziye zajongwa rhoqo 31,36 (Fig. 1). Ngokweenkqubo ezilandelayo, ezi ntlobo zazibekwe zodwa kwinkomo (n = 14), ixhama elibomvu (n = 16) kunye neehagu zasendle (n = 12) ukusuka kwi-2003 ukuya ku-2015: ukuqokelela nokuphatha izilwanyana ngokuhambelana nezikhokelo zeprothokholi ezicetyiswayo. phakathi. Iinkcubeko zifukanyelwa kwi-37 °C kwaye ukukhula kuhlolwa kanye ngeveki ubuncinane iiveki ezili-12. Iikoloni zigcinwa ngqo kwisisombululo se-glycerol kwi-80ºC. Kwi-Mycobacterium medium selective medium (Middlebrook 7H9, BD Diagnostics), iisampulu zangaphambili ezigciniweyo zagqithiswa ngendlela enye ye-vitro kwi-vitro ukufumana i-DNA yeprogram ye-WGS. Kule nto, isisombululo sesitokisi senkcubeko ekhenkcezisiweyo satyetyiswa nge-5% ye-sodium pyruvate kunye ne-10% ye-ADS (i-50 g albumin, i-20 g glucose, i-8.5 g ye-sodium chloride kwi-1 L yamanzi) kwi-Middlebrook 7H9 kwi-37 ° C Ukubuyisela kwakhona. Emva kweeveki ezi-4 zokukhula, i-medium yahlaziywa kwaye inkcubeko yayijongwa rhoqo de ukukhula kubonwe. Iiseli zavunwa nge-centrifugation, i-pellet yaphinda yamiswa kwi-500 µL ye-phosphate buffered saline (PBS), ifudunyezwe ku-99 °C imizuzu engama-30, i-centrifuged, kwaye i-supernatant yagcinwa kwi -20 °C de kube yi-WGS. Zonke iinkqubo zenziwa kwinqanaba le-3 lezibonelelo ze-biosafety.
Ilayibrari ye-WGS edibeneyo ye-genome ilungiswa ngokusebenzisa isalathisi esikhethekileyo sesampuli ye-DNA nganye, kwaye isebenzisa i-Illumina MiSeq (2 × 250 pb) (iisampuli ze-40) kunye ne-HiSeq (2 × 150 pb) (ezimbini zodwa) iteknoloji (Eurofins Genomics, eJamani) ngokulandelelana. Ngokwemiyalelo yomenzi, sebenzisa i-Illumina Genome Analyzer ene-attachment yemodyuli ephindwe kabini ukulandelelana kwe-DNA ye-genomic, kwaye usebenzise i-Nexera XT DNA Library Prep Kit esuka kwi-Illumina ukwakha ithala leencwadi.
Ukuthathela ingqalelo idatha efunyenwe kwi-SRA (n = 12), ukuchongwa kwe-clone complex ingasetyenziswa njengemetadata yoshicilelo oluhambelanayo 30, 41, 43. Xa kuqwalaselwa i-genome epheleleyo, ngaphandle kwe-Mycobacterium bovis AF2122/97 kunye ne-Mycobacterium bovis 3601, amalungu e-Eunal1 e-Eunal 3601 eyaziwayo kunye ne-Eunal1 I-29, ngokulandelanayo, iyafana ne-genome epheleleyo ye-Mycobacterium tuberculosis H37Rv (inombolo yokufikelela kwi-NCBI NC_000962.3). Ukulungelelaniswa kweGenome kwenziwa ngokusebenzisa i-MAFFT (iprogram yokulungelelanisa ezininzi ze-amino acid okanye ukulandelelana kwe-nucleotide, inguqulo ye-7.458) kunye ne-parameter -adfragments48. Emva koko, khangela ukungabikho kwee-clonal complexes ezahlukeneyo kunye / okanye ubukho beempawu ze-SNP.
I-Mycobacterium bovis esandul' ukulandelelana (n = 42) kunye nofundo lokuqala lwedrafti yegenome edibeneyo (n = 3) ilungelelanisa ikhompleksi kunye nereferensi genome Mycobacterium tuberculosis H37Rv ngombhobho we-vSNP kunye nobukho bokucinywa kunye/okanye iimpawu ze-SNP zee-clones ezahlukeneyo Ukhangelo lwenziwe.
Ukuqokelela ulwazi oluvela kukungabikho kweempawu kunye / okanye ubukho / ukungabikho kwe-SNP kunye neeprofayili ze-spoligotyping ukunika idatha ye-genomic kwi-clonal complex ehambelanayo. Kwiindibano ezine zoyilo, iprofayili ye-spoligotyping ayinakuchazwa, ngoko ke ifakwe kwiqela elithi "akukho bunzima".
I-bioinformatics workflow elandelwa ngulo msebenzi iqala kwindibano ye-de novo kunye nemephu ukuya kwisicwangciso sokubhekisela, ngenjongo yokuphonononga iziganeko zokudibanisa kunye neepolymorphisms ezithile ze-genome. Umzobo woku-1 ubonelela ngetshati yamanyathelo alandelweyo. Ukuhlalutya okudityanisiweyo, zonke iigenomes zisetyenziselwa ukunyusa ukomelela kweengqikelelo kunye nezalathi ezihambelanayo.
Ukuze kuncitshiswe iimpazamo ekuveliseni ulandelelwano lwe-genome consensus, saqala safumana indibano ye-de novo, saza safumana ulungelelwaniso oluninzi olungundoqo. Umbhobho we-Unicycler ufumaneka ngoku ku-https://github.com/rrwick/Unicycler49 kwaye usetyenziselwa ukwenza indibano ye-de novo yeegenomes ezilandelelanayo eziyi-54 (ama-42 asandul' ukulandelelana kunye neefayile eziyi-12 ze-fastq ezifunyenwe kwi-SRA). Ngamafutshane, ngaphambi kokuhlanganiswa ukusuka ekuqaleni, uhlalutyo lomgangatho wokufunda lwenziwa kwinguqulo ye-FastQC engu-0.11.7 (https://github.com/s-andrews/FastQC), kunye nenguqulo ye-Trimmomatic 0.36 (ukhetho "ukusika i-Adapter kunye nolunye ulandelelwano oluthile lokukhanya ukusuka kufundo" kwaye "Sika iziseko ezi-2, ukubamba iziseko ezi-2 ukusuka kwinqanaba elisezantsi, ukubamba iziseko ezi-2 isetyenzisiwe) (http://www.usadellab.org/cms/?page= trimmomatic) 50. Emva koko, i-SPAdes optimiser49 yayisetyenziselwa ukudibanisa i-genome, kwaye i-Pilon version 1.1851 yasetyenziselwa ukulungelelaniswa kwe-post-assembly. Kukhethwe imo yokuvala ibhulorho ukuthintela ukudityaniswa okungachanekanga, kwaye isayizi ye-k-mer yakhangelwa yaza yakhethwa phakathi kwe-20% kunye ne-95% yobude obufundwayo. Landela izikhokelo ze-SPAdes kwaye uqwalasele ubungakanani bokufunda, cima i-contigs encinci kune-300 bp, kwaye usethe i-20 yokufunda i-deep coverage cutoff ye-52. Kwisicwangciso sendibano ye-de novo, imimandla ye-genomic efana ne-proline-glutamate ephindaphinda kakhulu (PE) kunye ne-proline-proline glutamate (PPE) paralogs ayizange isuswe.
Umgangatho wendibano ye-de novo uvavanywa ngombhobho we-QUAST (http://quast.sourceforge.net/quast.html), oququzelela uhlaziyo lwe-contig kunye ne-M. bovis AF2122/97 genome yesalathiso (inombolo yofikelelo lwe-NCBI LT708304.1) Ukwenziwa kweMaphu (jonga iParamitha yeTheyibhile eyoNgezelelweyo1).
Ngoncedo lombhobho we-vSNP (https://github.com/USDA-VS/vSNP), ifayile ye-FASTQ ye-M. bovis esanda kulandelwa ngokulandelelana ukusuka kwi-Illumina ngokulandelelana ithelekiswa ne-M. bovis AF2122/97 i-genome ye-reference (LT708304.1)). Ngokwezona ngcebiso zisebenzayo zeGenome Analysis Toolkit (GATK) 53, 54, 55 sebenzisa iiparamitha zokucoca eziqhelekileyo okanye amanqaku obunzima ohlukeneyo okuhlaziya kwakhona. Iziphumo zihluzwa kusetyenziswa owona bunzima buphantsi be-SAMtools amanqaku e-150 kunye ne-AC = 2. Kwakhona sebenzisa i-Kraken (http://ccb.jhu.edu/software/kraken/) ukujonga ukufundwa ukukhupha ukungcola. Umbhobho we-vSNP osetyenziselwa ukwenza imephu ukulandelelanisa amaqhinga emsebenzini wethu uvavanya uluhlu lwee-SNP ezichaziweyo kunye nezinto ekujoliswe kuzo, kwaye kwakhona ayibandakanyi iimeko ezixubileyo zosulelo. Ufundo lwe-genome lungcono kune-99% (iTheyibhile eyoNgezelelweyo 1).
Ukuze ugweme iimpazamo zemephu kunye nee-SNP ezingalunganga, hluza ukwahluka kwezi meko zilandelayo: (1) ixhaswa ngokufunda okungaphantsi kwe-20, (2) ifunyenwe ngokuphindaphindiweyo ngaphantsi kwe-0.9, (3) ubuncinane kwi-Strains enye, kodwa ubuncinane kukho izikhewu kwelinye uhlobo. I-integrated genomics viewer (IGV) version 2.4.19 (http://software.broadinstitute.org/software/igv/)56 isetyenziswe ekuqinisekiseni ngokubonakalayo ii-SNP kunye nezikhundla ezineengxaki zemephu okanye zokulungelelanisa. Ekubeni i-proline-glutamate (PE) kunye ne-proline-proline glutamate (i-PPE) i-genes iphindwe kabini kwaye iyingxenye yentsapho ye-multi-gene, iqondwa lula ngokulandelelana kwe-Illumina kunye nokungahambi kakuhle, ngoko ke zikhethwa I-mycobacterial bioinformatics workflow isuswe amalungu e-tuberculosis complex xa usebenzisa isicwangciso sokulandelelana kwemephu ye-SNP ukuqinisekisa ukulandelelana. Ke ngoko, sihluze iijini ze-PE/PPE kunye ne-indels kuhlalutyo.
Ngokutsho kweBovilist (http://genolist.pasteur.fr/BoviList/), zonke ii-SNP zahlulwe ngokwamacandelo asebenzayo. Umbhobho we-SnpEff (https://pcingola.github.io/SnpEff/) usetyenziselwa ukuphembelela iziphumo ze-SNP (iinguqu ezifanayo okanye ezingezizo). Kwenziwa isiseko sedatha esitsha seMycobacterium bovis AF2122/97 genome (LT708304.1).
Ulungelelwaniso olungundoqo lwe-genome lwenziwe kusetyenziswa i-Parsnp v1.2, okwangoku ekhoyo ku-https://github.com/marbl/parsnp57, kusetyenziswa ii-69 ezipheleleyo ze-genomes/draft assemblies (kunye nenketho -c) kunye ne-M. bovis AF2122/97 (LT708304.1) isetyenziswa njengesalathiso. Ulungelelwaniso ezine ezingundoqo ezininzi zenziwe: kuphela amalungu e-Eu2 cloning complex (n = 37), kubandakanywa onke amalungu e-European cloning complex (n = 44), kubandakanywa indawo yokudibanisa i-European and African cloning complex (n = 51), kwaye ibandakanya yonke i-Mycobacterium bovis kolu cwaningo (n = 70).
Ulungelelwaniso olungundoqo oluveliswa yi-Parsnp lusetyenziselwa ukuqikelela umthi we-phylogenetic obona buninzi (ML) usebenzisa i-CIPRES Science Gateway v3.3 (http://www.phylo.org/) 58 usebenzisa i-RAxML, kwaye wenze ii-replications ezikhokelwayo ezili-1000.
Ii-algorithms ezintathu ezahlukeneyo kunye nezixhobo ze-bioinformatics zisetyenziselwa ukujonga ubukho beziganeko zokuphinda zihlanganiswe ngokufanayo: isofthiwe ye-SplitsTree4, i-Gubbins (umgca ongakhethiyo ngokudibanisa kwakhona kwi-nucleotide sequences) umbhobho, kunye ne-RDP4 (inkqubo yokufumanisa ukudibanisa kwakhona, i-software ye-beta 4.101) isofthiwe.
Indlela yokubola yokwahlula ephunyezwe kwi-SplitsTree4 v4.15.1 (http://www.splitstree.org/)59 isetyenziselwa ukubala uthungelwano lwe-phylogenetic olungenangcambu, kusetyenziswa uvavanyo lwe-Phi lokuqinisekisa ngeenkcukacha-manani, kwaye isiqalo sokubaluleka ngu-p = 0.05. Uhlalutyo oluphambili lwe-multi-alignment ye-Parsnp lusetyenziswa njengegalelo, kwaye ukuchithwa kokwahlula njengomgangatho wothungelwano kuyenziwa.
Umbhobho weGubbins v2.3.1 (https://github.com/sanger-pathogens/gubbins60 usebenza ngeeparamitha ezisilelayo njengenye indlela yokuvavanya impembelelo yokudibanisa kwakhona kwi-Mycobacterium bovis. Umgaqo-nkqubo ophunyeziweyo kumbhobho wakha ngokutsha umgca we-clone ofanelekileyo LT708304.1) zimacala omabini; kwaye ziskena indawo ye-SNP kwisebe ngalinye lomthi ukubona iqela le-SNP elimele isiganeko sokudibanisa kwakhona, oku kuthetha ukuba ii-SNP ezenzeka kwisebe kufuneka zisasazwe ngokulinganayo Iifayile ze-ParRA ezisetyenzisiweyo zisuka kwi-core yokulungelelanisa.
Ekugqibeleni, ukuze kuqinisekiswe isiganeko sohlengahlengiso olucetyiswe ngumbhobho we-Gubbins, i-algorithms ezintandathu eziphunyeziweyo kwi-RDP467 (RDP61, GENECONV62, Bootscan63, Maxchi64, Chimaera65 kunye ne-SiScan66) isetyenziswe kwi-Parsnp's core ulungelelwaniso oluninzi phantsi kwemimiselo engagqibekanga. Sinqume ukuba ubuncinane ezintathu ze-algorithms eziphunyeziweyo kwi-RDP4 kufuneka zibonise ngokuqhubekayo isignali ebalulekileyo yokuqinisekisa isiganeko ngasinye sokuphinda sihlanganiswe.
Ukuqwalasela ukuba zombini i-Gubbins kunye ne-software ye-RDP ijonge izibonakaliso zokudibanisa kwakhona ngokujonga ukulungelelaniswa okuphambili kwifestile ukuya kwi-500 bp, kunye nokuqinisekisa ukuba ukufakwa kweejene ze-PE / PPE ngexesha lendibano ye-de novo akuyi kuphazamisa izibonakaliso zokudibanisa kwakhona ezifunyenweyo, uhlalutyo olongezelelweyo lwenziwa nge-homolinearity Khangela indawo ekufutshane nesiganeko sokudibanisa i-recombination. Imephu ye-synlinear kusetyenziswa i-genome epheleleyo yakhiwa kusetyenziswa i-MAUVE-multi-genome alignment (http://darlinglab.org/mauve/mauve.html) ukukhuphela ngaphandle ukutshintshwa kwe-genome yendawo okanye ukuguqulwa. Ukongezelela, yonke i-genome yayisetyenziselwa ukwenza uhlalutyo lwe-homolinearity kulandelelwano lwe-amino acid ngokusebenzisa iseva yewebhu ye-SyntTax (https://archaea.i2bc.paris-saclay.fr/SyntTax/).
Uhlalutyo olunzulu lwedatha ye-genome efunyenwe kwi-Portuguese multi-host tuberculosis system kukujonga i-polymorphism yezakhi zofuzo ezikhankanywe kwiincwadi. Ezi zofuzo ziyi-37,38 kunye ne-gene encoding 3R efunyenwe zizinyanya zeMTBC ngokusebenzisa i-HGT (ukulungiswa kwe-DNA, ukuphindaphinda kunye nokudibanisa kwakhona) amacandelo enkqubo 39. Sebenzisa i-ClustalX v2.1 (http://www.clustal.org/clustal2/) kwaye usebenzise i-DnaSP v6.12.03 (http://u/edub. iyantlukwano ye-nucleotide (π) kunye ne-Tajima's D engathathi hlangothi yovavanyo lweparamitha yokufaka.
Ubuninzi obunokubakho (ML) umthi wephylogenetic osekelwe kwi-69 Mycoplasma bovis isolate kunye ne-reference genomes yafunyanwa (Figure 2A). Xa kuthelekiswa nemithi esekelwe kwi-gene okanye imithi emininzi, esi sicwangciso sivumela ukuveliswa kwemithi enamandla ngakumbi engabambi ukuhlukahluka kwe-genome yonke kwaye ngoko ke ibonise amandla aphantsi okucalula phakathi kweentlobo ze-68,69. Ubume be-topological bomthi we-ML buhlala buhambelana nokuhlelwa okuntsokothileyo kweeclones. I-genome ye-Eu2 idityaniswe kwisebe, kwaye i-genome ye-Af1 nayo idityaniswe kunye (Umfanekiso 2A). Isiphumo sikwahambelana nobudlelwane obaziwayo bokuvela kwe-Mycobacterium bovis, oko kukuthi, kukho umahluko omkhulu phakathi kwelungu le-Eu1 kunye neqela elibandakanya zonke ezinye ii-clonal complexes kunye ne-genomes, kodwa i-clonal complex 30 ayichazwanga. Ukungahambelani okuncinci phakathi kwe-clonal complex kunye nobudlelwane obubonwa kumthi we-phylogenetic bunokuchazwa yinyaniso yokuba i-clonal complex ichazwa ngokusekelwe kwimimandla ethile ye-genomic, ngelixa umthi we-phylogenetic usekelwe kwiindlela ezininzi zokulungelelaniswa kwee-core genome ezimele i-genome yonke.
Owona mthi unokwenzeka kakhulu wephylogenetic (GTR) wakhiwe ngokusekwe kulungelelwaniso lwegenome olungundoqo lweMycobacterium bovis genome phambi (A) nasemva (B) ukususwa kwendawo yokudibanisa kwakhona. Imibala yesebe imele iMycobacterium bovis clone complex: iYurophu 1 imfusa, iYurophu 2 ibomvu, iYurophu 3 iluhlaza okwesibhakabhaka, iAfrika 1 iorenji, kwaye iAfrika 2 iluhlaza. Umthi unqunyulwe kwaye utsalwe kwisikali, kwaye ubude besebe bulinganiswa njengokutshintshwa kwesiza ngasinye.
I-Mycobacterium tuberculosis complex ichazwa njenge-clonally evolved, kwaye ubungqina obuninzi obuqokelelwe kwiminyaka edlulileyo buxhasa ingcamango yokuba i-HGT eqhubekayo kunye neziganeko zokuphinda zenzeke aziyi kwenzeka kwinqanaba elibonakalayo le-MTBC15,17,18.
Umsebenzi wangaphambili ubonise ukuba kunokubakho ukudityaniswa okulinganiselweyo phakathi kwe-MTBC strains20,21, ngelixa abanye baye basilela ukuchonga iziganeko ezilinganisekayo zokudibanisa70,71. Phinda uxoxe ngalo mbandela ngokugxila kwi-Mycobacterium bovis, eyahlukileyo kumsebenzi wangaphambili owawuqwalasela kuphela i-Mycobacterium tuberculosis 70,71; okanye uthathele ingqalelo iMTBC iyonke, phantse akukho M. bovis emele 20; okanye uthathele ingqalelo kuphela amaqhezu eenkomo athintelweyo. I-dataset ye-mycobacteria, kulo msebenzi, kukho iintlobo ze-70 ezipheleleyo, ezimele zonke ii-clonal complexes, ezisetyenziselwa ukukhangela ukudibanisa kwakhona. Iseti yedatha ilinganiswa ngokwamanqanaba amane aqokelelweyo: (1) Amalungu e-Eu2, (2) onke amalungu e-clone aseYurophu (oko kukuthi iYurophu), (3) i-Clone complex yaseYurophu kunye ne-Afrika (i-Eu + Af) kunye (4) neNgqokelela yedatha yonke (kubandakanywa i-genomes engaqukwanga kuyo nayiphi na i-cloning complexes esele ichazwe).
Ukuqhubela phambili ukufundisisa le ngcamango, inethiwekhi yokuqhekeka kwe-split-decomposition yenzelwa ukuvavanya ukungabikho kweziganeko zokubuyisela kwakhona phakathi kwe-genomes, kuba le ndlela inokubona ubudlelwane bokhokho phakathi kwabantu kunye nokubonisa izibonakaliso ze-phylogenetic eziphikisanayo. Zonke iiseti ezine zedatha kuhlalutyo ziqinisekisile ubukho bee-loops kwinethiwekhi (oko kukuthi, iindawo ezingaguqukiyo zibe ngumthi omnye), kodwa uvavanyo lwe-Phi alunankxaso yezibalo (Eu2, p = 0.0956; iYurophu, p = 0.1637; Eu + Af p = 0.2774; yonke idatha iseti pganiza = 0.24 inika ubungqina obungakuhle ukuba i-pganiza 1) ibonelela ngeziganeko ezihluphekileyo ze-pganiza5 (i-0.24 okanye i-regional 5). 3A-D).
EYurophu i-2 genomes (n = 37) (A), i-genomes yaseYurophu (n = 44) (B), i-genomes yaseYurophu kunye ne-Afrika (n = 51) (C) kunye nedatha yonke (n = 70) (D).
Emva kolu hlalutyo, kunye nokuthathela ingqalelo ukuqwalaselwa kwe-cyclic kuwo wonke amanethiwekhi, i-algorithm yokwakhiwa ngokutsha ephunyezwe kumbhobho we-Gubbins isetyenzisiwe ukuze kwakhiwe ngokutsha umgca we-clonal kunye nokuncedisa uqikelelo lwesiphumo sokuhlanganiswa kwakhona kwi-genome ye-M. bovis. Qwalasela inani elongezelekayo leziganeko zokudityaniswa kwakhona, uninzi lwazo lwenzeka kumasebe e-terminal (oko kukuthi, kwigenome enye) (Itheyibhile 2). Ezi zalathi zibonisa ukuhambelana kwayo yonke isethi yedatha kwaye ibonisa ukuba ukuphindaphinda kweziganeko zokuphinda kuphindwe kuphindwe ngama-200 ukuya kuma-300 amaxesha okuguquka. Emva kokuba i-rho / theta ipharamitha emele imilinganiselo ehambelanayo yokudibanisa kunye neenguqu zengongoma kwisebe ibonakala phakathi kwe-0.0037 kunye ne-0.0056 (Itheyibhile 3). Kungekudala, umsebenzi opapashiweyo we-38 M. bovis strain ubonise ixabiso eliphezulu le-rho / theta (rho / theta = 0.1) kunokuba lifunyenwe kule datha, kodwa umsebenzi wePatané kunye noogxa basebenzise i-reference-based assembly to infer recombination parameters , Iinkcukacha zenkqubo, ngenxa yenkqubo yendibano, iye yadibaniswa nobuninzi becandelo le-terminal.
Okulandelayo, i-parameter ye-r / m imele i-diversity ratio ye-recombination kunye ne-mutation introduction, kunye nexabiso layo eliphakathi liphakathi kwe-0.025 kunye ne-0.037, ebonisa ukuba xa kuthelekiswa nokuguqulwa kwenguqu, ukuhlanganiswa kwakhona kunempembelelo ephantsi kwi-genetic diversity ye-M. bovis (Itheyibhile 3)). Uthelekiso olubanzi, indlela efanayo isetyenziselwe ukuqikelela i-r/m parameter ye-MTBC dataset eyenziwe ngama-23 genomes, ebonisa ixabiso elingumndilili we-0.48620, ngelixa i-Patané kunye noogxa babo '38 M. dataset ye-bovis, ibonakalise ixabiso eliphakathi ngu-0.98. Kuphononongo lokuqala, kuphela ezimbini ze-23 genomes ezibandakanyiweyo kumsebenzi we-M. bovis (M. bovis BCG kunye ne-reference strain), ngoko ke ixabiso elifunyenweyo linokuthi lithambekele ngenxa yokugqithiswa kwe-M. tuberculosis genome. Kwingxelo yesibini, uhlalutyo lwe-Mycobacterium bovis yafunyanwa ikakhulu e-United States kunye nemikhosi yemfuyo. Ngokwahlukileyo koko, kwiseti yethu yedatha, iindawo ezininzi zejografi kunye neentlobo zezilwanyana zimelwe, kwaye i-genomes ehlanganiswe kwii-clonal complexes ezineempawu ezahlukeneyo zofuzo zabemi nazo ziyasetyenziswa, ngaloo ndlela kuzuzwa ulwazi olunzulu nolubanzi lwabemi. Umahluko wexabiso eliphakathi kwe-r / m elifunyenweyo kunye neseti yethu yedatha ihambelana nengcamango yokuba iqondo lokuphinda liphinde liphinde lihluke kakhulu phakathi kwemigca eyabelwe uhlobo olufanayo lwe-taxonomic, ngoko ke ezi ziphumo zibonisa ukuba i-M. bovis clone complex ingabonisa ukungafani kwakhona Impembelelo iphinda iphakanyiswe nguDidelot & Maiden72. Nangona kunjalo, ukwandisa ngokubonakalayo le datha iseti ngokubandakanya inani elikhulu le-M. bovis genomes kuya kuvumela ukucaciswa ngakumbi kwale ngongoma. Zombini iiparamitha ze-r / m kunye ne-rho / theta zibonisa ukuhluka phakathi kwamasebe, kwaye esi siphumo sihambelana neengxelo kwezinye iintlobo zebhaktheriya72,73.
Ekugqibeleni, ukuze kuqinisekiswe iziganeko zohlengahlengiso ezichongiweyo ngumbhobho weGubbins, ii-algorithms ezintandathu ezahlukeneyo zisetyenzisiwe kwi-software ye-RDP4 ukuvavanya ngokuzimeleyo ukuthelekisa okuphambili okungafaniyo. Ehlabathini lonke, ngaphantsi kwesiqingatha seziganeko ezichongiweyo nguGubbins ziqinisekiswe yi-RDP4 (Iitheyibhile 4 kunye ne-5). Ukuqwalasela yonke isethi yedatha, iziganeko ezintathu zokudibanisa kwakhona ziqinisekisiwe, ezimbini ezibandakanya ii-nodes zangaphakathi kunye nezinye zibandakanya i-genome enye kwisebe le-terminal, apho ii-clonal complexes azikwazanga ukunikezelwa (I-Tables 4 kunye ne-5). Ukuchongwa kweziganeko kumasebe e-terminal kungabonisa ukuba ukudityaniswa kwakhona kusaqhuba kwiintlobo zangoku ze-M. bovis okanye iziphumo zibekwe endaweni engafanelekanga70. Kulo mmandla we-hypothetical recombination, malunga ne-20% yezikhundla zine-nucleotides ezingachazwanga (N), ngaloo ndlela zichaphazela isignali yokudibanisa (i-Supplementary Figure 2). Ukongeza, lo mmandla uchaphazela ijini ye-rrs, ukufaka ikhowudi ye-16S ribosomal RNA ekulindeleke ukuba igcinwe kakhulu, ngoko ke lo mqondiso wokudibanisa ubeka inokuba sisiphumo seempazamo zolandelelwano okanye ukungahambi kakuhle. Emva koko lonke ulungelelwaniso lwe-genome phakathi kwe-Mb0003 kunye ne-Mycobacterium bovis AF2122/97 lwenziwa, kwaye ubukho be-nucleotides engachazwanga kunye ne-SNP yaqinisekiswa, ngoko ke iingxaki ezinokwenzeka ezinxulumene nokulungelelaniswa okungalunganga azikho ngenxa yolwazi lwebhayoloji oluphunyeziweyo kulo msebenzi Wabonakala emva kokufunda inkqubo.
Akukho zithuba okanye i-nucleotide engachazwanga ifunyenwe kwimimandla yokudibanisa i-nodes yangaphakathi (Amanani 4 kunye ne-5). Ngokuphathelele ezi ziganeko, enye iqulethe kuphela i-Eu2 genome kwaye ichaphazela i-pks12 gene, edibanisa i-polyketide synthase enokwenzeka; ngelixa enye ibhaliswe kwi-Eu1 genome kwaye ichaphazela i-narX gene efaka i-nitrate reductase enokwenzeka (Itheyibhile 4). Ngokubanzi, uhlalutyo lokudityaniswa kwakhona lubonisa ukuba kukho inani elilinganiselweyo lamaqhekeza adityanisiweyo kunye nenkxaso yeenkcukacha-manani, kunye nezalathi ezichaziweyo zibonisa ukuba ukuphinda kudityaniswe kwakhona kunempembelelo ephantsi kumnombo we-M. bovis. Isiginali yokudibanisa kwakhona kulindeleke ukuba ibephantsi, kodwa kubalulekile ukwahlula isiginali yenguquko eyinyani kwingxolo yangasemva, engumsebenzi ocela umngeni. Ukuze kuncitshiswe umqondiso wengxolo oveliswe yindibano esekelwe kwireferensi kunye neengxaki ezingahambelaniyo 70, 71, zonke ezinye ngaphandle kwe-genome epheleleyo zahlanganiswa ukusuka ekuqaleni, kwaye umgangatho wokudibanisa uhlolwe kwaye waqinisekiswa ngokuhlalutya kwemibhobho ye-QUAST (Itheyibhile eyoNgezelelweyo 1). Ukongeza, uthotho lohlalutyo olongezelelweyo lwenziwa ukunika ukomelela nokuchaneka kovavanyo lulonke. Ke ngoko, umgangatho wolandelelwano lwe-narX kunye ne-pks12 yofuzo iye yavavanywa ngokufundwa kwemephu ngokuchasene ne-Mycobacterium bovis AF2122/97. Isikhundla esicetyiswayo se-SNP kwindawo yokudibanisa kwakhona siqinisekiswe ngokusebenzisa imigaqo ekhankanywe kwicandelo lendlela (ubuncinane ukufundwa kwe-20 kunye ne-0.9 yokutshintsha rhoqo). I-polymorphism ye-narX gene yaqinisekiswa ngokupheleleyo kwii-genomes ezimbini (Mb1792361 kunye ne-Mb7240415; 2.3%) kunye ne-genome ye-pks12 genome: Mb0891, Mb1711, Mb1789, Mb1870, Mb, Mb1766 kunye ne-M1704. Nangona kunjalo, kwi-genome ye-Mb2043, izikhundla ezithandathu kwezisibhozo azihlangabezani nomgaqo wokufunda ubunzulu ngenxa yokuba i-SNP ixhaswa ngobuninzi be-17 efundwayo, engaphantsi kwexabiso le-cutoff elimiselweyo le-20. Ngoko ke, ukudityaniswa kwakhona kwe-genomes ezintandathu (8.6%) kule ndawo ye-genome kunokuqinisekiswa (Amanani 4 kunye ne-5).
Umboniso oneenkcukacha wolungelelwaniso lommandla wodityaniso lwedatha yedatha ye-Mycobacterium bovis ichaphazela imfuza ye-narX efaka ikhowudi yokunciphisa i-nitrate enokwenzeka. Akukho zithuba okanye i-nucleotide engachazwanga ifunyenwe kwingingqi yokuhlanganiswa kweenodi zangaphakathi. Esi siganeko sibhaliswe kwi-Eu1 genome. Umgangatho wolandelelwano lwemfuza ye-narX yavavanywa ngokucwangcisa ukufundwa kweMycobacterium bovis AF2122/97. Qinisekisa indawo ekhuthazwayo ye-SNP kwindawo yokudibanisa kwakhona ngokusebenzisa imilinganiselo ekhankanywe kwicandelo lendlela (ubuncinane ukufundwa kwe-20 kunye ne-0.9 yokutshintsha rhoqo). I-polymorphism ye-narX gene yaqinisekiswa ngokupheleleyo kwi-genomes ye-Mb1792361 kunye ne-Mb7240415 (2.3%).
Umboniso oneenkcukacha wolungelelwaniso lwengingqi yeMycoplasma bovis dataset echaphazela i-pks12 gene. Akukho zithuba okanye i-nucleotide engachazwanga ifunyenwe kwingingqi yokuhlanganiswa kweenodi zangaphakathi. Ngokuphathelele isiganeko esichaphazela i-pks12 gene encoding enokwenzeka i-polyketide synthase, iqulethe kuphela i-Eu2 genome. Umgangatho wolandelelwano lwe-pks12 uvavanywe ngokufundwa kwemephu yeMycobacterium bovis AF2122/97. Qinisekisa indawo ekhuthazwayo ye-SNP kwindawo yokudibanisa kwakhona ngokusebenzisa imilinganiselo ekhankanywe kwicandelo lendlela (ubuncinane ukufundwa kwe-20 kunye ne-0.9 yokutshintsha rhoqo). Iipolymorphisms zegenomes Mb0891, Mb1711, Mb1789, Mb1870, Mb1758, Mb2043, kunye neMb1960 ziqinisekisiwe ngokupheleleyo.
I-PE kunye ne-PPE yofuzo inemimandla ephindaphindayo efundeka lula ngokulandelelana kwe-Illumina kunye nokungahambi kakuhle, ngoko ke ngokuqhelekileyo zicinywa kwi-bioinformatics workflow yamalungu e-M. tuberculosis kuphela xa usebenzisa isicwangciso sokucwangcisa-ukulandelelana. Inkcazo yeziganeko zokuphinda zisetyenziswe kulo msebenzi zisekelwe kwindibano ye-de novo ngaphandle kokucoca i-PE / PPE. Sikholelwa ukuba ngokuphumeza iindlela ezintathu ezahlukeneyo zokuncedisana kunye ne-algorithms ngokusebenzisa i-SplitsTree, ipayipi ye-Gubbins kunye ne-software ye-RDP4, izicwangciso ezisetyenzisiweyo zinamandla ekuqhubeni nasekuhluzeni imimandla ehlengahlengisiweyo ebangelwa iimpawu zephutha. Nangona kunjalo, ukwenzela ukuba ungabandakanyi ukuphazanyiswa kwe-PE / PPE gene kwi-Gubbins kunye ne-RDP4 software ukuchonga amaqela e-SNP, kwaye ngoko ke ukuchongwa kwemimandla yokudibanisa ecetywayo ukuchaphazela i-narX kunye ne-pks12 genes, ummelwane walezi gene zavavanywa (i-Supplementary Fig. 3-5). Kwi-M. bovis AF2122/97, i-narX gene yahlulwe yi-narK2 kunye ne-Mb1764c, ngelixa i-pks12 ijikelezwe ngu-Mb2075c e-Mb2073c (Umfanekiso owongezelelweyo 3-5). Imephu eveliswa kusetyenziswa imephu ye-synline ye-MAUVE ye-genome epheleleyo inikezela ngolwazi malunga nokugcinwa kolandelelwano lwemfuza kunye nokulungiswa kwakhona, ebonisa iibhloko ezine ze-collinear, kwaye akukho zibonakaliso ze-genome translocation okanye inversion. Ukongeza, uhlalutyo lokuzalisekisa kunye nolandelelwano lwe-amino acid lubonakalise i-homology kuzo zonke iigenomes ezipheleleyo, kwaye akukho PE/PPE yafunyanwa kwimimandla ekufutshane ye-narX okanye i-pks12. Kwi-narX, i-genome enye (i-Mb0030) inomlinganiselo ophantsi we-synonymy ngenxa yokuba i-narX gene yachongwa njengamaqhekeza amabini (amaqhekeza 1891 kunye ne-1890). Kwi-pks12, ngenxa yokufana, i-Mb0030 kunye ne-Mb003 ibonise amanqaku aphantsi e-sylinearity, ngelixa i-pks12 ichongiwe kwiinqununu ezimbini kunye nezintathu, ngokulandelanayo, ezimele imimandla eyahlukeneyo yeprotheni (i-Supplementary Figure 3-5). Ukuqwalasela olu lwazi, kunye ne-Gubbins kunye ne-RDP4 software zombini yenza uhlalutyo, jonga ulungelelwaniso oluninzi oluphambili lwe-500 bp ephezulu kwifestile, siqinisekisile ukuba i-PE / PPE gene ayiyi kuphazamisa isignali yokudibanisa echaphazela i-narX kunye ne-pks12.
Nangona imiqondiso yokudibanisa kwakhona efunyenwe kule sethi yedatha ingaqwalaselwa njengentsalela, kuyinyaniso ukuba ukuphinda kuhlanganiswe kwi-M. bovis akunakugwenywa, ngoko kufuneka kuqhubeke kumxholo wohlalutyo olongezelelweyo, apho ii-genomes ezipheleleyo ezivela kwiimeko ezahlukeneyo ze-epidemiological zilandelelana ukuya ezibalulekileyo.
Ukuthelekisa imithi ye-ML ye-phylogenetic efunyenwe ngaphambi nangemva kokulungiswa kwakhona (Umfanekiso 2A, B) ayizange iholele ekutshintsheni okubalulekileyo kwi-phylogenetic relationship inferred, kunye ne-M. bovis strains yahlanganiswa kwiqela elifanayo.
Emva kwemephu ye-42 esanda kulandelelwa i-M. bovis ifundeka kunye ne-reference genome ye-M. bovis AF2122/97, ulungelelwaniso lwe-SNP oluqulethe izithuba ze-polymorphic ze-1816 zafunyanwa. Uninzi lwe-SNP (87.1%) lufumaneka kwingingqi yekhowudi, kwaye izakhi zofuzo ezichaphazelekayo zibonakaliswe ngokweendidi zokusebenza eziboniswe kwi-Bovilist (Umfanekiso 6A, B). Ukuthathela ingqalelo inani elipheleleyo leemfuza kwicandelo ngalinye elisebenzayo, ijene kwicandelo elithi "lipid metabolism" libonise ii-SNP ezininzi, ezilandelwa "ludonga lweseli kunye neenkqubo zeseli" kunye "nemetabolism ephakathi kunye nokuphefumla", ebonisa ukuba kwi-M. bovis evolution.
Uhlalutyo lwemigangatho yedatha ye-M. bovis evela ePortugal (n = 42). Inani lilonke lee-SNP ezibhalisiweyo kunye neegene ezichaphazelekayo kwicandelo ngalinye elisebenzayo (A). Itotali yenani lotshintsho lwezithethantonye nezingezizo ezibhalisiweyo ngokwecandelo lomsebenzi (B).
Kwinqanaba lehlabathi, umlinganiselo we-dN / dS ungcono kune-1.5, ebonisa ukuba uxinzelelo lwehlabathi jikelele lokuziphendukela kwemvelo kukulahla urhulumente wokhokho kwaye imele i-positive (i-diversified or directed) kunye / okanye i-relaxed purification choice scenario. Kwiindidi ze "virulence, detoxification, adaptation", "ukufakwa ngokulandelelana kunye ne-phages", kunye "neprotheni ezilawulayo", ngaphezu kweyesibini kwisithathu se-SNP ayifani (Umfanekiso 6B).
Kuzo zonke iindidi, kukho izakhi zofuzo ezine-SNP ezininzi, okubangelwa ukuguqulwa kwesantya esiphakathi (oko kukuthi, umyinge we-SNP kwi-gene) enkulu kune-1 (Umfanekiso 6A). I-Pks12 (Mb2074c) kunye ne-15 SNPs kunye ne-fas (Mb2553c) kunye ne-8 SNP zinexabiso eliphezulu lokuguqula. Zombini ezi zofuzo zibandakanyeka kwi-fatty acid metabolism. I-pks gene ifaka i-polyketide synthase (PKS), eyi-enzyme esebenzayo ebandakanyekayo kwi-mycobacterial cell wall lipid biosynthesis74,75. Le mfuza ifaka i-polypeptide esebenzayo ebandakanyekayo kwi-synthesis ye-mycoketides74,76. I-fas gene ibandakanyeka kwi-synthesis ye-mycolic acid. Zombini ezi zakhi zofuzo zidlala indima ebalulekileyo kwi-biosynthesis yodonga lweseli ekudibaneni nomninimzi.
Ukuze uqhubele phambili ukufunda ngokuvela kweMycobacterium bovis, iiseti ezimbini zofuzo oluthile ziye zahlalutywa. Imisebenzi epapashwe ngaphambili isebenzisa ukubunjwa kolandelelwano kunye neendlela ze-phylogenetic ezichongiweyo zofuzo ezifunyenwe ngookhokho be-MTBC nge-HGT ngaphambi kokuhlukahluka37,38. Ezi genes zidweliswe kwi-Supplementary Table 2. Ukuhanjiswa kwe-SNP ye-77 genes enokuthi inxulumene ne-HGT yahlaziywa, kwaye iindawo ze-polymorphic ze-26 zachongwa, apho kwiimeko ezininzi (78%) zibangele utshintsho olungachazwanga (NS) (i-Supplementary Table 2). Umsebenzi wangaphambili kwi-MTBC genome ubonise ukuba ummandla we-HGT obekekileyo ubonisa umlinganiselo ophezulu we-NS SNP xa kuthelekiswa nayo yonke i-genome. Ukuba umntu ucinga ukuba le mimandla yokubuyisela ifunyenwe ngookhokho be-MTBC, kwaye ngoko ke, bagqithisa iipolymorphisms zamandulo, ngoko umlinganiselo weenguqu ezifanayo kulindeleke ukuba ube phezulu, kuba ukutshintshwa kwe-NS kulindeleke ukuba kupheliswe ngokukhetha okungalunganga ngenxa yeenguqu ze-amino acid Inokutshintsha umsebenzi weprotheni. Ke ngoko, iziphumo zethu zibonisa ukuba iziphumo zokusebenza zinokubangelwa kukutshintshwa kofuzo olufana ne-HGT, olubonisa ukubaluleka kweyantlukwano yemfuza eguqukayo.
Ngokuhambelana nolu hlalutyo, iijene ezifakela amacandelo e-3R (ukulungiswa kwe-DNA, ukuphindaphinda, kunye nokuphindaphinda) inkqubo zihlolwe ngokucokisekileyo ngokuhambelana noluhlu olupapashwe ngaphambili yi-dos Vultos kunye nabasebenzisana nabo (2008)39. Utshintshiselwano lwamaqhekeza e-DNA afanayo alukwazi ukubonwa ngokuthe ngqo, nangona ingaba yinkqubo rhoqo xa iibhaktheriya ezinxulumene ngokusondeleyo zibandakanyeka, njengakwimeko yale datha yedatha; ukongeza, le nkqubo inokuba ngundoqo kwiindlela zokulungisa i-DNA72, ngoko Dlala indima kwi-homologous recombination. Itotali yezithuba ze-polymorphic ze-26 ezisasazwa ngamajini angama-54 achongiwe (Itheyibhile eyoNgezelelweyo yesi-3). Kule setethi yezakhi zofuzo, utshintsho lwe-NS lubalelwa malunga ne-65% yemiphumo, ehambelana neengxelo zangaphambili kwi-Mycobacterium tuberculosis strains.
Ixesha lokuposa: Oct-21-2021