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Kutsatizana kwa ma genome kwatsitsimutsa gawo la kafukufuku wa matenda opatsirana, kuwulula miliri ya matenda, tizilombo toyambitsa matenda, kuyanjana kwa tizilombo toyambitsa matenda, ndi njira yachisinthiko yomwe imayikidwa pa tizilombo toyambitsa matenda. Mycobacterium tuberculosis complex (MTBC) imawona Mycobacterium bovis ngati imodzi mwa mamembala ake omwe amayambitsa chifuwa chachikulu cha TB (TB) mu zoyamwitsa zapadziko lapansi, ndipo ndi chitsanzo cha kusinthika kwa bakiteriya. Monga mamembala ena a MTBC, Mycobacterium bovis imaganiziridwa kuti ndi tizilombo toyambitsa matenda timene timatulutsa pang'onopang'ono, ndipo mwachiwonekere palibe chizindikiro chophatikizananso kapena kusamutsa majini opingasa. Mu ntchitoyi, timagwiritsa ntchito ma genomics ofananira ku gulu lonse la ma genome sequence (WGS) lopangidwa ndi ng'ombe 70 M. zochokera kumitundu yosiyanasiyana (Europe ndi Africa) kuti tidziwe za mitundu yosiyanasiyana ya ng'ombe M. Evolutionary power. Njira zitatu zosiyana zimagwiritsidwa ntchito poyerekezera zizindikiro za kukonzanso. Padziko lonse lapansi, zochitika zochepa zowonongeka zadziwika ndikutsimikiziridwa ndi njira ziwiri zodziimira ndi chithandizo cholimba. Komabe, poyerekeza ndi masinthidwe, kubwezeretsanso kumakhala ndi zotsatira zofooka pamitundu yosiyanasiyana ya M. bovis (yonse r/m = 0.037). Kusiyana kwa r/m pakati komwe kumapezeka mu clonal complex ya Mycobacterium bovis mu seti yathu ya data kumagwirizana ndi lingaliro lachidziwitso kuti kuchuluka kwa kuphatikizika kungasiyane kwambiri pakati pa mibadwo yoperekedwa ku mitundu yofanana ya taxonomic. Kutengera ndi ntchitoyi, kubwezeretsedwanso mu Mycobacterium bovis sikungathetsedwe, chifukwa chake iyenera kukhala nkhani yopitilira kafukufuku wamtsogolo wofananira wa ma genomics, momwe WGS yama data akulu amaseti kuchokera ku zochitika zosiyanasiyana za miliri padziko lonse lapansi ndizofunikira. Kusanthula kowonjezerako kunachitika pa data yaying'ono ya Mycobacterium bovis (n = 42) kuchokera kumagulu ambiri a TB, ndipo oposa 1,800 loci adadziwika, omwe pafupifupi mtundu umodzi umasonyeza nucleotide polymorphism (SNP) . Ambiri (87.1%) ali m'chigawo cholembera, ndipo chiwerengero cha padziko lonse cha kusintha kosafanana (dN / dS) kwa kusintha kofanana kumaposa 1.5, kusonyeza kuti kusankha bwino ndi mphamvu yofunikira yachisinthiko yomwe ikugwiritsidwa ntchito pa M. bovis. Kuchuluka kwa ma SNP kunapezeka m'majini olemera m'magulu ogwirira ntchito a "lipid metabolism", "cell wall and cellular process", ndi "metabolism yapakatikati ndi kupuma", kuwulula kuthekera kwawo mu biology ndi kusinthika kwa Mycobacterium bovis kufunika. Kuyang'anitsitsa majini a makolo a MTBC omwe amakonda kusamutsa jini yopingasa ndikuphatikizidwa mu dongosolo la 3R (kukonza DNA, kubwereza, ndi kubwezeretsanso) kukuwonetsani kuchuluka kwapadziko lonse lapansi kwa mayeso a Taijima a D, omwe akuwonetsa kusanthula kwakanthawi kosankhako
Mycobacterium tuberculosis complex (MTBC) ndi imodzi mwazochita zopambana kwambiri za tizilombo toyambitsa matenda komanso momwe mabakiteriya amasinthira. Mamembala ake amawonetsa modabwitsa kwambiri nucleotide pamlingo wa genomic (> 99%)1,2. Mitundu yosiyanasiyana ya MTBC ecotypes ingayambitse chifuwa chachikulu (TB), yomwe ndi matenda opatsirana a granulomatous, m'mitundu yambiri yamitundu yosiyanasiyana kuchokera ku zinyama zazing'ono kupita kwa anthu3,4,5. Pakadali pano, zovutazi zikuphatikizapo anthu [M. Tuberculosis (Mtb), Mycobacterium africanum] ndi tizilombo toyambitsa matenda (Mycobacterium bovis, Mycobacterium capitum, Mycobacterium pinnipedum, Mycobacterium microtobacter , Mycobacterium mongee, Mycobacterium miysani, Mycobacterium surika, "Bacillus chimpanzee")5, 6. M. canettii (wotchedwanso "Nodobacter glabrata") Chiwerengero cha nucleotide ndi mycobacteria yomwe tatchulayi ndi 98%, ndipo ntchito yofananitsa ya genomics yasonyeza kuti M. canettii ndi ena onse a MTBC asiyana posachedwapa ndi kholo wamba.7 Poganizira lingaliro ili, olemba ena a MT canetti 8 M.
MTBC imalongosoledwa mwadongosolo ngati gulu lolimba la ma clonal, ndipo kuchuluka kwa anthu kumayendetsedwa bwino ndi kuchepetsedwa kwa kusiyanasiyana, zolepheretsa, kusanthula kosankha, ndi ma genetic drift9,10. Kungotengera kusinthika kolimba kwa clonal, monga ma polymorphisms osowa sikungabwezeretsedwenso ndikuphatikizanso. Kutengera mfundoyi, zochitika zotsatizana za kuchotsedwa kwa ma genomic kwa dera losiyana (RD) ndi TbD1 (Mtb specific deletion 1 region) zaperekedwa ngati zolembera za MTBC evolution2,5,11. Comparative genomics and whole genome sequencing (WGS) ntchito imathandizira kugawidwa kwa mamembala osinthidwa ndi anthu kukhala mizere isanu ndi inayi (Mycobacterium tuberculosis L1 mpaka L4, L7, ndi L8; ndi Mycobacterium africanum L5, L6, ndi L9), mizere L2 mpaka L4 yogawana kufufuta TbD1,1312,1. Kuphatikiza apo, mamembala osinthidwa ndi nyama akufunsidwa kuti agawane kholo limodzi, lomwe limatanthauzidwa ndi kufufutidwa kwapadera mu RD7, RD8, RD9 ndi RD102, 5, ndi 14.
Kutengerapo kwa majini osakanikirana (HGT) ndi zochitika zophatikizanso zimawonedwa ngati zachilendo ndipo zimachitika m'makolo a MTBC, osati mbiri yosiyana ya membala wonse wa MTBC15,16,17. Malipoti awiri oyambirira a Hughes ndi ogwira nawo ntchito (2002) ndi Gutacker ndi othandizira (2006) adanena kuti zochitika zowonongeka zingathandize kupanga ma polymorphisms omwe amalemba malo enieni mu M. tuberculosis strains18,19. Zifukwa za kusowa kodziwikiratu kwa recombination mu MTBC ndi: (1) ndondomeko yamakina ndi kutaya mphamvu kwa HGT; (2) kupezeka kwa zochitika za HGT; (3) palibe mwayi wokonzanso zochitika mu MTBC niche14,17. Posachedwapa, kafukufuku wina wa genome sequencing (WGS) wogwiritsidwa ntchito ku MTBC strain 20 ndi Mycobacterium bovis 21 apereka umboni wakuphatikizanso, woyamba kuwonetsa kuti mitundu ya MTBC nthawi zambiri imasinthanitsa tizidutswa tating'ono ta DNA, koma chifukwa cha kusiyanasiyana kwa ma nucleotide, zochitika izi sizikudziwikabe.
Mycobacterium bovis ndiye membala wa MTBC yemwe amapezedwa kwambiri kuchokera ku ziweto (makamaka ng'ombe), ngakhale amathanso kupatulidwa ku nyama zakuthengo zomwe zimakhala zaulere komanso zotchingidwa ndi mipanda4,22,23,24. M. bovis adasinthika kukhala ma clonal complexes asanu [European 1 (Eu1), European 2 (Eu2), European 3 (Eu3), African 1 (Af1) ndi Africa 2 (Af2)], malinga ndi spoligotyping mbiri, kufufutika kwapadera Ndi ma polymorphisms amodzi a nucleotide (SNPs) 25, 27, 26, 92 mu genes yeniyeni, 26, 92, Ma clonal awa amawonetsa mawonekedwe osiyanasiyana a kuchuluka kwa Mycobacterium bovis komanso kuyanjana kwake ndi madera. Kuphatikiza apo, ntchito yaposachedwa ya WGS yochitidwa ndi Zimpel ndi othandizana nawo (2020) idapanga phylogeny kutengera SNP ya Mycobacterium bovis, yokhala ndi ma genome opitilira 1,900, zomwe zikuwonetsa kuti pali mizere inayi yosiyana (yotchedwa Lb1 mpaka Lb1 mpaka Lb4), sizingatsimikizidwenso kuti ndizovuta kwambiri, ngakhale kuti sizingafanane kwenikweni. Olemba awa adasanthula mosiyanasiyana za phylogeny ndi chibwenzi cha maselo, koma sanaphunzire kuyambiranso30.
Ntchito yam'mbuyo pogwiritsa ntchito njira zosiyanasiyana za maselo, monga spoligotyping, MIRU-VNTR (mycobacterial interspersed repeat unit-variable tandem repeat number), ndipo kulembera kwaposachedwa kwa SNP kunavumbula mlingo wina wa mitundu yosiyanasiyana ya majini pakati pa M. bovis strains 31,32,33, 34,35. Kusiyanitsa kwa kusiyanasiyana kwa ma genetic kwakhala chida chofunikira pofufuza za matenda a epidemiology, zomwe zimathandiza kumvetsetsa mozama za matenda, ma virus komanso kufalitsa matenda. Kuwonekera kwa njira ya WGS kumapereka mwayi wowonetsera zinthu zomwe zimayendetsedwa ndi Mycobacterium bovis genome pakukonzekera ndi kulimbikira kwa makamu osiyanasiyana ndi zochitika za miliri.
Mu ntchitoyi, timagwiritsa ntchito kusanthula kofananira kwa ma genomic pamitundu yosiyanasiyana ya Mycoplasma bovis (n=70), kuphatikiza zodzipatula kuchokera kumitundu yosiyanasiyana ya clonal, kuti tidziwe zambiri zakusintha kwa Mycoplasma bovis, makamaka kuthetsa maubwenzi a phylogenetic Ndi zochitika zophatikizanso. Monga chowonjezera pa kusanthula uku, gulu laling'ono la M. bovis limadzipatula (n = 42) lomwe linapezedwa kuchokera kumadera odziwika bwino a chifuwa chachikulu cha TB ku Portugal 31,36 linafufuzidwanso kuti liwonetsetse kuti palibe chidziwitso Chodziwika bwino pakati pa chiŵerengero cha chidziwitso (dN) ku synonymous (dS) nucleotide m'malo mwa ma genome, mabuku omwe amatchulidwa m'malo mwa genome. 37,38 yomwe inapezedwa ndi makolo a MTBC kudzera mu HGT, ndi encode 3R (DNA kukonza, replication, and recombination) gene gene components 39. Sankhani majini omwe amapezeka kudzera mu HGT chifukwa akhoza kuimira ma polymorphisms akale, kotero akuyembekezeka kuti akhale ndi gawo lalikulu la kusintha kofanana. Ma jini ophatikizidwa mu dongosolo la 3R adasankhidwa chifukwa ntchito yapitayi pa matenda a chifuwa chachikulu cha M. chifuwa chachikulu chinasonyeza zosankha zoipa / zoyeretsedwa zomwe zimagwira ntchito pa majiniwa, ndipo zingakhale ndi gawo lofunika kwambiri pa chisinthiko 39. Cholinga china cha ntchitoyi ndikuwonetsa kukhalapo kwa zochitika za kukonzanso. Pachifukwachi, poganizira kuti deta yathu yochokera ku Portugal ili ndi ma genome a European clone complex 2 ndi zovuta zomwe sizinapereke ma clone complex, tinaganiza zophatikizirapo zomwe zilipo poyera kuti tipeze woimira ma clone complexes, ndi Kupititsa patsogolo kulimba ndi kufalikira kwa zotsatira.
42 Mycoplasma bovis genomes yotsatiridwa kumene kuchokera ku Portugal endemic multi-host TB scene (zambiri pansipa), zodziwika bwino ndi miliri36, ndizomwe zili pakati pa ntchitoyi. Poganizira kuti zidziwitso zochokera ku Portugal zimangokhala ndi oimira European 2 clone complexes ndi zovuta zopanda malo osankhidwa, deta yopezeka pagulu ya ma genome athunthu awonjezedwa kukulitsa seti ya data yomwe ili ndi oyimira onse a M. bovis clone complexes. Choncho, magwero atatu otsatizana a ma genome adagwiritsidwa ntchito pa ntchitoyi: msonkhano wathunthu / wokonzekera ma genome, mpaka ma scaffolds a 10 osungidwa ku NCBI (National Center for Biotechnology Information) (n = 15 amapatula); kusungidwa mu SRA ( The Illumina fastq file of the sequence read archive) imayimira zovuta zosiyanasiyana za M. bovis clones (n = 12 isolate)30; ndi ma genome 42 otsatizana kumene ochokera ku Portugal. Mycobacterium bovis BCG (Bacille Calmette-Guerin) sanaphatikizidwe pakusaka kwa NCBI. M. bovis AF2122/97 nthawi zambiri imagwiritsidwa ntchito ngati genome yophatikizidwira mu dataset. Chifukwa cha kusapezeka kwagulu kwa mitundu yonse ya ma genome yoyimiridwa ndi gulu la African 1 cloning complex, komanso kuchepa kwa ma genomes ochokera ku mitundu yoyimilira ya Af2 ndi Eu1, deta yoyambirira yoperekedwa ndi SRA idagwiritsidwa ntchito pamilandu iyi. Ntchito ya Zimpel ndi ogwira nawo ntchito (2020) idathandizira kuzindikira ma genome kuchokera ku ma cloning omwe tawatchulawa ndipo adathandizira kusankha Mycobacterium bovis kuti alowe nawo mu seti ya data. Kwa Eu3, mtundu umodzi wokha wa genome ndi womwe ukufotokozedwa (Branger et al., 2020), kotero genome yomwe timaphatikiza ndi woyimira wosiyana ndi zovuta za Eu3.
Padziko lonse, deta iyi imaphatikizapo ng'ombe za 70 M. bovis zomwe zimalekanitsidwa ndi mitundu 8 yomwe ikukhala, yogawidwa m'mayiko 12 kuchokera ku 1985 mpaka 2016. Mitundu ya 36 imatchedwa Eu2, mitundu 7 ndi Eu1, 1 mitundu ndi Eu3, 3 mitundu ndi Af1, 4 mitundu ndi Af2 ndipo 19 siili pansi pa clonal yovuta. Zambiri (kuphatikiza nambala yolowa m'malo) ya Mycobacterium bovis yomwe yagwiritsidwa ntchito mu kafukufukuyu ikuwonetsedwa mu Table 1 ndi Supplementary Table 1.
Ma genomes 42 ongotsatizana kumene a Mycobacterium bovis ochokera ku Portugal komwe ali ndi chifuwa chachikulu cha nyama ku Portugal ndikugawidwa kwazaka zopitilira 12 ndiwo maziko a kafukufukuyu, popeza machitidwe a matenda a nyama zakuthengo akhala akuyang'aniridwa pafupipafupi 31,36 (Zowonjezera mkuyu. 1). Malinga ndi njira zotsatila, mitundu iyi idalekanitsidwa ndi ng'ombe (n = 14), nswala (n = 16) ndi nguluwe (n = 12) kuyambira 2003 mpaka 2015: sonkhanitsani ndi kusamalira nyama motsatira malangizo ovomerezeka a protocol. Zitsanzo za minofu zili mu OIE Terrestrial Animal Handbook ndipo zimayikidwa muzitsulo zolimba za Stonewepy ndi Lruvabrinkstein wapakati. Zikhalidwe zimakhazikika pa 37 ° C ndipo kukula kumafufuzidwa kamodzi pa sabata kwa masabata osachepera 12. Zigawozo zimasungidwa mwachindunji mu njira ya glycerol pa -80ºC. Mu Mycobacterium selective medium (Middlebrook 7H9, BD Diagnostics), zitsanzo zosungidwa zakale zidadutsa mundime imodzi ya in vitro kuti apeze DNA ya pulogalamu ya WGS. Pachifukwa ichi, njira yosungiramo madzi amtundu wachisanu idawonjezeredwa ndi 5% sodium pyruvate ndi 10% ADS (50 g albumin, 20 g glucose, 8.5 g sodium chloride m'madzi 1 L) pa Middlebrook 7H9 pa 37 ° C Retrain. Pambuyo pa masabata a 4 akukula, sing'angayo idakonzedwanso ndipo chikhalidwecho chimayang'aniridwa nthawi zonse mpaka kukula kukuwonekera. Maselo adakololedwa ndi centrifugation, pellet idakhazikitsidwanso mu 500 µL phosphate buffered saline (PBS), kutenthedwa pa 99 ° C kwa mphindi 30, centrifuged, ndipo supernatant idasungidwa pa -20 ° C mpaka WGS. Njira zonse zimachitika mu level 3 biosafety malo.
Laibulale ya ma genome ophatikizidwa a WGS imakonzedwa pogwiritsa ntchito cholozera chapadera cha sampuli iliyonse ya DNA, ndipo imagwiritsa ntchito Illumina MiSeq (2 × 250 pb) (zitsanzo 40) ndi ukadaulo wa HiSeq (2 × 150 pb) (ziwiri zodzipatula) (Eurofins Genomics, Germany) potsata ndondomeko. Malinga ndi malangizo a wopanga, gwiritsani ntchito Illumina Genome Analyzer yokhala ndi ma module okhala ndi mbali ziwiri kuti mutsatire ma genomic DNA, ndikugwiritsa ntchito Nextera XT DNA Library Prep Kit kuchokera ku Illumina kuti mumange laibulale.
Poganizira deta anachira ku SRA (n = 12), chizindikiritso cha choyerekeza zovuta angagwiritsidwe ntchito ngati metadata la lolingana buku 30, 41, 43. Poganizira matupi athu wathunthu, kupatula Mycobacterium bovis AF2122/97 ndi Mycobacterium bovis 3601, amene ndi Eu3 Eu21 zovuta, amene amazindikira Eu2 Eunal1 zovuta. 29, motsatira, ndizofanana ndi mtundu wathunthu wa Mycobacterium tuberculosis H37Rv (NCBI accession number NC_000962.3). Kuyanjanitsa kwa ma genome kumachitika pogwiritsa ntchito MAFFT (pulogalamu yambiri yolumikizira amino acid kapena ma nucleotide sequence, version 7.458) ndi parameter -addfragments48. Kenako, fufuzani kusapezeka kwamitundu yosiyanasiyana ya clonal ndi/kapena kupezeka kwa mawonekedwe a SNP.
Mycobacterium bovis (n = 42) yomwe yangotsatiridwa kumene (n = 42) ndi zowerengera zoyambira zomwe zasonkhanitsidwa (n = 3) zimagwirizanitsa zovutazo ndi genome Mycobacterium tuberculosis H37Rv kudzera papaipi ya vSNP ndi kupezeka kwa kufufutidwa ndi/kapena mawonekedwe a SNP amitundu yosiyanasiyana Kusaka kunachitika.
Sonkhanitsani zambiri kuchokera kukusowa kwa mawonekedwe ndi/kapena kupezeka/kusowa kwa SNP ndi mbiri ya spoligotyping kuti mugawire ma genomic data ku ma clonal complex. Pamisonkhano inayi yokonzekera, mbiri ya spoligotyping siyingaganizidwe, kotero imaphatikizidwa mu gulu la "palibe zovuta".
Mayendedwe a bioinformatics omwe amatsatiridwa ndi ntchitoyi amayambira pa msonkhano wa de novo ndi kupanga mapu kupita ku njira yofotokozera, ndi cholinga chowunikanso zochitika zophatikizananso ndi ma polymorphisms enaake a genome. Chithunzi 1 chikuwonetsa ndondomeko yotsatiridwa. Pakuwunikiranso, ma genome onse amagwiritsidwa ntchito kuti awonjezere kulimba kwa malingaliro ndi zizindikiro zofananira.
Pofuna kuchepetsa zolakwika pakupanga ma genome consensus sequences, tidapeza poyamba de novo assembly, kenako tidapeza ma core ma alignments. Mapaipi a Unicycler akupezeka pa https://github.com/rrwick/Unicycler49 ndipo amagwiritsidwa ntchito popanga ma genome otsatizana 54 (42 otsatiridwa kumene ndi mafayilo 12 a fastq omwe adapezedwa ku SRA). Mwachidule, tisanasonkhanitse kuyambira pachiyambi, kusanthula kwamtundu wowerengera kudachitika mu mtundu wa FastQC 0.11.7 (https://github.com/s-andrews/FastQC), ndi mtundu wa Trimmomatic 0.36 (njira "kudula ma Adapter ndi machitidwe ena owunikira" ndi "Dulani zoyambira, gwirani zoyambira" kugwiritsidwa ntchito) (http://www.usadellab.org/cms/?page= trimmomatic) 50. Kenaka, SPAdes optimiser49 inagwiritsidwa ntchito popanga ma genome, ndipo Pilon version 1.1851 inagwiritsidwa ntchito pokonzekera pambuyo pa msonkhano. Njira yotsekera mlatho idasankhidwa kuti apewe kusonkhanitsidwa kolakwika, ndipo kukula kwa k-mer kudafufuzidwa ndikusankhidwa pakati pa 20% ndi 95% ya kutalika kowerengedwa. Tsatirani malangizo a SPAdes ndikuganizirani kukula kwake, chotsani ma contigs ochepera 300 bp, ndikukhazikitsa 20 kuwerenga kwakuya kwa 52. Mu ndondomeko ya msonkhano wa de novo, zigawo za genomic monga mobwerezabwereza proline-glutamate (PE) ndi proline-proline glutamate (PPE) paralogs sizinachotsedwe.
Ubwino wa msonkhano wa de novo umawunikidwa kudzera papaipi ya QUAST (http://quast.sourceforge.net/quast.html), yomwe imathandizira kukonzanso kwa contig ndi M. bovis AF2122/97 reference genome (NCBI accession number LT708304.1) Mapping (onani 1 Zowonjezera Table parameter).
Mothandizidwa ndi mapaipi a vSNP (https://github.com/USDA-VS/vSNP), fayilo ya FASTQ ya M. bovis yotsatiridwa kumene kuchokera ku Illumina sequencing ikuyerekezedwa ndi M. bovis AF2122/97 reference genome (LT708304.1)). Malinga ndi malingaliro abwino a Genome Analysis Toolkit (GATK) 53, 54, 55 gwiritsani ntchito zosefera zokhazikika kapena kuchuluka kwamitundu yosiyanasiyana kuti muwerengenso. Zotsatira zimasefedwa pogwiritsa ntchito chiwerengero chotsika kwambiri cha SAMtools cha 150 ndi AC = 2. Gwiritsaninso ntchito Kraken (http://ccb.jhu.edu/software/kraken/) kuti muwone zowerengera kuti mupewe kuipitsidwa. Mapaipi a vSNP omwe amagwiritsidwa ntchito popanga mapu kuti atsatire njira muntchito yathu amawunika ma SNP angapo omwe amafotokozedwa ndi zomwe akufuna, komanso samaphatikizapo zochitika zosiyanasiyana za matenda. Kuwerenga kwa ma genome kuli bwino kuposa 99% (Table Supplementary Table 1).
Kuti mupewe zolakwika za mapu ndi ma SNP olakwika, zoseferani zosiyana pazochitika zotsatirazi: (1) zimathandizidwa ndi zowerengeka zosachepera 20, (2) zimapezeka pafupipafupi zosakwana 0.9, (3) zili mumtundu umodzi, koma pali mipata mumtundu wina. The Integrated genomics viewer (IGV) version 2.4.19 (http://software.broadinstitute.org/software/igv/)56 inagwiritsidwa ntchito kutsimikizira ma SNP ndi malo okhala ndi zovuta za mapu kapena kuyanjanitsa. Popeza kuti majeremusi a proline-glutamate (PE) ndi proline-proline glutamate (PPE) amapangidwa mowirikiza kwambiri ndipo ali mbali ya banja lamitundu yambiri, iwo samvetsetseka mosavuta ndi Illumina kutsatizana ndi zolakwika, kotero iwo amakonda The mycobacterial bioinformatics workflow anachotsa mamembala a chifuwa chachikulu cha chifuwa chachikulu pamene akugwiritsa ntchito njira yotsatizana ndi mapu a SNP. Chifukwa chake, tidasefa majini a PE/PPE ndi ma indels pakuwunika.
Malinga ndi Bovilist (http://genolist.pasteur.fr/BoviList/), ma SNP onse amagawidwa m'magulu ogwira ntchito. Mapaipi a SnpEff (https://pcingola.github.io/SnpEff/) amagwiritsidwa ntchito kutsimikizira zotsatira za SNP (zosintha zofanana kapena zosafanana). Anapanga nkhokwe yatsopano ya Mycobacterium bovis AF2122/97 genome (LT708304.1).
Kuyanjanitsa kwa ma genome angapo kunachitika pogwiritsa ntchito Parsnp v1.2, yomwe ikupezeka pano https://github.com/marbl/parsnp57, pogwiritsa ntchito ma genomes/draft assemblies 69 (ndi njira -c) ndi M. bovis AF2122/97 (LT708304.1) imagwiritsidwa ntchito ngati chilozero. Maiko anayi oyambira angapo adachitidwa: mamembala okha a Eu2 cloning complex (n = 37), kuphatikiza mamembala onse a European cloning complex (n = 44), kuphatikiza malo olumikizirana ndi European and African cloning complex (n = 51), ndikuphatikiza onse a Mycobacterium bovis mu phunziroli (n = 70).
Kuyanjanitsa kwapakati kopangidwa ndi Parsnp kumagwiritsidwa ntchito kuwonetsa kuthekera kwakukulu (ML) mtengo wa phylogenetic pogwiritsa ntchito CIPRES Science Gateway v3.3 (http://www.phylo.org/)58 pogwiritsa ntchito RAxML, ndikuchita 1000 motsogozedwa kubwereza.
Ma algorithms atatu osiyanasiyana ndi zida za bioinformatics zimagwiritsidwa ntchito poyang'ana kupezeka kwa zochitika zophatikizanso mofananira: SplitsTree4 software, Gubbins (mzera wosakondera kudzera pakuphatikizanso ma nucleotide sequences) mapaipi, ndi RDP4 (pulogalamu yozindikiranso, mtundu wa beta 4.101).
Njira yowola yogawanika yomwe yakhazikitsidwa mu SplitsTree4 v4.15.1 (http://www.splitstree.org/)59 imagwiritsidwa ntchito kuwerengetsa maukonde opanda mizu a phylogenetic, pogwiritsa ntchito mayeso a Phi potsimikizira ziwerengero, ndipo kufunikira kwake ndi p = 0.05. Kusanthula kwapakatikati kwa Parsnp kumagwiritsidwa ntchito ngati chothandizira, ndipo kuwonongeka kogawanika ngati mulingo wamaneti kumakwaniritsidwa.
Gubbins pipeline v2.3.1 (https://github.com/sanger-pathogens/gubbins60 imayenda ndi magawo osakhazikika ngati njira ina yowunikiranso mphamvu ya kuyanjananso pa Mycobacterium bovis. Njira yomwe yakhazikitsidwa paipiyi imamanganso mzere wofunikira wa clone LT708304.1) ndizogwirizana; ndikuyang'ana malo a SNP panthambi iliyonse yamtengo kuti azindikire gulu la SNP lomwe likuyimira chochitika chophatikizananso, kutanthauza kuti ma SNP omwe amapezeka panthambi ayenera kugawidwa mofanana Mafayilo amtundu wa ParRA akugwiritsidwa ntchito bwino kwambiri kuchokera ku mtengo wa ParRA.
Pomaliza, pofuna kutsimikizira chochitika chokonzanso chomwe chaperekedwa ndi mapaipi a Gubbins, ma aligorivimu asanu ndi limodzi omwe akhazikitsidwa mu RDP467 (RDP61, GENECONV62, Bootscan63, Maxchi64, Chimaera65 ndi SiScan66) amagwiritsidwa ntchito pamayendedwe angapo a Parsnp pansi pa zosintha zosasintha. Tidatsimikiza kuti osachepera atatu mwa ma aligorivimu omwe akhazikitsidwa mu RDP4 akuyenera kuwonetsa nthawi zonse chizindikiro chofunikira kutsimikizira chochitika chilichonse chophatikizanso.
Poganizira kuti mapulogalamu onse a Gubbins ndi RDP amayang'ana zizindikiro zobwezeretsanso poyang'ana zoyambira zingapo pawindo la 500 bp, ndikutsimikizira kuti kuphatikizika kwa majini a PE / PPE pa msonkhano wa de novo sikungasokoneze zizindikiro zophatikizanso zomwe zapezeka, kusanthula kwina kumachitidwa ndi homolinearity. Mapu a synlinear omwe amagwiritsa ntchito ma genome athunthu adapangidwa pogwiritsa ntchito kulumikizana kwa MAUVE-multi-genome (http://darlinglab.org/mauve/mauve.html) kuti asaphatikizepo kusintha kwa ma genome kapena kusinthika kwawo. Kuphatikiza apo, genome yonse idagwiritsidwa ntchito kusanthula homolinearity pamayendedwe a amino acid kudzera pa seva ya intaneti ya SyntTax (https://archaea.i2bc.paris-saclay.fr/SyntTax/).
Kusanthula mozama kwa deta ya genome yomwe imapezeka ku Chipwitikizi yamitundu yambiri ya chifuwa chachikulu ndiyo kufufuza polymorphism ya majini otchulidwa m'mabuku. Majini awa ndi 37,38 ndi jini encoding 3R yopezedwa ndi MTBC makolo kudzera mu HGT (DNA kukonza, replication ndi recombination) dongosolo zigawo 39. Gwiritsani ntchito ClustalX v2.1 (http://www.clustal.org/clustal2/) ndipo gwiritsani ntchito DnaSP v6.12.03 (http://u/dub divers calculator). ma nucleotide (π) ndi Tajima's D osalowerera ndale zoyeserera.
Kuthekera kwakukulu (ML) mtengo wa phylogenetic wozikidwa pa 69 Mycoplasma bovis isolates ndi ma genome ofotokozera adapezedwa (Chithunzi 2A). Poyerekeza ndi mitengo yokhala ndi jini imodzi kapena mitengo yamitundu yambiri, njirayi imalola kubadwa kwa mitengo yamphamvu kwambiri yomwe siigwira kusinthasintha kwa ma genome onse ndipo motero imasonyeza kutsika kwa kusiyana pakati pa mitundu 68,69. Mapangidwe apamwamba a mtengo wa ML nthawi zambiri amagwirizana ndi magulu ovuta a ma clones. Genome ya Eu2 imalumikizidwa munthambi, ndipo genome ya Af1 imalumikizidwanso palimodzi (Chithunzi 2A). Zotsatira zake zimagwirizananso ndi ubale wodziwika bwino wa chisinthiko wa Mycobacterium bovis, ndiko kuti, pali kusiyana kwakukulu pakati pa membala wa Eu1 ndi gulu lomwe lili ndi ma clonal complexes ndi ma genome, koma clonal complex 30 sinatchulidwe. Kusagwirizana kwakung'ono pakati pa zovuta za clonal ndi ubale womwe umawonedwa pa mtengo wa phylogenetic ukhoza kufotokozedwa ndi mfundo yakuti zovuta za clonal zimafotokozedwa kutengera zigawo za genomic, pamene mtengo wa phylogenetic umachokera kumagulu angapo a ma genome omwe amaimira mtundu wonse wa genome.
Mtengo waukulu wa phylogenetic (GTR) umapangidwa potengera momwe ma genome amayendera a Mycobacterium bovis genome isanachitike (A) ndi pambuyo (B) kuchotsedwa kwa malo ophatikizanso. Mitundu ya nthambiyi imayimira Mycobacterium bovis clone complex: Europe 1 ndi yofiirira, Europe 2 ndi yofiira, Europe 3 ndi buluu, Africa 1 ndi lalanje, ndipo Africa 2 ndi yobiriwira. Mtengowo umazika mizu ndi kukokeredwa ku sikelo, ndipo kutalika kwa nthambi kumayezedwa ngati m'malo mwa malo aliwonse.
Mycobacterium tuberculosis complex imafotokozedwa kuti idasinthika mokhazikika, ndipo maumboni ambiri omwe adasonkhanitsidwa pazaka zambiri amathandizira lingaliro lakuti HGT yomwe ikupitilira ndi zochitika zophatikizananso sizidzachitika pamlingo wodziwika wa MTBC15,17,18.
Ntchito yam'mbuyomu yawonetsa kuti pangakhale kuyanjananso pang'ono pakati pa MTBC strain20,21, pomwe ena alephera kuzindikira zochitika zoyezeka zobwerezabwereza70,71. Kambirananinso nkhaniyi molunjika pa Mycobacterium bovis, yomwe ili yosiyana ndi ntchito yapitayi yomwe imangoganizira za Mycobacterium tuberculosis 70,71; kapena kuganizira MTBC yonse, popanda pafupifupi M. bovis kuimira 20; kapena kungoganizira magawo oletsa ng'ombe. Dongosolo la mycobacteria, mu ntchitoyi, pali mitundu yonse ya 70, yomwe ikuyimira ma clonal complexes, omwe amagwiritsidwa ntchito kuwunikira kuti agwirizanenso. Deta imayikidwa molingana ndi milingo inayi yophatikizika: (1) Mamembala a Eu2, (2) mamembala onse a European clone complex (ie Europe), (3) European and African clone complex (Eu + Af) ndi (4) Zosonkhanitsira deta yonse (kuphatikiza ma genomes omwe sanaphatikizidwe muzopanga zilizonse zomwe zafotokozedwa kale).
Kuti mupitirize kuphunzira lingaliro ili, kugawanika-kuwonongeka kwa intaneti kunachitidwa kuti awone kusapezeka kwa zochitika zobwerezabwereza pakati pa ma genome, chifukwa njirayi ikhoza kuwonetsa ubale wa makolo pakati pa anthu ndikuwonetsa zizindikiro zotsutsana za phylogenetic. Ma data onse anayi pakuwunika adatsimikizira kukhalapo kwa malupu pamaneti (ndiko kuti, madera omwe sasintha kukhala mtengo umodzi), koma mayeso a Phi alibe thandizo lachiwerengero (Eu2, p = 0.0956; Europe, p = 0.1637; Eu + Af p = 0.2774; deta yonseyi idakhazikitsidwa p = 0.2), zomwe zimapereka umboni wosauka wa pganiza5, zomwe zimapereka umboni wa 0.2. 3A-D).
Ku Ulaya 2 genomes (n = 37) (A), European genomes (n = 44) (B), European and African genomes (n = 51) (C) ndi deta yonse (n = 70) (D).
Pambuyo pa kusanthula uku, ndikuganiziranso zowonera m'maukonde onse, njira yomanganso yomwe idakhazikitsidwa mupaipi ya Gubbins idagwiritsidwa ntchito pomanganso mzere wa clonal ndikuwonjezera kuyerekeza kwa zotsatira za kuyanjananso pamtundu wa M. bovis genome. Onetsani kuchuluka kwa zochitika zophatikizanso, zomwe zambiri zidachitika munthambi zomaliza (ndiko kuti, mumtundu umodzi wamtundu) (Table 2). Zizindikirozi zimasonyeza kugwirizana kwa deta yonse ndikuwonetsa kuti maulendo obwerezabwereza ndi 200 mpaka 300 nthawi ya masinthidwe. Kamodzi rho / theta parameter yomwe ikuyimira chiwerengero cha recombination ndi kusintha kwa mfundo pa nthambi ikuwoneka kuti ili pakati pa 0.0037 ndi 0.0056 (Table 3). Posachedwapa, ntchito yofalitsidwa ya 38 M. bovis strain inasonyeza mtengo wapamwamba wa rho / theta (rho / theta = 0.1) kuposa zomwe zinapezedwa mu deta iyi, koma ntchito ya Patané ndi anzake adagwiritsa ntchito msonkhano wokhudzana ndi zolemba kuti afotokoze magawo obwerezabwereza , Tsatanetsatane wa ndondomeko, chifukwa cha ndondomeko ya msonkhano, yakhala ikugwirizana ndi kuwonjezereka kwa zochitika zanthambi.
Kenako, r/m parameter imayimira kusiyanasiyana kwa kuyanjananso ndi kuyambika kwa masinthidwe, ndipo mtengo wake wapakati uli pakati pa 0.025 ndi 0.037, kusonyeza kuti poyerekeza ndi masinthidwe, kubwezeretsanso kumakhala ndi zotsatira zochepa pamitundu yosiyanasiyana yamtundu wa M. bovis (Table 3) . Poyerekeza kwambiri, njira yofananayi idagwiritsidwa ntchito kuyerekezera r/m pa dataset ya MTBC yopangidwa ndi ma genome 23, kuwonetsa mtengo wapakati wa 0.48620, pomwe pa Patané ndi anzawo '38 M. bovis dataset, zidatsimikizira kuti mtengo wake ndi 0.98. Pakafukufuku woyamba, ma genomes awiri okha mwa 23 omwe akuphatikizidwa mu ntchito ya M. bovis (M. bovis BCG ndi vuto lachidziwitso), kotero kuti mtengo wopezeka ukhoza kukhala wokondera chifukwa cha kuchulukitsidwa kwa mtundu wa M. chifuwa chachikulu. Mu lipoti lachiwiri, kuchuluka kwa Mycobacterium bovis komwe kufufuzidwa kunapezeka makamaka ku United States komanso komwe kuli ziweto. Mosiyana ndi zimenezi, m'magulu athu a deta, malo ochulukirapo komanso mitundu yomwe ikukhalamo imayimiridwa, ndipo ma genome omwe amagawidwa m'magulu osiyanasiyana omwe ali ndi makhalidwe osiyanasiyana amtundu wa anthu amagwiritsidwanso ntchito, motero amapeza chidziwitso chozama komanso chochuluka cha Chiwerengero cha anthu. Kusiyana kwa r / m pafupifupi mtengo wopezedwa ndi seti yathu ya deta ikugwirizana ndi lingaliro lakuti mlingo wa recombination umasiyana kwambiri pakati pa mibadwo yomwe imaperekedwa ku mitundu yofanana ya taxonomic, kotero zotsatirazi zimasonyeza kuti M. bovis clone complex ikhoza kusonyeza kusiyana kwa recombination Zomwe zimakhudzidwanso ndi momwe Didelot & Maiden72 anafotokozera. Komabe, kukulitsa kwambiri deta iyi mwa kuphatikiza chiwerengero chokulirapo cha ma genomes a M. bovis kudzalola kumveketsa bwino mfundoyi. Magawo onse a r/m ndi rho/theta amasonyeza kusiyana pakati pa nthambi, ndipo zotsatira zake zimagwirizana ndi malipoti a mitundu ina ya mabakiteriya72,73.
Pomaliza, pofuna kutsimikizira zochitika zakukonzanso zomwe zidazindikirika ndi mapaipi a Gubbins, ma aligorivimu osiyanasiyana asanu ndi limodzi adagwiritsidwa ntchito mu pulogalamu ya RDP4 kuyesa paokha mafananidwe osiyanasiyana apakati. Padziko lonse lapansi, zosakwana theka la zochitika zomwe Gubbins adazizindikira zidatsimikiziridwa ndi RDP4 (Matebulo 4 ndi 5). Poganizira za deta yonse, zochitika zitatu zogwirizanitsa zinatsimikiziridwa, ziwiri zomwe zikuphatikizapo mfundo zamkati ndi zina zomwe zimaphatikizapo genome imodzi munthambi yotsiriza, yomwe ma clonal complexes sakanakhoza kuperekedwa (Tables 4 ndi 5). Kuzindikiritsa zochitika m'nthambi zomaliza kungasonyeze kuti kugwirizanitsa kudakali mkati mwa mitundu yamakono ya M. bovis kapena zotsatira zake zatayika70. M'chigawo chophatikizana choyerekeza ichi, pafupifupi 20% ya malo ali ndi ma nucleotides (N), motero amakhudza chizindikiro chogwirizanitsa (Supplementary Figure 2). Kuphatikiza apo, derali limakhudza jini ya rrs, kuyika 16S ribosomal RNA yomwe ikuyembekezeka kusungidwa bwino, kotero chizindikiro cholumikizira ichi chikhoza kukhala chifukwa cha zolakwika zotsatizana kapena kusanja molakwika. Ndiye lonse matupi athu mayikidwe pakati Mb0003 ndi Mycobacterium bovis AF2122/97 unkachitika, ndi kukhalapo undefined nucleotides ndi SNPs anatsimikizira, kotero mavuto zotheka okhudzana ndi mayikidwe olakwika sanali chifukwa cha zamoyo zambiri akuyendera mu ntchito imeneyi Anaonekera pambuyo kuphunzira pulogalamu.
Palibe mipata kapena ma nucleotide osadziwika omwe adapezeka m'zigawo zophatikizananso zamkati (Zithunzi 4 ndi 5). Ponena za zochitika izi, imodzi ili ndi Eu2 genome yokha ndipo imakhudza jini ya pks12, yomwe imayika zotheka polyketide synthase; pomwe winayo adalembetsedwa mu genome ya Eu1 ndipo amakhudza jini ya narX yomwe imatsekera zotheka kuchepetsa nitrate (Table 4). Kawirikawiri, kusanthula kwa recombination kumasonyeza kuti pali chiwerengero chochepa cha zidutswa zowonongeka ndi chithandizo cha ziwerengero, ndipo zizindikiro zowonongeka zimasonyeza kuti kubwezeretsanso kumakhala ndi zotsatira zochepa pa mzere wa M. bovis. Chizindikiro chophatikizanso chikuyembekezeka kukhala chochepa, koma ndikofunikira kusiyanitsa chizindikiro chenicheni cha chisinthiko ndi phokoso lakumbuyo, lomwe ndi ntchito yovuta. Pofuna kuchepetsa phokoso la phokoso lomwe limayambitsidwa ndi zovuta zogwirizanitsa ndi zolakwika 70, 71, ena onse kupatula matupi athunthu anasonkhanitsidwa kuyambira pachiyambi, ndipo khalidwe la msonkhano linayang'aniridwa ndikutsimikiziridwa ndi kusanthula mapaipi a QUAST (Table Supplementary Table 1). Kuphatikiza apo, kafukufuku wowonjezera wowonjezera adachitidwa kuti apereke kulimba komanso kulondola kwa kafukufukuyu. Chifukwa chake, kusanja kwamtundu wa narX ndi pks12 kudawunikidwa powerenga mapu motsutsana ndi Mycobacterium bovis AF2122/97. Malo ovomerezeka a SNP m'dera la recombination adatsimikiziridwa pogwiritsa ntchito njira zomwe zatchulidwa mu gawo la njira (osachepera 20 kuwerenga ndi 0.9 kusintha pafupipafupi). Polymorphism ya jini ya narX idatsimikizika kwathunthu mumitundu iwiri (Mb1792361 ndi Mb7240415; 2.3%) ndi ma genome a pks12 genome: Mb0891, Mb1711, Mb1789, Mb1870, Mb, Mb1706 ndi M1704 Komabe, kwa ma genome Mb2043, asanu ndi atatu mwa asanu ndi atatuwa samakumana ndi chiwerengero chowerengera chakuya chifukwa SNP imathandizidwa ndi chiwerengero cha 17 chowerengera, chomwe chili pansi pa mtengo wokhazikitsidwa wa cutoff wa 20. Choncho, kuyanjananso kwa ma genomes asanu ndi limodzi (8.6%) pa tsamba la genome akhoza kutsimikiziridwa (Zithunzi 4 ndi 5).
Kuwonetseratu mwatsatanetsatane kwa kulumikizananso kwa chigawo cha Mycobacterium bovis dataset kumakhudza jini ya narX yomwe imayika zotheka kuchepetsa nitrate. Palibe mipata kapena ma nucleotide osadziwika omwe adapezeka m'chigawo chophatikizananso chamkati. Chochitikachi chalembetsedwa mu Eu1 genome. Makhalidwe otsatizana a jini ya narX adawunikidwa pokonza zowerengera za Mycobacterium bovis AF2122/97. Tsimikizirani malo ovomerezeka a SNP m'dera lophatikizanso pogwiritsa ntchito njira zomwe zatchulidwa mu gawo la njira (osachepera 20 kuwerengera ndi 0.9 kusintha pafupipafupi). Polymorphism ya jini ya narX idatsimikiziridwa mokwanira mu ma genomes a Mb1792361 ndi Mb7240415 (2.3%).
Kuwonera mwatsatanetsatane kugwirizanitsanso chigawo cha dataset ya Mycoplasma bovis yomwe ikukhudza pks12 jini. Palibe mipata kapena ma nucleotide osadziwika omwe adapezeka m'chigawo chophatikizananso chamkati. Ponena za chochitika chomwe chikukhudza pks12 gene encoding zotheka polyketide synthase, ili ndi Eu2 genome yokha. Mkhalidwe wotsatizana wa pks12 unawunikidwa ndi mapu owerengedwa a Mycobacterium bovis AF2122/97. Tsimikizirani malo ovomerezeka a SNP m'dera lophatikizanso pogwiritsa ntchito njira zomwe zatchulidwa mu gawo la njira (osachepera 20 kuwerengera ndi 0.9 kusintha pafupipafupi). Ma polymorphisms a genomes Mb0891, Mb1711, Mb1789, Mb1870, Mb1758, Mb2043, ndi Mb1960 atsimikiziridwa mokwanira.
Majini a PE ndi PPE ali ndi zigawo zobwerezabwereza zomwe zimasokonekera mosavuta ndi kutsatizana kwa Illumina ndikulakwitsa, kotero nthawi zambiri amachotsedwa ku bioinformatics workflow ya mamembala a M. chifuwa chachikulu pokhapokha akugwiritsa ntchito mapu-to-sequence strategy. Lingaliro la zochitika zobwerezabwereza zomwe zimagwiritsidwa ntchito mu ntchitoyi zakhazikitsidwa pa msonkhano wa de novo popanda kusefa PE/PPE. Timakhulupirira kuti pogwiritsa ntchito njira zitatu zosiyana zowonjezera ndi ma aligorivimu kudzera mu SplitsTree, Gubbins pipeline ndi RDP4 software, njira zomwe zimagwiritsidwa ntchito zimakhala zolimba pokonza ndi kusefa madera omwe adakonzedwanso chifukwa cha zizindikiro zolakwika. Komabe, pofuna kuti asasokoneze kusokoneza kwa jini ya PE / PPE pa Gubbins ndi pulogalamu ya RDP4 kuti azindikire magulu a SNP, choncho chizindikiritso cha zigawo zophatikizananso zomwe zimakhudzidwa kuti zikhudze majini a narX ndi pks12, dera la majiniwa linayesedwa (Supplementary Fig. 3-5). Mu M. bovis AF2122/97, jini ya narX imasiyanitsidwa ndi narK2 ndi Mb1764c, pamene pks12 ikuzunguliridwa ndi Mb2075c e Mb2073c (Chithunzi Chowonjezera 3-5). Mapu opangidwa pogwiritsa ntchito mapu a synline a MAUVE a ma genome athunthu amapereka chidziwitso chokhudza kasungidwe ka jini ndi kukonzanso, kuwonetsa midadada inayi, ndipo palibe zizindikiro zakusintha kwa ma genome kapena kusintha. Kuphatikiza apo, kusanthula kokwanira ndi katsatidwe ka amino acid kunatsimikizira homology mumitundu yonse yathunthu, ndipo palibe PE/PPE yomwe idapezeka kumadera oyandikana nawo a narX kapena pks12. Kwa narX, genome imodzi (Mb0030) imakhala ndi mawu ofanana chifukwa jini ya narX idadziwika ngati zidutswa ziwiri (zidutswa 1891 ndi 1890). Kwa pks12, chifukwa chofanana, Mb0030 ndi Mb003 adawonetsa ziwerengero zochepa za synlinearity, pomwe pks12 idadziwika mu zidutswa ziwiri ndi zitatu, motsatana, zomwe zikuyimira madera osiyanasiyana a protein (Supplementary Figure 3-5). Poganizira za izi, ndi mapulogalamu a Gubbins ndi RDP4 onse amasanthula, fufuzani mayendedwe angapo apakati pa 500 bp pawindo, tinatsimikizira kuti jini ya PE / PPE sidzasokoneza chizindikiro cha recombination chomwe chimakhudza narX ndi pks12.
Ngakhale zizindikiro zophatikizanso zomwe zapezeka mu seti iyi zitha kuonedwa ngati zotsalira, ndizowona kuti kubwezeretsanso mu M. bovis sikungatheke, kotero ziyenera kupitiliza kukhala nkhani yowunikiranso, momwe ma genomes onse ochokera ku zochitika zosiyanasiyana za epidemiological amatsatiridwa kukhala Zofunika.
Kuyerekeza mitengo ya ML phylogenetic yomwe inapezedwa isanayambe komanso itatha kukonzedwanso (Chithunzi 2A, B) sichinapangitse kusintha kwakukulu mu chiyanjano cha phylogenetic, ndipo zovuta za M. bovis zinaphatikizidwa mu gulu lomwelo.
Pambuyo pojambula mapu 42 otsatiridwa kumene a M. bovis amawerengedwa ndi genome ya M. bovis AF2122/97, kuyanjanitsa kwa SNP komwe kuli 1816 malo a polymorphic kunapezedwa. Ma SNP ambiri (87.1%) ali m'chigawo cholembera, ndipo majini okhudzidwa amadziwika molingana ndi magulu ogwira ntchito omwe akuwonetsedwa mu Bovilist (Chithunzi 6A, B). Poganizira chiwerengero cha majini m'gulu lililonse la ntchito, jini mu "lipid metabolism" gulu anasonyeza SNPs zambiri, kenako "maselo khoma ndi selo njira" ndi "wapakatikati kagayidwe ndi kupuma", kuwulula kuti ali M. bovis chisinthiko.
Kusanthula kwadongosolo la dataseti ya M. bovis kuchokera ku Portugal (n = 42). Chiwerengero chonse cha ma SNP olembetsedwa ndi majini okhudzidwa pagulu lililonse logwira ntchito (A). Chiwerengero chonse cha zosintha zofananira ndi zosafananizidwa zolembetsedwa ndi gulu la magwiridwe antchito (B).
Padziko lonse lapansi, chiŵerengero cha dN / dS ndi chabwino kuposa 1.5, chomwe chimasonyeza kuti chiwopsezo cha chisinthiko cha dziko lonse ndicho kuchotsa chikhalidwe cha makolo ndipo chimayimira chisankho chabwino (chosiyana kapena chowongolera) ndi / kapena chomasuka. M'magulu a "virulence, detoxification, adaptation", "insertion sequence and phages", ndi "regulatory proteins", oposa magawo awiri pa atatu a SNPs sali ofanana (Chithunzi 6B).
M'magulu onse, pali majini omwe ali ndi ma SNP angapo, zomwe zimapangitsa kuti pakhale kusintha kwapakati (ndiko kuti, SNP pamtundu uliwonse) wamkulu kuposa 1 (Chithunzi 6A). Pks12 (Mb2074c) yokhala ndi 15 SNPs ndi fas (Mb2553c) yokhala ndi 8 SNPs ili ndi masinthidwe apamwamba kwambiri. Ma jini onsewa amakhudzidwa ndi kagayidwe ka mafuta acid. The pks gene encodes polyketide synthase (PKS), yomwe ndi michere yambiri yomwe imakhudzidwa ndi mycobacterial cell wall lipid biosynthesis74,75. Jini iyi imayika polypeptide yogwira ntchito zambiri yomwe imakhudzidwa ndi kaphatikizidwe ka mycoketides74,76. Fas jini imakhudzidwa ndi kaphatikizidwe ka mycolic acid. Ma jini onsewa amatenga gawo lofunikira mu biosynthesis ya khoma la cell polumikizana ndi wolandila.
Kuti mupitirize kuphunzira za kusinthika kwa Mycobacterium bovis, magulu awiri a majini enieni adawunikidwa. Ntchito zomwe zidasindikizidwa m'mbuyomo pogwiritsa ntchito njira zotsatizana komanso njira za phylogenetic zomwe zidapezeka ndi makolo a MTBC kudzera pa HGT isanachitike37,38. Majiniwa amalembedwa mu Supplementary Table 2. Kugawidwa kwa SNP kwa chiwerengero cha majini a 77 omwe angakhale okhudzana ndi HGT adafufuzidwa, ndipo malo a 26 a polymorphic adadziwika, omwe nthawi zambiri (78%) adayambitsa kusintha kosafanana (NS) (Supplementary Table 2). Ntchito yam'mbuyomu pa genome ya MTBC idawonetsa kuti dera lokhazikika la HGT limawonetsa kuchuluka kwa NS SNP poyerekeza ndi ma genome ena onse. Ngati wina akuganiza kuti zigawo za recombination izi zinapezedwa ndi makolo a MTBC, choncho, amaimira ma polymorphisms akale, ndiye kuti chiwerengero cha kusintha kofanana chikuyembekezeka kukhala chapamwamba, chifukwa m'malo mwa NS akuyembekezeka kuthetsedwa ndi kusankha kolakwika chifukwa cha kusintha kwa amino acid Kukhoza kusintha ntchito ya mapuloteni. Chifukwa chake, zotsatira zathu zikuwonetsa kuti zotsatira zake zitha kuchitika chifukwa cholowa m'malo mwa jini ngati HGT, zomwe zikuwonetsa kufunikira kwawo pakusiyanasiyana kwamitundu yosinthika.
Mogwirizana ndi kusanthula uku, majini omwe amalemba zigawo za 3R (DNA kukonza, kubwereza, ndi kubwezeretsanso) dongosolo adawunikidwa bwino malinga ndi mndandanda womwe unasindikizidwa kale ndi dos Vultos ndi othandizira (2008)39. Kusinthana kwa zidutswa zofanana za DNA sikungathe kuwonedwa mwachindunji, ngakhale kuti zikhoza kukhala zochitika kawirikawiri pamene mabakiteriya ogwirizana kwambiri akukhudzidwa, monga momwe zilili ndi deta iyi; kuonjezera apo, ndondomekoyi ingakhale chinsinsi cha njira zokonzetsera DNA72, kotero Sewerani gawo pakuphatikizana kofanana. Malo okwana 26 a polymorphic omwe amagawidwa ndi majini a 54 adadziwika (Supplementary Table 3). Mu seti iyi ya majini, kusintha kwa NS kunatenga pafupifupi 65% ya zotsatira zake, zomwe zimagwirizana ndi malipoti am'mbuyomu a matenda a chifuwa chachikulu cha Mycobacterium.
Nthawi yotumiza: Oct-21-2021