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Ua ho'ōla hou ka Genome sequencing i ke kahua o ka noiʻi ʻana i nā maʻi infectious, e hōʻike ana i ka maʻi epidemiology, pathogenesis, host-pathogen interactions, a me ke kaʻina evolutionary i kau ʻia i nā pathogens. Manaʻo ka Mycobacterium tuberculosis complex (MTBC) i ka Mycobacterium bovis ma ke ʻano he lālā hoʻololi holoholona e kumu ai ka maʻi maʻi maʻi (TB) i nā mammals terrestrial, a he kumu hoʻohālike maʻamau o ka evolution bacteria. E like me nā lālā ʻē aʻe o ka MTBC, ua manaʻo ʻia ʻo Mycobacterium bovis he pathogen i hoʻopaʻa ʻia, e ulu mālie ana, a ʻaʻohe hōʻailona o ka hoʻohui hou ʻana a i ʻole ka hoʻololi ʻana o ke ʻano. Ma kēia hana, hoʻohana mākou i nā genomics hoʻohālikelike i kahi pūʻulu genome holoʻokoʻa (WGS) i haku ʻia me 70 pipi M. mai nā moʻopuna like ʻole (Europe a me ʻApelika) e loaʻa ai nā ʻike i ka ʻokoʻa genetic o nā pipi M. Evolutionary power. Hoʻohana ʻia ʻekolu mau ʻano like ʻole e koho i nā hōʻailona o ka hoʻonohonoho hou ʻana. Ma ke ao holoʻokoʻa, ua ʻike ʻia a hōʻoia ʻia kahi helu liʻiliʻi o nā hanana recombination e ʻelua mau ala kūʻokoʻa me ke kākoʻo paʻa. Eia naʻe, ke hoʻohālikelike ʻia me nā hoʻololi ʻana, ʻoi aku ka nāwaliwali o ka recombination i ka ʻokoʻa o M. bovis (r / m = 0.037 holoʻokoʻa). ʻO ka ʻokoʻa r/m awelika i loaʻa i loko o ka clonal complex o Mycobacterium bovis i kā mākou hoʻonohonoho ʻikepili e kūlike me ka manaʻo maʻamau e ʻokoʻa nui ka degere o ka hui hou ʻana ma waena o nā laina i hāʻawi ʻia i nā ʻano taxonomic like. Ma muli o kēia hana, ʻaʻole hiki ke hoʻokuʻu ʻia ka hui hou ʻana i ka Mycobacterium bovis, no laila e lilo ia i kumu no ka hoʻoikaika hou ʻana i ka noiʻi hoʻohālikelike hoʻohālikelike e hiki mai ana, kahi WGS o nā pūʻulu ʻikepili nui mai nā hiʻohiʻona epidemiological like ʻole a puni ka honua. A laila ua hana ʻia kahi loiloi hou ma ka Mycobacterium bovis data set (n = 42) mai ka maʻi TB multi-host, a ʻoi aku ma mua o 1,800 loci i ʻike ʻia, a ma kahi o hoʻokahi kānana i hōʻike i kahi nucleotide polymorphism (SNP). Loaʻa ka hapa nui (87.1%) i loko o ka ʻāpana coding, a ʻo ka ratio honua o nā hoʻololi like ʻole (dN/dS) o nā hoʻololi like ʻoi aku ma mua o 1.5, e hōʻike ana i ke koho maikaʻi ʻana he ikaika evolutionary koʻikoʻi i hana ʻia ma M. bovis. Ua ʻike ʻia kahi hapa kiʻekiʻe o nā SNP i nā genes waiwai i nā ʻano hana o "lipid metabolism", "cell wall and cellular process", a me "intermediate metabolism and respiration", e hōʻike ana i ko lākou hiki i ka biology a me ka ulu ʻana o ka Mycobacterium bovis koʻikoʻi. ʻO ka nānā pono ʻana i nā genes i loko o nā kūpuna MTBC i maʻalahi i ka hoʻololi ʻana i nā genes horizontal a hoʻokomo ʻia i loko o ka ʻōnaehana 3R (DNA repair, replication, and recombination) e hōʻike ana i ka waiwai maikaʻi ʻole o ka hoʻāʻo ʻana o Taijima's D neutral test.
ʻO ka Mycobacterium tuberculosis complex (MTBC) kekahi o nā ʻauhau kūleʻa loa o nā maʻi bacterial a me kahi hihia maʻamau o ka evolution bacteria. Hōʻike kona mau lālā i ka ʻike nucleotide kiʻekiʻe ma ka pae genomic (> 99%)1,2. Hiki i nā ecotypes MTBC like ʻole ke hoʻoulu i ka maʻi maʻi (TB), kahi maʻi granulomatous infectious, ma kahi ākea o nā ʻano mea hoʻokipa mai nā micro-mammals a hiki i ke kanaka3,4,5. I kēia manawa, aia ka paʻakikī i nā kānaka [M. Maʻi maʻi (Mtb), Mycobacterium africanum] a me nā maʻi pathogens i hoʻololi ʻia e nā holoholona (Mycobacterium bovis, Mycobacterium capitum, Mycobacterium pinnipedum, Mycobacterium microtobacter , Mycobacterium mongee, Mycobacterium miysani, Mycobacterium surikampanzee" a me "6" M. canettii (nodobacter glabrata) ʻO ka ʻike nucleotide maʻamau me ka mycobacteria i haʻi ʻia ma mua he 98%, a ua hōʻike nā hana genomics comparative ua hoʻokaʻawale ʻo M. canettii a me ke koena o MTBC mai ka kupuna maʻamau.
Ua wehewehe ʻia ʻo MTBC ma ke ʻano he clonal paʻakikī, a ua hoʻomalu ʻia kona ʻano heluna kanaka e ka hoʻemi ʻia ʻana o nā ʻano like ʻole, nā bottlenecks, nā koho koho, a me ka genetic drift9,10. Ke manaʻo nei ʻaʻole hiki ke hoʻihoʻi hou ʻia e ka hui hou ʻana, e like me nā polymorphism i nalowale. Ma muli o kēia manaʻo, ua manaʻo ʻia nā hanana like ʻole o ka holoi ʻana i ka genomic o ka ʻāpana ʻokoʻa (RD) a me TbD1 (Mtb kikoʻī hoʻopau 1 ʻāina) i manaʻo ʻia e like me nā māka molekala o MTBC evolution2,5,11. Kākoʻo ka hana comparative genomics and whole genome sequencing (WGS) i ka mahele ʻana o nā lālā i hoʻololi ʻia e ke kanaka i ʻeiwa mau laina (Mycobacterium tuberculosis L1 a L4, L7, a me L8; a me Mycobacterium africanum L5, L6, a me L9), nā laina L2 a L4 i hoʻokaʻawale i ka TbD1 region2,11. Eia hou, ua noi ʻia nā lālā i hoʻololi ʻia i nā holoholona e kaʻana like i kahi kupuna maʻamau, i wehewehe ʻia e nā holoi kikoʻī clade ma RD7, RD8, RD9 a me RD102, 5, a me 14.
ʻO ka hoʻololi ʻana o ke ʻano hoʻopaʻa (HGT) a me nā hanana hoʻohui hou i manaʻo ʻia he kakaikahi a hiki i nā kūpuna o MTBC, ma mua o ka moʻolelo like ʻole o ka lālā MTBC holoʻokoʻa15,16,17. ʻElua mau hōʻike mua a Hughes and collaborators (2002) a me Gutacker and collaborators (2006) i manaʻo e hiki i nā hanana hui hou ke kōkua i ke ʻano polymorphisms e hōʻailona ai i nā loci kikoʻī i M. tuberculosis strains18,19. ʻO nā kumu o ka maopopo ʻole o ka recombination ma MTBC: (1) ke kaʻina hana mechanical a me ka nalowale o ka hiki o HGT; (2) ka liʻiliʻi o nā hanana HGT; (3) ʻaʻohe manawa o nā hanana recombination ma ka MTBC niche14,17. I kēia mau lā, ua hoʻohana ʻia kekahi mau haʻawina holoʻokoʻa-genome sequencing (WGS) i ka MTBC strain 20 a me Mycobacterium bovis 21 i hōʻike i ka hoʻohui hou ʻana, ʻo ka mea mua e hōʻike i ka hoʻololi pinepine ʻana o MTBC i nā ʻāpana DNA liʻiliʻi, akā ma muli o ka liʻiliʻi o ka hoʻololi o ke kaʻina nucleotide, ʻaʻole i ʻike ʻia kēia mau hanana.
ʻO ka Mycobacterium bovis ka lālā MTBC i hoʻihoʻi pinepine ʻia mai nā holoholona (ʻoi aku ka nui o nā pipi), ʻoiai hiki ke hoʻokaʻawale ʻia mai nā holoholona hihiu manuahi a paʻa i ka pā4,22,23,24. M. bovis evolved i elima nui clonal complexes [European 1 (Eu1), European 2 (Eu2), European 3 (Eu3), African 1 (Af1) a me Africa 2 (Af2)], e like me ka spoligotyping profile, kiko'ī holoiʻia A me hookahi nucleotide polymorphisms (SNPs) 25, 92 kiko'ī ma 8, 25, 26. Hōʻike kēia mau clonal complexes i ke ʻano like ʻole o ka heluna Mycobacterium bovis a me kona hui pū ʻana me nā wahi ʻāina. Eia kekahi, ʻo ka hana WGS hou i hana ʻia e Zimpel a me nā hoa hana (2020) i hoʻolālā i kahi phylogeny e pili ana i ka SNP o Mycobacterium bovis, me ka ʻoi aku o 1,900 genomes, e hōʻike ana aia ma kahi o ʻehā mau laina like ʻole (i kapa ʻia ʻo Lb1 a Lb1 a Lb4), ʻaʻole hiki ke hoʻohālikelike ʻia me ka kikoʻī kikoʻī kikoʻī. Ua hana kēia mau mea kākau i ka loiloi ʻokoʻa o ka phylogeny a me ka molecular dating, akā ʻaʻole i aʻo i ka recombination30.
ʻO ka hana mua me ka hoʻohana ʻana i nā ʻenehana molekala like ʻole, e like me ka spoligotyping, MIRU-VNTR (mycobacterial interspersed repeat unit-variable tandem repeat number), a me ka paʻi ʻana i ka SNP hou i hōʻike ʻia i kahi pae o ka ʻokoʻa genetic ma waena o M. bovis strains 31,32,33, 34,35. Ua lilo ka hoʻokaʻawale ʻana o ka hoʻololi genetic i mea hana koʻikoʻi i ke aʻo ʻana i ka maʻi epidemiology, he mea kōkua no ka hoʻomaopopo hohonu ʻana i ka pathogenesis, virulence a me ka lawe ʻana i nā maʻi. ʻO ka puka ʻana o ke ʻano WGS e hāʻawi i ka hiki ke hōʻike i nā mea hoʻokele evolutionary i kau ʻia e ka Mycobacterium bovis genome i ke kaʻina o ka hoʻololi ʻana a me ka hoʻomau ʻana i nā pūʻali like ʻole a me nā hiʻohiʻona epidemiological.
Ma kēia hana, hoʻohana mākou i ka loiloi genomic hoʻohālikelike i nā ʻikepili Mycoplasma bovis like ʻole (n=70), me nā mea hoʻokaʻawale mai nā clonal complexes like ʻole, e loaʻa ai nā ʻike i ke kaʻina evolutionary o Mycoplasma bovis, ʻoi aku ka hoʻoholo ʻana i nā pilina phylogenetic A me nā hanana recombination. Ma ke ʻano he hoʻohui i kēia loiloi, ua ʻimi hou ʻia kahi pūʻulu ʻikepili o M. bovis isolates (n = 42) i loaʻa mai kahi maʻi maʻi maʻi multi-host ma Portugal 31,36 i ʻimi hou ʻia e infer non-identity ʻO ke kaulike ma waena o ka ratio pili o ka manaʻo (dN) i ka synonymous (dS) nucleotide substitutions. 37,38 i loaʻa e nā kūpuna MTBC ma o HGT, a hoʻopili i ka 3R (DNA repair, replication, and recombination) system gene component 39. E koho i nā genes i loaʻa ma o HGT no ka mea hiki ke hōʻike i nā polymorphisms kahiko, no laila ua manaʻo ʻia aia paha i loko o ka hapa nui o nā loli like. Ua koho ʻia nā genes i loko o ka ʻōnaehana 3R no ka mea, ʻo ka hana mua ma M. tuberculosis strains i hōʻike ʻia i nā koho ʻino / hoʻomaʻemaʻe maʻamau e hana ana i kēia mau genes, a he hana koʻikoʻi paha lākou i ka evolution 39. ʻO kekahi pahuhopu o kēia hana ʻo ia ka infer i ke ola ʻana o nā hanana hoʻonohonoho hou. No kēia kumu, me ka noʻonoʻo ʻana i kā mākou ʻikepili mai Pokukala wale nō nā genomes o ʻEulopa clone complex 2 a me nā ʻano ʻaʻole i hāʻawi i ka paʻakikī clone, ua hoʻoholo mākou e hoʻokomo i ka ʻikepili genome i loaʻa i ka lehulehu e loaʻa i kahi ʻelele o nā paʻakikī clone āpau, a hoʻomaikaʻi i ka ikaika a me ka laulā o nā hopena.
ʻO 42 nā genomes Mycoplasma bovis hou mai ka Pukiki endemic multi-host tuberculosis scene (nā kikoʻī ma lalo), i hōʻike mua ʻia mai kahi hiʻohiʻona epidemiological36, ke kikowaena o kēia hana. Ke noʻonoʻo nei i ka ʻikepili i hoʻonohonoho ʻia mai Pokukala he mau ʻelele wale nō o ka European 2 clone complexes a me nā kānana me ka ʻole o nā complexes i koho ʻia, ua hoʻohui ʻia i ka lehulehu ka ʻikepili sequencing genome holoʻokoʻa e hoʻonui i ka pūʻulu ʻikepili e komo pū ana nā ʻelele a pau o ka M. bovis clone complexes. No laila, ua hoʻohana ʻia ʻekolu mau kumu ʻikepili sequencing holoʻokoʻa i loko o kēia hana: ka hui genome piha / draft, a hiki i ka 10 scaffolds i mālama ʻia ma NCBI (National Center for Biotechnology Information) (n = 15 isolates); mālama ʻia ma SRA ( ʻO ka Illumina fastq file o ka sequence read archive) e hōʻike ana i ka ʻokoʻa paʻakikī o nā clones M. bovis (n = 12 isolates)30; a me 42 mau genomes hou mai Pokukala. Ua kāpae ʻia ka Mycobacterium bovis BCG (Bacille Calmette-Guerin) mai ka ʻimi NCBI. Hoʻohana mau ʻia ʻo M. bovis AF2122/97 ma ke ʻano he genome kuhikuhi e hoʻokomo ʻia i loko o ka dataset. Ma muli o ka loaʻa ʻole o ka lehulehu o ke kaʻina genome holoʻokoʻa i hōʻike ʻia e ka ʻApelika 1 cloning complex, a me ka helu liʻiliʻi o nā genome mai nā ʻano kikoʻī o Af2 a me Eu1, ua hoʻohana ʻia ka ʻikepili sequencing kumu i hāʻawi ʻia e SRA i kēia mau hihia. ʻO ka hana a Zimpel a me kāna mau hoa hana (2020) i kōkua i ka ʻike ʻana i ka genome mai ka cloning complex i ʻōlelo ʻia a kōkua i ke koho ʻana i ka Mycobacterium bovis no ka hoʻokomo ʻana i ka ʻikepili. No Eu3, hoʻokahi wale nō ʻano o ka genome i wehewehe ʻia (Branger et al., 2020), no laila, ʻo ka genome a mākou e hoʻokomo ai he ʻelele ʻokoʻa ia o ka complex Eu3.
Ma ke ao holoʻokoʻa, ʻo kēia pūʻulu ʻikepili he 70 pipi M. bovis i hoʻokaʻawale ʻia mai nā ʻano hoʻokipa 8, i māhele ʻia ma nā ʻāina 12 mai 1985 a 2016. Ua koho ʻia nā ʻano he 36 e like me Eu2, 7 mau ʻano ʻo Eu1, 1 ʻano he Eu3, 3 mau ʻano he Af1, 4 mau ʻano he Af2 a me 19 ʻaʻole i pili i nā clonal complex (ma lalo o kekahi clonal complex). Hōʻike ʻia ka ʻike kikoʻī (me ka helu komo) o Mycobacterium bovis i hoʻohana ʻia i kēia haʻawina ma ka Papa 1 a me ka Papa Hoʻohui 1.
42 hou sequenced genomes holoʻokoʻa o Mycobacterium bovis mai Pokukala's holoholona tuberculosis piko wela a puunaue no oi ma mua o 12 makahiki ke kikowaena o keia haʻawina, e like me ka hiki holoholona hihiu-holoholona maʻi nenoai ua nānā mau 31,36 (Supplementary Fig. 1). Wahi a nā kaʻina hana ma hope mai, ua hoʻokaʻawale ʻia kēia mau ʻano mai nā pipi (n = 14), deer ʻulaʻula (n = 16) a me ka puaʻa hihiu (n = 12) mai 2003 a 2015: hōʻiliʻili a mālama i nā holoholona e like me nā alakaʻi protocol i ʻōlelo ʻia ʻO nā ʻāpana kiko i loko o ka OIE Terrestrial Animal Handbook a ua hoʻopaʻa ʻia ma luna o Stonebrinesteink-and solid medium medium. Hoʻokomo ʻia nā moʻomeheu ma 37 °C a nānā ʻia ka ulu ʻana i hoʻokahi manawa i ka pule no 12 mau pule. Hoʻopaʻa pololei ʻia nā kolo i loko o kahi hopena glycerol ma -80ºC. I loko o ka Mycobacterium selective medium (Middlebrook 7H9, BD Diagnostics), ua hoʻoili ʻia nā mea hoʻohālike kumu i hoʻopaʻa ʻia i loko o ka pauku in vitro hoʻokahi in vitro e loaʻa ai ka DNA o ka papahana WGS. No kēia, ua hoʻonui ʻia ka solution stock culture paʻa me 5% sodium pyruvate a me 10% ADS (50 g albumin, 20 g glucose, 8.5 g sodium chloride i 1 L wai) ma Middlebrook 7H9 ma 37°C Retrain. Ma hope o 4 mau pule o ka ulu ʻana, ua hoʻololi hou ʻia ka mea waena a nānā mau ʻia ka moʻomeheu a hiki i ka ʻike ʻia ʻana o ka ulu. Ua hōʻiliʻiliʻia nā pūnaewele e ka centrifugation, ua hoʻokuʻu houʻia ka pellet i 500 µL phosphate buffered saline (PBS), i hoʻomehanaʻia ma 99 ° C no nā minuke 30, centrifuged, a ua mālamaʻia ka supernatant ma -20 ° C a hiki i ka WGS. Hoʻokō ʻia nā kaʻina hana a pau i nā keʻena biosafety pae 3.
Hoʻomākaukau ʻia ka WGS paired-end genome library me ka hoʻohana ʻana i ka ʻike kikoʻī o kēlā me kēia DNA sample, a hoʻohana iā Illumina MiSeq (2 × 250 pb) (40 samples) a me HiSeq (2 × 150 pb) (ʻelua isolates) ʻenehana (Eurofins Genomics, Kelemānia) no ka hoʻonohonoho ʻana. E like me nā ʻōlelo a ka mea hana, e hoʻohana i ka Illumina Genome Analyzer me ka hoʻopili ʻana i ʻelua module e hoʻonohonoho i ka genomic DNA, a hoʻohana i ka Nextera XT DNA Library Prep Kit mai Illumina e kūkulu i ka waihona.
Ma ka noʻonoʻo ʻana i ka ʻikepili i hoʻihoʻi ʻia mai ka SRA (n = 12), hiki ke hoʻohana ʻia ka ʻike ʻana o ka clone complex e like me ka metadata o ka paʻi pili 30, 41, 43. Ke noʻonoʻo nei i ka genome holoʻokoʻa, koe wale nō Mycobacterium bovis AF2122/97 a me Mycobacterium bovis 3601 Mycobacterium bovis 3601, ʻo ia ka cloud1 a me nā lālā paʻakikī o ka cloud1 a me nā lālā. 29, kēlā me kēia, ua like ia me ka genome piha o Mycobacterium tuberculosis H37Rv (ka helu komo NCBI NC_000962.3). Hana ʻia ka alignment genome me ka hoʻohana ʻana i ka MAFFT (polokalamu hoʻohālikelike lehulehu o ka amino acid a i ʻole ka nucleotide sequence, version 7.458) a me ka parameter -addfragments48. A laila, e ʻimi no ka loaʻa ʻole o nā ʻano clonal complexes a / a i ʻole ke alo o nā hiʻohiʻona SNP.
ʻO ka Mycobacterium bovis hou (n = 42) a me ka heluhelu mua ʻana o ke kiʻi genome i hui ʻia (n = 3) e hoʻohālikelike i ka paʻakikī me ka genome kuhikuhi Mycobacterium tuberculosis H37Rv ma o ka vSNP pipeline a me ke alo o ka holoi ʻana a/a i ʻole nā hiʻohiʻona SNP o nā clones like ʻole Ua hana ʻia kahi hulina.
E hōʻiliʻili i ka ʻike mai ka nele o nā hiʻohiʻona a/a i ʻole ka loaʻa ʻole o ka SNP a me ka spoligotyping profiles e hāʻawi i ka ʻikepili genomic i ka paʻakikī clonal pili. No nā hui kikoʻī ʻehā, ʻaʻole hiki ke ʻike ʻia ka ʻaoʻao spoligotyping, no laila ua hoʻokomo ʻia lākou i ka hui "ʻaʻohe paʻakikī".
Hoʻomaka ka bioinformatics workflow i ukali ʻia e kēia hana mai ka hui de novo a me ka palapala ʻāina i kahi hoʻolālā kuhikuhi, me ka pahuhopu e ʻimi i nā hanana recombination a me nā polymorphism genome kikoʻī. Hāʻawi ka Figure 1 i kahi papa kuhikuhi o nā ʻanuʻu i ukali ʻia. No ka hoʻopili hou ʻana, hoʻohana ʻia nā genome a pau e hoʻonui i ka paʻa o nā inferences a me nā hōʻailona pili.
I mea e hōʻemi ai i nā hewa i ka hoʻokumu ʻana i nā kaʻina kuʻikahi genome, ua loaʻa mua mākou i ka hui de novo, a laila loaʻa nā alignments nui. Loaʻa ka pipeline Unicycler i kēia manawa ma https://github.com/rrwick/Unicycler49 a hoʻohana ʻia e hana i ka hui de novo o 54 sequenced genomes (42 sequenced hou a me 12 fastq files i hoʻihoʻi ʻia mai SRA). I ka pōkole, ma mua o ka hui ʻana mai ka ʻōpala, heluhelu ʻia ka nānā ʻana i ka maikaʻi ma FastQC version 0.11.7 (https://github.com/s-andrews/FastQC), a me Trimmomatic version 0.36 (koho "cutting Adapters and other lighting-specific sequences from reads" a me "Oki i nā kumu mai ka hopena o ka paepae" he haʻahaʻa ka maikaʻi. (http://www.usadellab.org/cms/?page= trimmomatic) 50. A laila, ua hoʻohana ʻia ʻo SPAdes optimiser49 no ka hui genome, a ua hoʻohana ʻia ʻo Pilon version 1.1851 no ka hoʻoponopono ʻana ma hope o ka hui. Ua koho ʻia ke ʻano conservative bridging e pale aku i ka hui hewa ʻana, a ua ʻimi ʻia ka nui k-mer a koho ʻia ma waena o 20% a me 95% o ka lōʻihi heluhelu. E hahai i nā alakaʻi SPAdes a e noʻonoʻo i ka nui heluhelu, e holoi i nā contigs liʻiliʻi ma mua o 300 bp, a e hoʻokumu i kahi ʻoki kikoʻī 20 heluhelu hohonu o 52. Ma ka papahana hui de novo, ʻaʻole i wehe ʻia nā ʻāpana genomic e like me ka proline-glutamate (PE) a me ka proline-proline glutamate (PPE) paralogs.
ʻIke ʻia ka maikaʻi o ka hui de novo ma o ka pipeline QUAST (http://quast.sourceforge.net/quast.html), e hoʻomaʻamaʻa i ka hana hou o ka contig a me ka M. bovis AF2122/97 reference genome (NCBI accession number LT708304.1) Mapping (e nānā i ka Papa Hoʻohui 1 no nā ʻāpana maikaʻi).
Me ke kōkua o ka vSNP pipeline (https://github.com/USDA-VS/vSNP), ua hoʻohālikelike ʻia ka faila FASTQ o ka M. bovis hou mai Illumina sequencing me ka M. bovis AF2122/97 reference genome (LT708304.1)). Wahi a nā ʻōlelo aʻoaʻo hoʻomaʻamaʻa maikaʻi loa o ka Genome Analysis Toolkit (GATK) 53, 54, 55 e hoʻopili i nā ʻāpana kānana maʻamau a i ʻole nā helu nuipaʻa like ʻole no ka hoʻoponopono hou ʻana. Hoʻopili ʻia nā hopena me ka hoʻohana ʻana i ka helu haʻahaʻa haʻahaʻa loa o SAMtools o 150 a me AC = 2. E hoʻohana pū i Kraken (http://ccb.jhu.edu/software/kraken/) e nānā i nā heluhelu no ka pale ʻana i ka haumia. ʻO ka pipeline vSNP i hoʻohana ʻia no ka palapala ʻana i nā hoʻolālā hoʻolālā i kā mākou hana e nānā i ke ʻano o nā SNP i wehewehe ʻia a me nā pahuhopu, a kāpae pū kekahi i nā hiʻohiʻona maʻi huikau. ʻOi aku ka maikaʻi o ka uhi ʻana o ka genome ma mua o 99% (Paʻi Hoʻohui 1).
No ka pale ʻana i nā hewa palapala palapala a me nā SNP hewa, kānana i kahi ʻano like ʻole i kēia mau hihia: (1) kākoʻo ʻia ia e ka liʻiliʻi ma mua o 20 mau heluhelu, (2) ʻike ʻia me ke alapine o ka liʻiliʻi ma mua o 0.9, (3) aia ma ka liʻiliʻi loa i hoʻokahi Strains, akā ma ka liʻiliʻi loa he mau hakahaka ma kahi ala ʻē aʻe. Ua hoʻohana ʻia ka mana o ka integrated genomics viewer (IGV) version 2.4.19 (http://software.broadinstitute.org/software/igv/)56 no ka hōʻoia ʻana i nā SNP a me nā kūlana me ka palapala palapala a i ʻole nā pilikia alignment. No ka mea, ua hoʻohālikelike nui ʻia nā genes proline-glutamate (PE) a me ka proline-proline glutamate (PPE) a he ʻāpana o ka ʻohana multi-gene, maʻalahi lākou e kuhi hewa ʻia e ka Illumina sequencing a mismapping, no laila ua makemake ʻia lākou ʻO ka mycobacterial bioinformatics workflow wehe i nā lālā o ka paʻakikī tuberculosis i ka wā e hoʻohana ai i ka hoʻolālā o ka hoʻopaʻa ʻana i ka SNP i ka sequence. No laila, ua kānana mākou i nā genes PE/PPE a me nā indels mai ka nānā ʻana.
Wahi a Bovilist (http://genolist.pasteur.fr/BoviList/), ua hoʻokaʻawale ʻia nā SNP āpau i nā ʻāpana hana. Hoʻohana ʻia ka pipeline SnpEff (https://pcingola.github.io/SnpEff/) no ka hoʻoholo ʻana i nā hopena SNP (nā hoʻololi like a i ʻole nā hoʻololi like ʻole). Hana ʻia kahi waihona hou o Mycobacterium bovis AF2122/97 genome (LT708304.1).
Ua hoʻohana ʻia ka alignment nui genome me ka hoʻohana ʻana iā Parsnp v1.2, i loaʻa i kēia manawa ma https://github.com/marbl/parsnp57, me ka hoʻohana ʻana i 69 genomes/draft assemblies (me ke koho -c) a hoʻohana ʻia ʻo M. bovis AF2122/97 (LT708304.1) ma ke ʻano he kuhikuhi. ʻEhā mau hoʻolikelike nui i hana ʻia: ʻo nā lālā wale nō o ka Eu2 cloning complex (n = 37), me nā lālā a pau o ka European cloning complex (n = 44), me ka junction point o ka European and African cloning complex (n = 51), a hoʻokomo i nā Mycobacterium bovis āpau i kēia haʻawina (n = 70).
Hoʻohana ʻia ka alignment koʻikoʻi i hana ʻia e Parsnp no ka hoʻohana ʻana i ka lāʻau phylogenetic likelihood (ML) me ka hoʻohana ʻana iā CIPRES Science Gateway v3.3 (http://www.phylo.org/)58 me ka RAxML, a hana i 1000 replications alakaʻi.
Hoʻohana ʻia ʻekolu mau algorithms a me nā mea hana bioinformatics e nānā i ke ʻano o nā hanana recombination i ka like: SplitsTree4 software, Gubbins (unbiased lineage through recombination in nucleotide sequences) pipeline, a me RDP4 (recombination detection program, version beta 4.101) lako polokalamu.
Hoʻohana ʻia ke ʻano decomposition split i SplitsTree4 v4.15.1 (http://www.splitstree.org/)59 no ka helu ʻana i ka pūnaewele phylogenetic rootless, me ka hoʻohana ʻana i ka Phi no ka hōʻoia helu helu, a ʻo ka paepae koʻikoʻi he p = 0.05. Hoʻohana ʻia ka loiloi multi-alignment koʻikoʻi o Parsnp ma ke ʻano he hoʻokomo, a ʻike ʻia ka hoʻokaʻawale ʻana ma ke ʻano he maʻamau pūnaewele.
ʻO Gubbins pipeline v2.3.1 (https://github.com/sanger-pathogens/gubbins60 e holo ana me nā palena paʻamau ma ke ʻano he ala ʻē aʻe e loiloi i ka hopena o ka recombination ma Mycobacterium bovis. ʻO ka algorithm i hoʻokō ʻia i ka pipeline e kūkulu hou i ka laina clone kūpono ʻO ka hui genome/draft piha o kā mākou hoʻonohonoho ʻikepili a me ka reference genome/97 (bovine bovid2 genome) LT708304.1) i kekahi;
ʻO ka mea hope loa, i mea e hōʻoia ai i ka hanana hoʻonohonoho hou i manaʻo ʻia e ka Gubbins pipeline, ua hoʻohana ʻia nā algorithm ʻeono i hoʻokō ʻia ma RDP467 (RDP61, GENECONV62, Bootscan63, Maxchi64, Chimaera65 a me SiScan66) i hoʻopili ʻia i ka alignment nui o Parsnp ma lalo o nā hoʻonohonoho paʻamau. Ua hoʻoholo mākou he ʻekolu o nā algorithms i hoʻokō ʻia ma RDP4 e hōʻike mau i kahi hōʻailona koʻikoʻi e hōʻoia i kēlā me kēia hanana recombination.
Ke noʻonoʻo nei ʻo Gubbins a me RDP lako polokalamu e ʻimi i nā hōʻailona recombination ma ka nānā ʻana i ka nui o nā alignments nui i ka puka makani a hiki i 500 bp, a me ka hōʻoia ʻana ʻaʻole e hoʻopilikia ka hoʻopili ʻana o nā genes PE/PPE i ka wā o ka hui de novo i nā hōʻailona recombination i loaʻa, hana hou ʻia e ka homolinearity. Ua kūkulu ʻia ka palapala synlinear e hoʻohana ana i ka genome holoʻokoʻa me ka hoʻohana ʻana i ka MAUVE-multi-genome alignment (http://darlinglab.org/mauve/mauve.html) e kāpae i nā unuhi genome kūloko a i ʻole nā hoʻololi. Eia hou, ua hoʻohana ʻia ka genome holoʻokoʻa e hana i ka homolinearity analysis ma ke kaʻina amino acid ma o ka pūnaewele pūnaewele SyntTax (https://archaea.i2bc.paris-saclay.fr/SyntTax/).
ʻO ka ʻike hohonu loa o ka ʻikepili genome i loaʻa mai ka Pukiki multi-host tuberculosis system e nānā i ka polymorphism o nā genes i ʻōlelo ʻia ma ka palapala. ʻO kēia mau genes he 37,38 a me ka gene encoding 3R i loaʻa e nā kūpuna MTBC ma o ka HGT (DNA repair, replication and recombination) system components 39. E hoʻohana i ka ClustalX v2.1 (http://www.clustal.org/clustal2/) a e hoʻohana i ka DnaSP v6.12.03 (http://www.ub.edu/edu/d nucleotide genevers) (π) a me Tajima's D neutral test parameter input.
Ua loaʻa ka lāʻau phylogenetic likelihood (ML) ma muli o 69 Mycoplasma bovis isolates a me nā genomes kuhikuhi (Figure 2A). Ke hoʻohālikelikeʻia me nā lāʻau hoʻokahi-gene a iʻole nā lāʻau multi-locus-based, hiki i kēia hoʻolālā ke hana i nā kumu lāʻau ikaikaʻole i hopuʻole i ka loli o ka genome holoʻokoʻa a no laila e hōʻike ana i ka haʻahaʻa haʻahaʻa e hoʻokaʻawale i waena o nāʻano 68,69. ʻO ke ʻano topological o ka lāʻau ML e kūlike me ka hoʻohālikelike paʻakikī o nā clones. Hoʻopili ʻia ka genome o Eu2 i kahi lālā, a hui pū ʻia ka genome o Af1 (Figure 2A). Ua like pū ka hopena me ka pilina evolutionary i ʻike ʻia o Mycobacterium bovis, ʻo ia hoʻi, aia ka ʻokoʻa nui ma waena o ka lālā Eu1 a me ka hui i loaʻa nā clonal complexes a me nā genomes, akā ʻaʻole i kuhikuhi ʻia ka clonal complex 30. Hiki ke wehewehe ʻia ka liʻiliʻi like ʻole ma waena o ka clonal complex a me ka pilina i ʻike ʻia ma ka lāʻau phylogenetic ma ke ʻano o ka wehewehe ʻana o ka clonal complex ma muli o nā ʻāpana genomic kikoʻī, ʻoiai ke kumu o ka lāʻau phylogenetic ma luna o nā alignments he nui o nā genome kumu e hōʻike ana i ka genome holoʻokoʻa.
Kūkulu ʻia ka lāʻau phylogenetic likelihood nui loa (GTR) ma muli o ka alignment genome kumu o ka Mycobacterium bovis genome ma mua o (A) a ma hope o (B) ka wehe ʻana i ka pūnaewele recombination. Hōʻike nā kala lālā i ka Mycobacterium bovis clone complex: ʻEulopa 1 he poni, ʻEulopa 2 he ʻulaʻula, ʻEulopa 3 he uliuli, ʻApelika 1 he ʻalani, a ʻo ʻApelika 2 he ʻōmaʻomaʻo. Hoʻokumu ʻia ka lāʻau a huki ʻia i ka pālākiō, a ua ana ʻia ka lōʻihi o ka lālā ma ke ʻano he pani no kēlā me kēia kahua.
ʻO ka Mycobacterium tuberculosis complex i wehewehe ʻia e like me ka clonally evolved, a ʻo ka hapa nui o nā hōʻike i hōʻiliʻili ʻia i nā makahiki e kākoʻo ana i ka manaʻo ʻaʻole hiki ke hoʻomau ʻia nā hanana HGT a me ka recombination i ka pae ʻike ʻia o MTBC15,17,18.
Ua hōʻike ʻia nā hana mua e hiki ke kaupalena ʻia ka recombination ma waena o MTBC strains20,21, aʻo nā mea ʻē aʻe ʻaʻole i ʻike i nā hanana recombination hiki ke ana ʻia70,71. E kūkākūkā hou i kēia pilikia me ka nānā ʻana i ka Mycobacterium bovis, he ʻokoʻa ia mai ka hana mua i manaʻo wale ʻia ʻo Mycobacterium tuberculosis 70,71; a i ʻole e noʻonoʻo i ka MTBC holoʻokoʻa, ʻaneʻane ʻaʻohe M. bovis e hōʻike ana i ka 20; a i ʻole e noʻonoʻo wale i nā hakina pipi kaohi. ʻO ka ʻikepili mycobacteria, i loko o kēia hana, he 70 ka nui o nā kānana, e hōʻike ana i nā paʻakikī clonal āpau, i hoʻohana ʻia e pale no ka hui hou ʻana. Hoʻonui ʻia ka hoʻonohonoho ʻikepili e like me nā pae kumulative ʻehā: (1) nā lālā Eu2, (2) nā lālā paʻakikī clone ʻEulopa a pau (ʻo ia hoʻi ʻo ʻEulopa), (3) ʻEulopa a me ʻApelika clone complex (Eu + Af) a me (4) nā hōʻiliʻili ʻikepili holoʻokoʻa (me nā genomes ʻaʻole i hoʻokomo ʻia i loko o nā complex cloning i wehewehe mua ʻia).
No ke aʻo hou ʻana i kēia kuhiakau, ua hana ʻia kahi ʻupena hoʻokaʻawale-decomposition e loiloi i ka ʻole o nā hanana recombination ma waena o nā genomes, no ka mea hiki i kēia ʻano ke ʻike i ka pilina kupuna ma waena o nā kānaka a hōʻike i nā hōʻailona phylogenetic kū'ē. Ua hōʻoia ʻia nā ʻikepili ʻehā a pau i ka nānā ʻana i ka noho ʻana o nā puka lou i ka pūnaewele (ʻo ia hoʻi, nā wahi i hui ʻole ʻia i hoʻokahi lāʻau), akā ʻaʻohe kākoʻo helu helu Phi (Eu2, p = 0.0956; ʻEulopa, p = 0.1637; Eu + Af p = 0.2774; ʻo ka hōʻike holoʻokoʻa p = 0.2451 hōʻike hōʻike hōʻike maikaʻi ʻole). 3A-D).
Ma ʻEulopa 2 genomes (n = 37) (A), European genomes (n = 44) (B), European and African genomes (n = 51) (C) a me ka ʻikepili holoʻokoʻa (n = 70) (D).
Ma hope o kēia loiloi, a me ka noʻonoʻo ʻana i nā ʻike cyclic i nā pūnaewele āpau, ua noi ʻia ka algorithm reconstruction i hoʻokō ʻia i ka pipeline Gubbins e hana hou i ka laina clonal a me ka hoʻohui ʻana i ka manaʻo o ka hopena o ka recombination ma ka M. bovis genome. E hoʻohālikelike i ka helu kumulative o nā hanana hoʻohui hou, ʻo ka hapa nui o ia mau mea i hana ʻia ma nā lālā hope (ʻo ia hoʻi, i loko o kahi genome hoʻokahi) (Papa 2). Hōʻike kēia mau hōʻailona i ke kūlike o ka hoʻonohonoho ʻikepili holoʻokoʻa a hōʻike i ka pinepine o nā hanana recombination he 200 a 300 mau manawa o nā mutations. I ka manawa e ʻike ʻia ai ka ʻāpana rho/theta e hōʻike ana i nā helu pili o ka hoʻohui hou ʻana a me ka hoʻololi ʻana o ke kiko ma ka lālā ma waena o 0.0037 a me 0.0056 (Papa 3). I kēia mau lā, ua hōʻike ka hana i paʻi ʻia o ka 38 M. bovis strain i kahi kiʻekiʻe rho / theta waiwai (rho / theta = 0.1) ma mua o ka mea i loaʻa i kēia dataset, akā ʻo ka hana a Patané a me nā hoahana i hoʻohana i ka ʻaha kūkā e hoʻohālikelike ai i nā ʻāpana recombination , ʻO kahi kikoʻī kaʻina hana, ma muli o ke kaʻina hana, ua pili pū me ka recombination o nā hanana.
A laila, ʻo ka r / m parameter e hōʻike ana i ka lākiō like ʻole o ka recombination a me ka hoʻomaka ʻana o ka mutation, a ʻo kona awelika waiwai ma waena o 0.025 a me 0.037, e hōʻike ana i ka hoʻohālikelike ʻia me nā mutations, ʻoi aku ka haʻahaʻa o ka recombination i ka ʻokoʻa genetic o M. bovis (Table 3)). No ka hoʻohālikelike nui ʻana, ua hoʻohana ʻia kahi ʻano like no ka koho ʻana i ka ʻāpana r/m no ka dataset MTBC i haku ʻia me 23 genomes, e hōʻike ana i ka waiwai awelika o 0.48620, ʻoiai no ka ʻikepili 38 M. bovis ʻo Patané a me nā hoa, ua hōʻike ʻia ka waiwai awelika ʻo 0.98. Ma ka haʻawina mua, ʻelua wale nō o nā genomes 23 i hoʻokomo ʻia i ka hana a M. bovis (M. bovis BCG a me ka reference strain), no laila hiki ke hoʻohālikelike ʻia ka waiwai i loaʻa ma muli o ka overexpression o ka M. tuberculosis genome. Ma ka hōʻike ʻelua, ua loaʻa ka nui o ka heluna Mycobacterium bovis i kālailai ʻia mai ʻAmelika Hui Pū ʻIa a me nā pūʻali holoholona. ʻO ka ʻokoʻa, ma kā mākou pūʻulu ʻikepili, ʻoi aku ka nui o nā wahi kikoʻī a me nā ʻano mea hoʻokipa, a ua hoʻohana pū ʻia nā genomes i hui pū ʻia i loko o nā paʻakikī clonal like ʻole me nā ʻano genetic o ka heluna kanaka, no laila e loaʻa ai kahi ʻike hohonu a ākea. ʻO ka ʻokoʻa r/m awelika waiwai i loaʻa me kā mākou pūʻulu ʻikepili ua kūlike me ka manaʻo e ʻokoʻa nui ke degere o ka hui hou ʻana ma waena o nā laina i hāʻawi ʻia i nā ʻano taxonomic hoʻokahi, no laila e hōʻike ana kēia mau hopena e hōʻike ana ka M. bovis clone complex i nā ʻokoʻa recombination ʻO ka hopena e like me ka manaʻo o Didelot & Maiden72. Eia nō naʻe, ʻo ka hoʻonui nui ʻana i kēia ʻikepili i hoʻonohonoho ʻia me ka hoʻokomo ʻana i kahi helu nui o M. bovis genomes e ʻae i ka wehewehe hou ʻana i kēia wahi. Hōʻike nā ʻāpana r/m a me rho/theta i ka ʻokoʻa ma waena o nā lālā, a ua kūlike kēia hopena me nā hōʻike e pili ana i nā ʻano bacteria ʻē aʻe72,73.
ʻO ka mea hope loa, i mea e hōʻoia ai i nā hanana hoʻonohonoho hou i ʻike ʻia e ka pipeline Gubbins, ua hoʻohana ʻia nā algorithms ʻokoʻa ʻeono i ka polokalamu RDP4 e hoʻāʻo kūʻokoʻa i nā ʻano like ʻole multi-comparisons. Ma ke ao holoʻokoʻa, emi iho ka hapalua o nā hanana i ʻike ʻia e Gubbins i hōʻoia ʻia e RDP4 (Nā Papa 4 a me 5). Ma ka noʻonoʻo ʻana i ka hoʻonohonoho ʻikepili holoʻokoʻa, ua hōʻoia ʻia ʻekolu mau hanana recombination, ʻelua e pili ana i nā nodes i loko a ʻo kekahi e pili ana i kahi genome hoʻokahi i loko o kahi lālā terminal, kahi i hiki ʻole ai ke hāʻawi ʻia i nā clonal complexes (Table 4 a me 5). ʻO ka ʻike ʻia ʻana o nā hanana ma nā lālā hope e hōʻike ana i ka holomua ʻana o ka hui hou ʻana i nā kuʻuna M. bovis o kēia manawa a i ʻole ke kuhi hewa ʻia ka hopena70. Ma kēia ʻāpana hoʻohui hypothetical, ma kahi o 20% o nā kūlana i ʻike ʻole ʻia nā nucleotides (N), no laila e pili ana i ka hōʻailona recombination (Supplementary Figure 2). Eia kekahi, pili kēia māhele i ka gene rrs, e hoʻopili ana i ka 16S ribosomal RNA i manaʻo ʻia e mālama nui ʻia, no laila ʻo kēia hōʻailona hoʻohui hou paha ka hopena o ka hoʻonohonoho ʻana i nā hewa a i ʻole misalignment. A laila ua hana ʻia ka genome alignment a pau ma waena o Mb0003 a me Mycobacterium bovis AF2122/97, a ua hōʻoia ʻia ka noho ʻana o nā nucleotides a me nā SNP i ʻike ʻole ʻia, no laila, ʻaʻole nā pilikia e pili ana i ka alignment hewa ma muli o ka ʻike biological i hoʻokō ʻia ma kēia hana Ua ʻike ʻia ma hope o ke aʻo ʻana i ka papahana.
ʻAʻole i ʻike ʻia nā ʻāpana a i ʻole nā nucleotides i ʻike ʻole ʻia ma nā wahi recombination o nā nodes kūloko (Figure 4 a me 5). E pili ana i kēia mau hanana, aia wale nō ka genome Eu2 a pili i ka gene pks12, kahi e hoʻopili ai i ka polyketide synthase; ʻoiai ua hoʻopaʻa inoa ʻia ka mea ʻē aʻe i ka genome Eu1 a pili i ka gene narX e hoʻopili ana i kahi reductase nitrate hiki ke loaʻa (Papa 4). Ma keʻano laulā, hōʻike ka hōʻuluʻulu hoʻohui houʻana he palena iki o nā'āpana hoʻohui hou me ke kākoʻo helu helu, aʻo nā hōʻailona i manaʻoʻia e hōʻike ana he haʻahaʻa ka hopena o ka hui houʻana i ka moʻokūʻauhau M. bovis. He haʻahaʻa ka hōʻailona recombination, akā he mea nui e ʻike i ka hōʻailona evolution maoli mai ka walaʻau hope, he hana paʻakikī. I mea e hōʻemi ai i ka leo leo hōʻailona i hoʻokomo ʻia e ka hui kuhikuhi a me nā pilikia mismatch 70, 71, ʻo ke koena a pau koe ka genome piha i hōʻuluʻulu ʻia mai ka ʻohi ʻana, a ua nānā ʻia ka maikaʻi o ka hui a hōʻoia ʻia e ka QUAST pipeline analysis (Supplementary Table 1). Eia hou, ua mālama ʻia kekahi pūʻulu o nā loiloi hou e hāʻawi i ka paʻa a me ka pololei o ka anamanaʻo holoʻokoʻa. No laila, ua loiloi ʻia ka maikaʻi o nā genes narX a me pks12 e ka heluhelu palapala ʻāina kūʻē iā Mycobacterium bovis AF2122/97. Ua hōʻoia ʻia ke kūlana SNP i manaʻo ʻia ma ka ʻāina recombination ma o ka hoʻohana ʻana i nā koina i ʻōlelo ʻia ma ka ʻāpana hana (ma ka liʻiliʻi he 20 heluhelu a me 0.9 hoʻololi pinepine). Ua hoʻokūpaʻa pihaʻia ka polymorphism o ka gene narX i nā genomesʻelua (Mb1792361 a me Mb7240415; 2.3%) a me nā genomes o ka pks12 genome: Mb0891, Mb1711, Mb1789, Mb1870, Mb17046, Mb17046, Mb17046, Mb17046, Mb Eia naʻe, no ka genome Mb2043, ʻeono o nā kūlana ʻewalu ʻaʻole i kūpono i ka helu hohonu heluhelu no ka mea ua kākoʻo ʻia ka SNP e ka nui o 17 heluhelu, aia ma lalo o ka waiwai cutoff i hoʻokumu ʻia o 20. No laila, hiki ke hōʻoia ʻia ka hui hou ʻana o ʻeono genomes (8.6%) ma kēia pūnaewele genome (Figures 4 a me 5).
ʻO ka hiʻohiʻona kikoʻī o ka hoʻolikelike ʻana o ka ʻāina hoʻohuihui o ka ʻikepili Mycobacterium bovis e pili ana i ka gene narX e hoʻopili ana i kahi reductase nitrate. ʻAʻole i ʻike ʻia nā ʻāpana a i ʻole nā nucleotides i ʻike ʻole ʻia ma ka ʻāpana hui hou o nā nodes kūloko. Hoʻopaʻa inoa ʻia kēia hanana kūikawā ma ka genome Eu1. Ua loiloi ʻia ka maikaʻi o ke kaʻina narX ma ka hoʻolālā ʻana i nā heluhelu o Mycobacterium bovis AF2122/97. E hōʻoia i ka wahi SNP i manaʻo ʻia ma ka wahi hoʻohui hou ma o ka hoʻohana ʻana i nā koina i ʻōlelo ʻia ma ka ʻāpana hana (ma ka liʻiliʻi he 20 heluhelu a me ka 0.9 hoʻololi pinepine). Ua hoʻokūpaʻa pihaʻia ka polymorphism o ka gene narX i nā genomes o Mb1792361 a me Mb7240415 (2.3%).
ʻO ka ʻike kikoʻī o ka hoʻonohonoho ʻana o ka ʻāina recombination o ka ʻikepili Mycoplasma bovis e pili ana i ka gene pks12. ʻAʻole i ʻike ʻia nā ʻāpana a i ʻole nā nucleotides i ʻike ʻole ʻia ma ka ʻāpana hui hou o nā nodes kūloko. E pili ana i ka hanana e pili ana i ka pks12 gene encoding possible polyketide synthase, aia wale no ka Eu2 genome. Ua loiloi ʻia ka maikaʻi o ka hoʻonohonoho ʻana o pks12 e ka heluhelu palapala ʻana o Mycobacterium bovis AF2122/97. E hōʻoia i ka wahi SNP i manaʻo ʻia ma ka wahi hoʻohui hou ma o ka hoʻohana ʻana i nā koina i ʻōlelo ʻia ma ka ʻāpana hana (ma ka liʻiliʻi he 20 heluhelu a me ka 0.9 hoʻololi pinepine). Ua hōʻoia piha ʻia nā polymorphisms o nā genomes Mb0891, Mb1711, Mb1789, Mb1870, Mb1758, Mb2043, a me Mb1960.
Loaʻa i nā genes PE a me PPE nā ʻāpana i hiki ke heluhelu hewa ʻia e ka Illumina sequencing a mismapping, no laila e holoi pinepine ʻia lākou mai ka bioinformatics workflow o nā lālā M. tuberculosis wale nō i ka wā e hoʻohana ai i ka hoʻolālā mapping-to-sequence. Hoʻokumu ʻia ka inference o nā hanana hui hou i kēia hana ma ka hui de novo me ka kānana ʻole ʻana i ka PE/PPE. Ke manaʻoʻiʻo nei mākou ma ka hoʻokō ʻana i ʻekolu mau ʻano hoʻohui like ʻole a me nā algorithms ma o SplitsTree, Gubbins pipeline a me RDP4 lako polokalamu, ua paʻa nā hoʻolālā i hoʻohana ʻia i ka hoʻoponopono ʻana a me ka kānana ʻana i nā wahi i hoʻonohonoho hou ʻia e nā hōʻailona hewa. Eia naʻe, i mea e hoʻokaʻawale ai i ka hoʻopili ʻana o ka gene PE / PPE ma nā polokalamu Gubbins a me RDP4 e ʻike ai i nā pūʻulu SNP, a no laila ʻo ka ʻike ʻana i nā ʻāpana recombination i manaʻo ʻia e hoʻopilikia i nā genes narX a me pks12, ua nānā ʻia ke kaiāulu o kēia mau genes (Supplementary Fig. 3-5). Ma M. bovis AF2122/97, ua hoʻokaʻawale ʻia ka gene narX e narK2 a me Mb1764c, ʻoiai ua hoʻopuni ʻia ʻo pks12 e Mb2075c e Mb2073c (Hui 3-5). ʻO ka palapala ʻāina i hana ʻia me ka palapala ʻāina MAUVE synline o ka genome holoʻokoʻa e hāʻawi i ka ʻike e pili ana i ka mālama ʻana a me ka hoʻonohonoho hou ʻana o ka gene, e hōʻike ana i ʻehā mau ʻāpana collinear, a ʻaʻohe hōʻailona o ka hoʻololi ʻana a i ʻole ka hoʻohuli ʻana. Eia kekahi, ua hōʻoia ka hoʻopili hoʻohui me ka ʻāpana amino acid i ka homology i nā genomes piha, a ʻaʻohe PE/PPE i loaʻa ma nā wahi pili o narX a i ʻole pks12. No ka narX, hoʻokahi genome (Mb0030) he haʻahaʻa haʻahaʻa like ʻole no ka mea ua ʻike ʻia ka gene narX he ʻelua ʻāpana (mau ʻāpana 1891 a me 1890). No ka pks12, ma muli o nā mea like, hōʻike ʻo Mb0030 a me Mb003 i nā helu synlinearity haʻahaʻa, aʻo pks12 i ʻike ʻia i ʻelua a ʻekolu mau ʻāpana, kēlā me kēia, e hōʻike ana i nā kikowaena like ʻole o ka protein (Supplementary Figure 3-5). Ma ka noʻonoʻo ʻana i kēia ʻike, a hana ʻo Gubbins a me RDP4 lako polokalamu i ka nānā ʻana, e nānā i ka alignment nui nui o ka 500 bp kiʻekiʻe ma ka puka makani, ua hōʻoia mākou ʻaʻole e hoʻopilikia ka PE / PPE i ka hōʻailona recombination e pili ana i ka narX a me pks12.
ʻOiai hiki ke noʻonoʻo ʻia nā hōʻailona recombination i ʻike ʻia ma kēia pūʻulu ʻikepili he koena, he ʻoiaʻiʻo ʻaʻole hiki ke hoʻokuʻu ʻia ka hui hou ʻana ma M. bovis, no laila pono e hoʻomau i ke kumuhana o ka nānā hou ʻana, kahi e hoʻonohonoho ʻia ai nā genomes holoʻokoʻa mai nā hiʻohiʻona epidemiological like ʻole i Mea nui.
ʻO ka hoʻohālikelike ʻana i nā kumulāʻau phylogenetic ML i loaʻa ma mua a ma hope o ka hoʻoponopono hou ʻana (Figure 2A, B) ʻaʻole i alakaʻi i nā loli nui i ka pilina phylogenetic inferred, a ua hui pū ʻia nā ʻano M. bovis i ka hui like.
Ma hope o ka palapala 'ana i ka helu 42 M. bovis i ho'onohonoho hou 'ia me ka genome kuhikuhi o M. bovis AF2122/97, ua loa'a mai kahi alignment SNP i loa'a nā kūlana polymorphic 1816. ʻO ka hapa nui o nā SNP (87.1%) aia ma ka ʻāpana coding, a ua ʻike ʻia nā genes i hoʻopili ʻia e like me nā ʻano hana i hōʻike ʻia ma Bovilist (Figure 6A, B). Ma ka helu ʻana i ka huina o nā genes i kēlā me kēia ʻano hana, ua hōʻike nā genes i ka ʻāpana "lipid metabolism" i nā SNP hou aʻe, a ukali ʻia e "ka pa a me nā kaʻina cell cell" a me "metabolism intermediate a me ka hanu", e hōʻike ana aia lākou i M. bovis evolution.
Ka nānā 'ana i ka hierarchical o ka M. bovis dataset mai Pokukala (n = 42). ʻO ka huina o nā SNP i hoʻopaʻa ʻia a me nā genes i hoʻopili ʻia no kēlā me kēia ʻano hana (A). ʻO ka huina o nā hoʻololi like ʻole a me nā hoʻololi like ʻole i hoʻopaʻa inoa ʻia e ka ʻāpana hana (B).
Ma ke ao holoʻokoʻa, ʻoi aku ka maikaʻi o ka ratio dN/dS ma mua o 1.5, ʻo ia ka mea e hōʻike ana ʻo ke kaomi hoʻololi honua e hoʻopau i ka mokuʻāina kupuna a hōʻike i kahi hiʻohiʻona koho hoʻomaʻemaʻe maikaʻi (diversified a kuhikuhi ʻia) a hoʻomaha hoʻi. Ma nā ʻano o "virulence, detoxification, adaptation", "insertion sequences and phages", a me "regulatory proteins", ʻoi aku ma mua o ʻelua hapakolu o nā SNP ʻaʻole like (Figure 6B).
Ma nā māhele a pau, aia nā genes me nā SNP he nui, e hopena i ka awelika mutation rate (ʻo ia hoʻi, ka awelika SNP no kēlā me kēia gene) i ʻoi aku ma mua o 1 (Figure 6A). ʻO Pks12 (Mb2074c) me 15 SNPs a me fas (Mb2553c) me 8 SNP i loaʻa nā waiwai mutation kiʻekiʻe. Hoʻopili ʻia kēia mau genes ʻelua i ka metabolism fatty acid. Hoʻopili ka gene pks i ka polyketide synthase (PKS), he enzyme multifunctional i komo i loko o ka mycobacterial cell wall lipid biosynthesis74,75. Hoʻopili kēia gene i kahi polypeptide multifunctional i komo i ka synthesis o mycoketides74,76. Hoʻokomo ka gene fas i ka synthesis o ka mycolic acid. He kuleana koʻikoʻi kēia mau genes ʻelua i ka biosynthesis o ka paia o ke kelepona e pili ana me ka mea hoʻokipa.
I mea e aʻo hou ai i ka ulu ʻana o Mycobacterium bovis, ʻelua pūʻulu o nā genes kikoʻī i kālai ʻia. ʻO nā hana i hoʻopuka mua ʻia me ka hoʻohana ʻana i ka haku mele a me nā ʻano phylogenetic i ʻike ʻia i nā ʻano genes i loaʻa e nā kūpuna MTBC ma o HGT ma mua o ka diversification37,38. Ua heluʻia kēia mau genes ma ka Pākuʻi Pākuʻi 2. Ua nānāʻia ka māhele SNP o ka nui o nā genes 77 e pili ana i ka HGT, a uaʻikeʻia nā pūnaewele polymorphic 26, a ma ka hapanui o nā hihia (78%) i loaʻa i nā loli non-synonymous (NS) (Supplementary Table 2). Ua hōʻike ʻia ka hana mua ma ka genome MTBC e hōʻike ana ka ʻāpana HGT putative i kahi kiʻekiʻe NS SNP ratio i hoʻohālikelike ʻia i ke koena o ka genome. Inā manaʻo kekahi ua loaʻa kēia mau ʻāina recombination e nā kūpuna MTBC, a no laila, overrepresent lākou i nā polymorphisms kahiko, a laila ʻoi aku ka kiʻekiʻe o ka nui o nā hoʻololi like, no ka mea, ua manaʻo ʻia e hoʻopau ʻia nā pani ʻana o NS ma muli o ka hoʻololi ʻana o ka waikawa amino. No laila, ke hōʻike nei kā mākou mau hopena i nā hopena hana mai ka hoʻololi ʻana o nā genes like HGT, e hōʻike ana i ko lākou koʻikoʻi no ka ʻokoʻa genetic adaptive waiwai.
I ka like me keia anaana, ua nana pono ia na genes e hoopili ana i na mea o ka 3R (DNA repair, replication, and recombination) system e like me ka papa inoa i hoopuka mua ia e dos Vultos and collaborators (2008)39. ʻAʻole hiki ke nānā pono ʻia ka hoʻololi ʻana o nā ʻāpana DNA like, ʻoiai he hana pinepine ia i ka wā e pili ana i nā bacteria pili pili, e like me ke ʻano o kēia ʻikepili; Eia kekahi, ʻo kēia kaʻina hana ke kī i nā kaʻina hana hoʻoponopono DNA72, no laila e pāʻani i kahi hana i ka homologous recombination. Ua ʻike ʻia he 26 mau kūlana polymorphic i puʻunaue ʻia e nā genes 54 (Paʻi Hoʻohui 3). Ma kēia pūʻulu o nā genes, ua helu ʻia nā hoʻololi NS ma kahi o 65% o nā hopena, i kūlike me nā hōʻike mua e pili ana i nā maʻi Mycobacterium tuberculosis.
Ka manawa hoʻouna: Oct-21-2021