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Tsarin kwayoyin halitta ya sake farfado da fannin binciken cututtuka masu yaduwa, bayyanar cututtuka na cututtuka, cututtuka, hulɗar kwayoyin cuta, da tsarin juyin halitta wanda aka sanya akan ƙwayoyin cuta. Ƙungiyar tarin fuka ta Mycobacterium (MTBC) tana ɗaukar Mycobacterium bovis a matsayin ɗaya daga cikin mambobi na dabba wanda ke haifar da tarin fuka (TB) a cikin dabbobi masu shayarwa na duniya, kuma shi ne nau'i na al'ada na juyin halittar kwayan cuta. Kamar sauran membobin MTBC, Mycobacterium bovis ana ɗauka a matsayin ɗanɗano mai ƙarfi, mai haɓaka ƙwayoyin cuta a hankali, kuma babu shakka babu alamar sake haɗuwa ko canja wuri a kwance. A cikin wannan aikin, mun yi amfani da kwatancen genomics zuwa ga jimlar genome series (WGS) dataset wanda ya ƙunshi shanu 70 M. daga sassa daban-daban (Turai da Afirka) don samun fahimtar bambancin jinsin dabbobi M. Ƙarfin Juyin Halitta. Ana amfani da hanyoyi daban-daban guda uku don kimanta alamun sake tsarawa. A duniya, an gano ƙananan abubuwan sake haɗuwa kuma an tabbatar da su ta hanyoyi biyu masu zaman kansu tare da goyon baya mai ƙarfi. Duk da haka, idan aka kwatanta da maye gurbi, sake haɗuwa yana da rauni mai rauni akan bambancin M. bovis (gaba ɗaya r/m = 0.037). Bambancin r / m matsakaicin da aka samu a cikin hadaddun clonal na Mycobacterium bovis a cikin saitin bayanan mu ya yi daidai da ra'ayi na gabaɗaya cewa matakin sake haɗuwa na iya bambanta sosai tsakanin layin da aka ba wa nau'in taxonomic iri ɗaya. Dangane da wannan aikin, sake haɗawa a cikin Mycobacterium bovis ba za a iya kawar da shi ba, don haka ya kamata ya zama batun ƙarin yunƙuri a cikin binciken kwatancen genomics na gaba, wanda WGS na manyan bayanan da suka fito daga yanayin yanayin annoba daban-daban a duniya yana da mahimmanci. Bayan haka an yi ƙarin bincike akan ƙananan bayanan Mycobacterium bovis (n = 42) daga yawancin tarin tarin fuka, kuma an gano fiye da 1,800 loci, wanda aƙalla nau'i ɗaya ya nuna nau'in nucleotide polymorphism (SNP). Yawancin (87.1%) suna cikin yanki na coding, kuma rabon duniya na canje-canje marasa daidaituwa (dN/dS) na canje-canje masu kama da juna ya wuce 1.5, yana nuna cewa zaɓi mai kyau shine muhimmin ƙarfin juyin halitta wanda aka yi akan M. bovis. An gano mafi girma na SNPs a cikin kwayoyin halitta masu arziki a cikin nau'o'in ayyuka na "lipid metabolism", "bangon tantanin halitta da tsarin salula", da "tsakanin metabolism da numfashi", yana nuna yiwuwar su a cikin ilmin halitta da juyin halitta na Mycobacterium bovis muhimmancin. Idan aka yi la’akari da ƙwayoyin halittar kakannin kakannin MTBC waɗanda ke da saurin canja wurin kwayar halitta a kwance kuma an haɗa su cikin tsarin 3R (gyaran DNA, maimaitawa, da sake haɗawa) yana nuna matsakaicin ƙarancin ƙimar gwajin tsaka-tsakin duniya na Taijima's D, wanda ke nuna zaɓen da ya gabata.
Mycobacterium tarin fuka hadaddun (MTBC) yana daya daga cikin mafi nasara haraji na ƙwayoyin cuta pathogens da kuma al'ada hali na kwayoyin juyin halitta. Membobinta suna nuna girman girman nucleotide mai ban mamaki a matakin genomic (> 99%)1,2. Daban-daban na MTBC ecotypes na iya haifar da tarin fuka (TB), wanda shine cututtukan granulomatous mai yaduwa, a cikin nau'in nau'in nau'i mai yawa daga ƙananan dabbobi masu shayarwa zuwa mutane3,4,5. A halin yanzu, hadaddun ya haɗa da mutane [M. Tuberculosis (Mtb), Mycobacterium africanum] da kuma dabbobin da suka dace da dabbobi (Mycobacterium bovis, Mycobacterium capitum, Mycobacterium pinnipedum, Mycobacterium microtobacter, Mycobacterium mongee, Mycobacterium miysani, Mycobacterium surika, "Bacillus chimpanzee" da "dassie"). M. canettii (wanda aka fi sani da "Nodobacter glabrata") Matsakaicin asalin nucleotide tare da mycobacteria da aka ambata shine 98%, kuma aikin kwatancen genomics ya nuna cewa M. canettii da sauran MTBC kwanan nan sun bambanta daga kakanni na kowa.7 Bisa la'akari da wannan ra'ayi, wasu marubutan BC suna kiran M.
MTBC an siffanta shi da tsari azaman ƙaƙƙarfan hadaddun clonal, kuma tsarin yawan jama'a ana sarrafa shi a fili ta hanyar rage bambance-bambance, kwalabe, zaɓen sikanin, da drift genetic drift9,10. Tsammanin haɗaɗɗen ingantaccen juyin halitta na clonal, kamar ɓacewar polymorphisms ba za a iya dawo da su ta hanyar haɗuwa ba. Dangane da wannan jigo, abubuwan da suka faru a jere na gogewar genomic na yanki daban-daban (RD) da TbD1 (yankin Mtb ƙayyadaddun sharewa 1) an gabatar da su azaman alamomin kwayoyin halitta na MTBC evolution2,5,11. Kwatanta kwayoyin halitta da tsarin tsarin kwayoyin halitta gaba daya (WGS) yana goyan bayan rarrabuwar membobin da suka dace da mutum zuwa zuriya tara (Mycobacterium tarin fuka L1 zuwa L4, L7, da L8; da Mycobacterium africanum L5, L6, da L9), layin L2 zuwa L4 sun raba share yankin TbD12,131,12 Bugu da ƙari, an ba da shawarar membobin da suka dace da dabba don raba kakanni ɗaya, wanda aka bayyana ta takamaiman gogewa a cikin RD7, RD8, RD9 da RD102, 5, da 14.
Canja wurin kwayoyin halitta a kwance (HGT) da abubuwan sake hadewa ana daukarsu da wuya kuma suna faruwa a cikin kakannin MTBC, maimakon tarihin daban-daban na memba na MTBC gaba daya15,16,17. Rahotanni na farko guda biyu na Hughes da masu haɗin gwiwa (2002) da Gutacker da masu haɗin gwiwa (2006) sun nuna cewa abubuwan da suka faru na sake haɗuwa na iya taimakawa wajen tsara nau'in polymorphisms wanda ke nuna takamaiman loci a cikin ƙwayar tarin fuka na M. tuberculosis18,19. Dalilan rashin sake haduwa a fili a cikin MTBC sune: (1) tsarin injina da asarar iyawar HGT; (2) ƙarancin abubuwan HGT; (3) babu damar sake haɗuwa da abubuwan da suka faru a cikin MTBC niche14,17. Kwanan nan, wasu binciken da aka yi amfani da su a kan nau'in MTBC na 20 da Mycobacterium bovis 21 sun ba da shaida na sake haɗuwa, na farko da ya nuna cewa nau'in MTBC yakan canza ƙananan sassan DNA, amma saboda iyakanceccen nau'in nucleotide, waɗannan abubuwan har yanzu ba a lura ba.
Mycobacterium bovis shine memba na MTBC da aka fi ganowa daga dabbobi (mafi yawan shanu), kodayake kuma ana iya keɓance shi daga namun daji masu kyauta da shinge4,22,23,24. M. Bovis ya samo asali ne cikin manyan rikice-rikice guda biyar [Turai 1 (EU1), Tarayyar Turai (EU1), SNPS) 25, 27, 29, 29, 29, 29, 29, 29, 29, 29, 29, 29. Waɗannan rukunin clonal suna nuna nau'ikan tsari na yawan Mycobacterium bovis da haɗin gwiwa tare da yankuna. Bugu da ƙari, aikin WGS na baya-bayan nan da Zimpel da masu haɗin gwiwar (2020) suka yi sun tsara wani nau'i na phylogeny dangane da SNP na Mycobacterium bovis, tare da fiye da 1,900 genomes, yana nuna cewa akwai akalla nau'i hudu daban-daban (mai suna Lb1 zuwa Lb1 zuwa Lb4), ba su da cikakkiyar daidaituwa tare da ƙayyadaddun ƙayyadaddun 3 ko da yake ana iya tabbatar da su a baya. Waɗannan marubutan sun yi nazari na banbance-banbance game da phylogeny da dating na kwayoyin halitta, amma ba su yi nazarin sake haɗuwa30 ba.
Ayyukan da suka gabata ta amfani da fasaha daban-daban na kwayoyin halitta, irin su spoligotyping, MIRU-VNTR (mycobacterial interspersed repeat unit-variable tandem repeat number), da kuma kwanan nan SNP buga buga wani mataki na bambancin kwayoyin halitta tsakanin M. bovis damuwa 31,32,33, 34,35. Bambance-bambancen kwayoyin halitta ya zama kayan aiki mai mahimmanci a cikin nazarin cututtukan cututtuka, wanda ke taimakawa wajen fahimtar zurfin fahimtar cututtuka, cututtuka da cututtuka. Fitowar hanyar WGS tana ba da damar bayyana abubuwan tuƙi na juyin halitta wanda Mycobacterium bovis genome ya sanya a cikin tsarin daidaitawa da dagewa ga runduna daban-daban da yanayin cututtukan annoba.
A cikin wannan aikin, muna amfani da bincike na kwatankwacin genomic akan daban-daban Mycoplasma bovis datasets (n=70), gami da keɓancewa daga rukunin clonal daban-daban, don samun fahimtar tsarin juyin halitta na Mycoplasma bovis, musamman don warware alaƙar phylogenetic da abubuwan sake haɗuwa. A matsayin kari ga wannan bincike, wani sub-data sa na M. bovis ware (n = 42) samu daga wani m hali Multi-host tuberculosis yankin a Portugal 31,36 aka kara binciko zuwa infer rashin ganewa Ma'auni tsakanin dangi rabo na ma'ana (dN) zuwa synonymous (dS) nucleotide maye gurbinsu, kazalika da takamaiman wallafe-wallafen da aka ambata a cikin juyin halitta. 37,38 da kakannin MTBC suka samu ta hanyar HGT, kuma suna ɓoye 3R (gyaran DNA, maimaitawa, da sake haɗawa) sassan tsarin tsarin 39. Zaɓi kwayoyin halittar da aka samu ta hanyar HGT saboda suna iya wakiltar tsohuwar polymorphisms, don haka ana tsammanin za su iya ƙunsar mafi girman adadin canje-canje masu kama da juna. An zaɓi kwayoyin halittar da ke cikin tsarin 3R saboda aikin da aka yi a baya akan nau'in tarin fuka na M. ya nuna babban zaɓi mara kyau / tsarkakewa wanda ke aiki a kan waɗannan kwayoyin halitta, kuma suna iya taka muhimmiyar rawa a cikin juyin halitta 39. Wani makasudin wannan aikin shine don ganin wanzuwar abubuwan sake tsarawa. A saboda wannan dalili, la'akari da cewa mu dataset daga Portugal kawai ya ƙunshi genomes na Turai clone hadaddun 2 da damuwa da cewa ba a sanya clone hadaddun, mun yanke shawarar hada da jama'a samuwa bayanai genome a karshe samu wakilin duk clone gidaje, da kuma inganta ƙarfi da fadi da sakamakon.
42 sabon jerin Mycoplasma bovis genomes daga yanayin cutar tarin fuka da yawa na Portugal (bayanan da ke ƙasa), wanda aka kwatanta a baya daga hangen nesa na annoba36, sune cibiyar wannan aikin. Idan akai la'akari da cewa bayanan da aka saita daga Portugal kawai suna da wakilai na rukunin clone na Turai 2 da damuwa ba tare da ƙayyadaddun ƙayyadaddun ƙayyadaddun bayanai ba, an ƙara yawan bayanan da aka samu a bainar jama'a don faɗaɗa saitin bayanan da ya haɗa da duk wakilai na M. bovis clone complexes. Sabili da haka, an yi amfani da maɓuɓɓukan bayanan bayanan gaba ɗaya-genome guda uku a cikin wannan aikin: cikakke / daftarin tsarin genome, har zuwa 10 scaffolds adana a NCBI (National Center for Biotechnology Information) (n = 15 ware); adana a cikin SRA (Fayil ɗin Illumina fastq na jerin abubuwan da aka karanta tarihin) yana wakiltar bambance-bambancen bambancin M. bovis clones (n = 12 ware)30; da sabbin nau'ikan kwayoyin halitta 42 daga Portugal. An cire Mycobacterium bovis BCG (Bacille Calmette-Guerin) daga binciken NCBI. M. bovis AF2122/97 yawanci ana amfani dashi azaman kwayar halitta don haɗawa a cikin saitin bayanai. Saboda rashin samun jama'a na dukkanin jerin kwayoyin halittar da ke wakilta ta hanyar haɗin gwiwar 1 na Afirka, da ƙananan adadin kwayoyin halitta daga nau'ikan nau'ikan nau'ikan Af2 da Eu1, an yi amfani da ainihin bayanan jeri da SRA ta bayar a waɗannan lokuta. Ayyukan Zimpel da abokan aikinsa (2020) sun taimaka wajen gano kwayoyin halitta daga rukunin cloning da aka ambata a baya kuma sun taimaka zaɓi Mycobacterium bovis don haɗawa a cikin saitin bayanai. Don Eu3, nau'in kwayar halitta guda ɗaya ne aka kwatanta (Branger et al., 2020), don haka kwayoyin halittar da muka haɗa shine keɓantaccen wakilin Eu3.
A duk duniya, wannan bayanan ya ƙunshi shanu 70 M. bovis ware daga 8 nau'in masauki, rarraba a cikin kasashe 12 daga 1985 zuwa 2016. 36 jinsunan an sanya su a matsayin Eu2, 7 jinsunan Eu1, 1 jinsuna ne Eu3, 3 jinsunan ne Af1, 4 jinsunan ne Af2 da 19 ba hadaddun a kasa (details). Cikakkun bayanai (ciki har da lambar shiga) na Mycobacterium bovis da aka yi amfani da su a wannan binciken ana nuna su a cikin Tebu 1 da Ƙarin Tebu 1.
42 sabbin jerin nau'ikan kwayoyin halittar Mycobacterium bovis daga wuraren cutar tarin fuka na dabba na Portugal da aka rarraba sama da shekaru 12 sune cibiyar wannan binciken, kamar yadda ake kula da tsarin cututtukan daji da dabbobi akai-akai 31,36 (Ƙarin Hoto 1). Dangane da hanyoyin da suka biyo baya, waɗannan nau'ikan an keɓe su daga shanu (n = 14), jajayen barewa (n = 16) da boar daji (n = 12) daga 2003 zuwa 2015: tattara da sarrafa dabbobi daidai da ka'idodin ka'idojin da aka ba da shawarar Samfuran nama suna cikin littafin OIE Terrestrial Animal Handbook kuma ana cikin su tare da stein-Jense da ƙwanƙwasa akan Littafin Hannun Dabbobi na OIE. ruwa matsakaici. Ana shuka al'adun a zazzabi na 37 ° C kuma ana bincika girma sau ɗaya a mako na akalla makonni 12. Ana adana mazaunan kai tsaye a cikin maganin glycerol a -80ºC. A cikin zaɓin zaɓi na Mycobacterium (Middlebrook 7H9, BD Diagnostics), ainihin samfuran da aka adana an wuce ta hanyar in vitro guda ɗaya a cikin vitro don samun DNA na shirin WGS. Don wannan, an wadatar da maganin al'ada daskararre tare da 5% sodium pyruvate da 10% ADS (50 g albumin, 20 g glucose, 8.5 g sodium chloride a cikin 1 L ruwa) akan Middlebrook 7H9 a 37°C Sakewa. Bayan makonni 4 na girma, an sabunta matsakaici kuma ana kula da al'ada akai-akai har sai an sami girma. An girbe sel ta hanyar centrifugation, an sake dawo da pellet a cikin 500 µL phosphate buffered saline (PBS), mai zafi a 99 ° C na minti 30, a tsakiya, kuma an adana supernatant a -20 ° C har zuwa WGS. Ana aiwatar da duk hanyoyin a matakin 3 na kayan kare lafiyar halittu.
An shirya ɗakin karatu na WGS guda biyu-ƙarshen genome ta amfani da keɓaɓɓen fihirisar kowane samfurin DNA, kuma yana amfani da Illumina MiSeq (2 × 250 pb) (samfuran 40) da HiSeq (2 × 150 pb) (keɓancewa biyu) fasaha (Eurofins Genomics, Jamus) don yin jerin gwano. Bisa ga umarnin masana'anta, yi amfani da Illumina Genome Analyzer tare da haɗe-haɗe na module mai ƙare biyu don jera DNA ɗin kwayoyin halitta, kuma amfani da Nextera XT DNA Prep Kit daga Illumina don gina ɗakin karatu.
Yin la'akari da bayanan da aka samo daga SRA (n = 12), za'a iya amfani da ganewa na hadaddun clone a matsayin metadata na 30, 41, 43. Lokacin da aka yi la'akari da cikakkiyar kwayar halitta, sai dai Mycobacterium bovis AF2122/97 da Mycobacterium bovis 3601, wanda aka gane 12 da E5, da kuma mambobi na E5. bi da bi, daidai yake da cikakken kwayar cutar ta Mycobacterium tarin fuka H37Rv (lambar shiga NCBI NC_000962.3). Ana yin daidaitawar kwayoyin halitta ta amfani da MAFFT (tsarin daidaitawa da yawa na amino acid ko jerin nucleotide, sigar 7.458) da siga -addfragments48. Sa'an nan, bincika rashi na daban-daban clonal complexes da/ko gaban siffofin SNP.
Sabbin jerin Mycobacterium bovis (n = 42) da kuma ainihin karantawa na daftarin kwayoyin halitta (n = 3) sun daidaita hadaddun tare da ma'anar kwayar halittar Mycobacterium tarin fuka H37Rv ta bututun vSNP da kasancewar gogewa da / ko SNP fasali na clones daban-daban An gudanar da bincike.
Tattara bayanai daga rashin fasalulluka da/ko kasancewar/rashin SNP da bayanan spoligotyping don sanya bayanan genomic zuwa hadaddun clonal daidai. Don daftarin majalissar guda huɗu, ba za a iya yin la'akari da bayanan spoligotyping ba, don haka an haɗa su cikin rukunin "babu rikitarwa".
Gudun aikin bioinformatics wanda wannan aikin ya biyo baya yana farawa daga taron de novo da taswira zuwa dabarun tunani, tare da manufar bincika abubuwan sake haɗuwa da takamaiman nau'ikan polymorphisms na genome. Hoto na 1 yana ba da tsarin tafiyar matakai da ake bi. Don nazarin sake haɗuwa, ana amfani da duk kwayoyin halitta don ƙara ƙarfin abubuwan da aka ambata da alamun da ke da alaƙa.
Domin rage kurakurai a cikin tsararrun jerin jigogi na genome, mun fara samun taron de novo, sannan mu sami alignments masu yawa na asali. A halin yanzu ana samun bututun Unicycler a https://github.com/rrwick/Unicycler49 kuma ana amfani dashi don yin taron de novo na genomes 54 (sabbin jerin 42 da fayilolin fastq 12 da aka dawo dasu daga SRA). A takaice, kafin haɗuwa daga karce, an yi nazarin ingancin karantawa a cikin sigar FastQC 0.11.7 (https://github.com/s-andrews/FastQC), da Trimmomatic version 0.36 (zaɓin "yanke Adafta da sauran takamaiman jerin haske daga karantawa" da "Yanke tushe daga ƙarshen ingancin karatun 2" idan an karanta 0. (http://www.usadellab.org/cms/?page= trimmomatic) 50. Sa'an nan, SPAdes optimiser49 da aka yi amfani da genome taro, da kuma Pilon version 1.1851 da aka yi amfani domin inganta bayan taro. An zaɓi yanayin haɗaɗɗiyar ra'ayin mazan jiya don guje wa taron da ba daidai ba, kuma an bincika girman k-mer kuma an zaɓi tsakanin 20% da 95% na tsawon karatun. Bi ka'idodin SPAdes kuma kuyi la'akari da girman karantawa, share abubuwan da ke ƙasa da 300 bp, kuma kafa 20 karanta zurfin ɗaukar hoto yanke na 52. A cikin tsarin tsarin taron de novo, yankunan genomic irin su proline-glutamate (PE) da proline-proline glutamate (PPE) paralogs ba a cire su ba.
Ana kimanta ingancin taron de novo ta hanyar bututun QUAST (http://quast.sourceforge.net/quast.html), wanda ke sauƙaƙe sabuntawar haɗin gwiwa da kuma M. bovis AF2122/97 genome (lambar shiga NCBI LT708304.1) Taswira (duba Taswirar Ƙari na 1 don ƙimar inganci).
Tare da taimakon bututun vSNP (https://github.com/USDA-VS/vSNP), fayil ɗin FASTQ na sabon tsarin M. bovis daga Illumina sequencing an kwatanta shi da M. bovis AF2122/97 reference genome (LT708304.1)) . Dangane da ingantattun shawarwarin aiki na Kayan aikin Nazarin Halittu (GATK) 53, 54, 55 yi amfani da daidaitattun sigogin tacewa ko bambance-bambancen makin taro don sake daidaitawa. Ana tace sakamakon ta amfani da mafi ƙanƙanta SAMtools mass score na 150 da AC = 2. Hakanan yi amfani da Kraken (http://ccb.jhu.edu/software/kraken/) don duba karatun don kawar da gurɓatawa. Bututun vSNP da aka yi amfani da shi don taswira don tsara dabaru a cikin aikinmu yana nazarin jerin ma'anar SNPs da maƙasudi, sannan kuma ya keɓance yanayin yanayin kamuwa da cuta. Rubutun genome da aka karanta ya fi 99% (Ƙarin Teburin 1).
Don kauce wa kurakuran taswira da kuskuren SNPs, tace wani bambance-bambance a cikin waɗannan lokuta: (1) yana tallafawa da ƙasa da karatun 20, (2) ana samun shi tare da mitar ƙasa da 0.9, (3) yana cikin aƙalla nau'i ɗaya, amma aƙalla akwai rata a cikin wani nau'in. An yi amfani da sigar 2.4.19 (http://software.broadinstitute.org/software/igv/)56 mai duba genomics viewer (IGV) don tabbatar da gani na SNPs da matsayi tare da taswira ko matsalolin daidaitawa. Tun da kwayoyin proline-glutamate (PE) da proline-proline glutamate (PPE) suna da kwafi sosai kuma suna cikin dangin dangi da yawa, ana iya fahimtar su cikin sauƙi ta hanyar tsarin Illumina da rashin taswira, don haka an fi son aikin aikin mycobacterial bioinformatics da aka cire membobin tarin tarin fuka sun tabbatar da hadaddun lokacin amfani da dabarun taswira zuwa jerin SNP. Saboda haka, mun tace kwayoyin PE/PPE da indels daga bincike.
A cewar Bovilist (http://genolist.pasteur.fr/BoviList/), duk SNPs an raba su zuwa nau'ikan aiki. Ana amfani da bututun SnpEff (https://pcingola.github.io/SnpEff/) don haifar da sakamakon SNP (canje-canje masu kama da juna ko mara ma'ana). Ƙirƙiri sabon bayanan Mycobacterium bovis AF2122/97 genome (LT708304.1).
An yi alignment mahara genome ta hanyar amfani da Parsnp v1.2, a halin yanzu ana samuwa a https://github.com/marbl/parsnp57, ta amfani da 69 cikakken genomes / zane-zane (tare da zaɓi -c) da M. bovis AF2122/97 (LT708304.1) ana amfani dashi azaman tunani. An yi jeri guda huɗu masu mahimmanci: kawai mambobi na Eu2 cloning complex (n = 37), ciki har da duk mambobi na tsarin cloning na Turai (n = 44), ciki har da mahaɗin haɗin haɗin gwiwar Turai da Afirka (n = 51), kuma sun haɗa da duk Mycobacterium bovis a cikin wannan binciken (n = 70).
Ainihin daidaitawar da Parsnp ya haifar ana amfani da shi don samar da mafi girman yuwuwar (ML) bishiyar phylogenetic ta amfani da CIPRES Science Gateway v3.3 (http://www.phylo.org/)58 ta amfani da RaxML, da aiwatar da 1000 kwafi.
Ana amfani da algorithms daban-daban guda uku da kayan aikin bioinformatics don bincika kasancewar abubuwan sake haɗawa a cikin layi ɗaya: SplitsTree4 software, Gubbins (launi marar son kai ta hanyar sake haɗuwa a cikin jerin nucleotide) bututun bututun, da RDP4 (shirin ganowa na sake haɗawa, sigar beta 4.101) software.
Hanyar lalatawar da aka aiwatar a cikin SplitsTree4 v4.15.1 (http://www.splitstree.org/) 59 ana amfani da ita don ƙididdige hanyar sadarwar phylogenetic mara tushe, ta amfani da gwajin Phi don tabbatar da ƙididdiga, kuma mahimmin kofa shine p = 0.05. Ana amfani da ainihin ƙididdigar daidaitawa da yawa na Parsnp azaman shigarwa, kuma an gane bazuwar ɓarna azaman ma'aunin cibiyar sadarwa.
Gubbins bututun v2.3.1 (https://github.com/sanger-pathogens/gubbins60 yana gudana tare da sigogi na asali a matsayin wata hanya don kimanta tasirin sake haɗuwa akan Mycobacterium bovis. Algorithm da aka aiwatar a cikin bututun yana sake gina layin clone da ya dace da cikakken genome/draft taro na bayanan bayanan mu da kuma genome AF721) LT708304.1).
A ƙarshe, don tabbatar da taron sake tsarawa da bututun Gubbins ya ba da shawara, algorithms shida da aka aiwatar a cikin RDP467 (RDP61, GENECONV62, Bootscan63, Maxchi64, Chimaera65 da SiScan66) ana amfani da su zuwa ginshiƙi da yawa na Parsnp a ƙarƙashin saitunan tsoho. Mun ƙaddara cewa aƙalla uku na algorithms da aka aiwatar a cikin RDP4 dole ne su ci gaba da nuna sigina mai mahimmanci don tabbatar da kowane taron sake haɗuwa.
Idan akai la'akari da cewa duka Gubbins da software na RDP suna neman siginar sake haɗawa ta hanyar duba ainihin ma'auni masu yawa a cikin taga har zuwa 500 bp, da kuma tabbatar da cewa hada kwayoyin PE/PPE a lokacin taron de novo ba zai tsoma baki tare da siginar sake haɗuwa da aka samu ba, ana yin ƙarin bincike ta hanyar homolinearity Duba kusa da kwayar halitta ta gano abin da ya faru.com. Taswirar synlinear ta amfani da cikakkiyar kwayar halitta an gina ta ta amfani da daidaitawar MAUVE-multi-genome (http://darlinglab.org/mauve/mauve.html) don ware jujjuyawar kwayoyin halitta na gida ko jujjuyawa. Bugu da kari, an yi amfani da dukkan kwayoyin halitta don yin nazarin homolinearity akan jerin amino acid ta hanyar sabar yanar gizo ta SyntTax (https://archaea.i2bc.paris-saclay.fr/SyntTax/).
Ƙarin zurfin bincike na bayanan kwayoyin halitta da aka samo daga tsarin tarin fuka mai yawa na Portuguese shine duba polymorphism na kwayoyin halitta da aka ambata a cikin wallafe-wallafe. Wadannan kwayoyin halitta sune 37,38 da kwayar halittar 3R da aka samu ta kakannin MTBC ta hanyar HGT (gyaran DNA, maimaitawa da sake hadewa) sassan tsarin 39. Yi amfani da ClustalX v2.1 (http://www.clustal.org/clustal2/) da kuma amfani da DnaSP v6.12.03 (http://wwwsp.ub) diversity da nucleide (π) da Tajima's D tsaka-tsakin ma'aunin gwaji.
Matsakaicin yuwuwar (ML) bishiyar phylogenetic bisa 69 Mycoplasma bovis keɓewa da ma'anar kwayoyin halitta an samu (Hoto 2A). Idan aka kwatanta da bishiyoyi masu tushe guda ɗaya ko bishiyoyi masu yawa, wannan dabarar tana ba da damar samar da bishiyoyi masu ƙarfi waɗanda ba su kama da sauye-sauyen dukkanin kwayoyin halitta ba don haka suna nuna ƙananan ikon nuna bambanci tsakanin nau'in 68,69. Tsarin topological na bishiyar ML yawanci yana daidai da hadadden rarrabuwa na clones. Kwayoyin halittar Eu2 an taru a cikin reshe, kuma genome na Af1 shima yana tattare tare (Hoto 2A). Sakamakon kuma ya yi daidai da sanannen alaƙar juyin halitta na Mycobacterium bovis, wato, akwai babban bambanci tsakanin memba na Eu1 da ƙungiyar da ta ƙunshi duk sauran rukunin clonal da kwayoyin halitta, amma ba a fayyace rukunin clonal 30 ba. Ƙananan rashin daidaituwa tsakanin hadaddun clonal da dangantakar da aka lura a kan bishiyar phylogenetic za a iya bayyana su ta hanyar gaskiyar cewa an kwatanta hadaddun clonal bisa takamaiman yankuna na kwayoyin halitta, yayin da bishiyar phylogenetic ta dogara ne akan alignments masu yawa na kwayoyin kwayoyin halitta masu wakiltar dukkanin kwayoyin halitta.
Matsakaicin yuwuwar bishiyar phylogenetic (GTR) an gina ta bisa tushen daidaitawar kwayoyin halittar Mycobacterium bovis genome kafin (A) da bayan (B) kawar da wurin sake hadewa. Launukan reshe suna wakiltar hadaddun ƙwayar cuta ta Mycobacterium bovis: Turai 1 shuɗi ne, Turai 2 ja ne, Turai 3 shuɗi ne, Afirka 1 orange ne, Afirka 2 kuma kore ce. An dasa itacen kuma an zana shi zuwa ma'auni, kuma ana auna tsayin reshen a matsayin maye gurbin kowane wuri.
An kwatanta hadadden tarin tarin fuka na Mycobacterium a matsayin wanda ya samo asali, kuma yawancin shaidun da aka tara a tsawon shekaru suna goyan bayan ra'ayin cewa HGT mai gudana da abubuwan sake haɗuwa ba za su faru a matakin ganowa na MTBC15,17,18 ba.
Ayyukan da suka gabata sun nuna cewa za a iya samun iyakancewar haɗuwa tsakanin nau'ikan MTBC20,21, yayin da wasu suka kasa gano abubuwan sake haɗuwa da aunawa70,71. Sake tattauna wannan batu tare da mayar da hankali kan Mycobacterium bovis, wanda ya bambanta da aikin da ya gabata wanda kawai ya yi la'akari da tarin fuka na Mycobacterium 70,71; ko yi la'akari da MTBC gabaɗaya, tare da kusan babu M. bovis da ke wakiltar 20; ko kawai la'akari da ƙayyadaddun ɓangarorin shanu. Bayanin Mycebceria na Mycebceria, a cikin wannan aikin, akwai juzu'i na 70, suna wakiltar dukkan hadaddun murabba'i, wanda aka yi amfani da shi wajen tallatawa. An ƙididdige saitin bayanan bisa ga matakan tarawa huɗu: (1) membobin Eu2, (2) duk membobi hadaddun clone na Turai (watau Turai), (3) hadaddun clone na Turai da Afirka (Eu + Af) da (4) Duk Tarin bayanai (ciki har da kwayoyin halittar da ba a haɗa su cikin kowane rukunin cloning da aka riga aka kwatanta).
Don ci gaba da nazarin wannan hasashe, an yi hanyar sadarwa mai tsaga-bazuwar don tantance rashin sake haduwa tsakanin kwayoyin halitta, saboda wannan hanya na iya hango alakar kakanni tsakanin mutane da kuma nuna siginar dabi'ar halitta masu karo da juna. Duk bayanan bayanan guda huɗu a cikin bincike sun tabbatar da kasancewar madaukai a cikin hanyar sadarwa (wato, wuraren da ba su haɗuwa a cikin bishiya ɗaya), amma gwajin Phi ba shi da goyon bayan ƙididdiga (Eu2, p = 0.0956; Turai, p = 0.1637; Eu + Af p = 0.2774; duk bayanan da aka saita p1 = 0.245) wanda ya ba da shaida mara kyau ga abubuwan da suka faru. 3 A-D).
A cikin Turai 2 genomes (n = 37) (A), kwayoyin halittar Turai (n = 44) (B), Turai da Afirka (n = 51) (C) da duk bayanan da aka saita (n = 70) (D).
Bayan wannan bincike, da kuma yin la'akari da abubuwan lura na cyclic a cikin dukkanin cibiyoyin sadarwa, an yi amfani da algorithm na sake ginawa a cikin bututun Gubbins don sake gina layin clonal kuma don ƙarin kimanta tasirin sake haɗuwa a kan M. bovis genome. Nuna adadin adadin abubuwan da suka faru na sake haɗawa, yawancin waɗanda suka faru a cikin rassan ƙarshen (wato, a cikin kwayar halitta guda ɗaya) (Table 2). Waɗannan alamun suna nuna daidaiton duk saitin bayanai kuma suna nuna cewa yawan abubuwan sake haɗuwa shine sau 200 zuwa 300 na maye gurbi. Da zarar ma'aunin rho/theta wanda ke wakiltar ƙimar dangi na sake haɗuwa da ma'anar maye gurbi akan reshe ya bayyana tsakanin 0.0037 da 0.0056 (Table 3). Kwanan nan, aikin da aka buga na nau'in 38 M. bovis ya nuna darajar rho / theta (rho / theta = 0.1) fiye da abin da aka samu a cikin wannan bayanan, amma aikin Patane da abokan aiki sunyi amfani da taro na tushen tunani don ƙaddamar da sigogi na sake haɗawa , A tsarin daki-daki, saboda tsarin taro, an hade shi tare da abubuwan da suka faru na rebination da yawa.
Na gaba, ma'auni na r / m yana wakiltar nau'in bambancin sake haɗuwa da gabatarwar maye gurbi, kuma matsakaicin ƙimarsa yana tsakanin 0.025 da 0.037, yana nuna cewa idan aka kwatanta da maye gurbi, sake haɗuwa yana da ƙananan tasiri na gaba ɗaya akan nau'in jinsin M. bovis (Table 3)). Don kwatance mai yawa, an yi amfani da irin wannan hanyar don ƙididdige ma'aunin r/m na MTBC dataset wanda ya ƙunshi nau'ikan kwayoyin halitta 23, yana nuna matsakaicin ƙimar 0.48620, yayin da Patane da abokan aiki' 38 M. bovis dataset, ya tabbatar da matsakaicin ƙimar shine 0.98. A cikin binciken farko, kawai biyu daga cikin kwayoyin halitta na 23 da aka haɗa a cikin aikin M. bovis (M. bovis BCG da kuma ma'anar tunani), don haka ƙimar da aka samu na iya zama mai banƙyama saboda ƙaddamar da ƙwayar ƙwayar cuta ta M. tuberculosis. A cikin rahoton na biyu, an gano yawan mutanen Mycobacterium bovis da aka yi nazari a kai daga Amurka da kuma dabbobi. Sabanin haka, a cikin saitin bayanan mu, ana wakilta ƙarin wurare na yanki da nau'ikan masu masaukin baki, kuma ana amfani da kwayoyin halittar da aka haɗa su cikin rukunin clonal daban-daban waɗanda ke da halaye iri-iri na yawan jama'a, don haka samun zurfin ilimin Yawan jama'a. Bambancin r / m matsakaicin ƙimar da aka samu tare da saitin bayanan mu ya dace da ra'ayi cewa matakin sake haɗuwa ya bambanta sosai tsakanin layin da aka ba da nau'in nau'in taxonomic guda ɗaya, don haka waɗannan sakamakon sun nuna cewa M. bovis clone complex na iya nuna bambance-bambancen sake haɗuwa da tasiri kuma kamar yadda Didelot & Maiden72 suka nuna. Duk da haka, faɗaɗa wannan bayanan da aka saita ta hanyar haɗa da adadi mai yawa na M. bovis genomes zai ba da damar ƙarin fayyace wannan batu. Dukansu sigogin r / m da rho / theta suna nuna bambancin tsakanin rassan, kuma wannan sakamakon ya dace da rahotanni game da wasu nau'in kwayoyin cuta72,73.
A ƙarshe, don tabbatar da abubuwan da suka faru na sake tsarawa da bututun Gubbins suka gano, an yi amfani da algorithms daban-daban guda shida a cikin software na RDP4 don gwada kansu daban-daban na kwatance-kwatance daban-daban. A duk duniya, ƙasa da rabin abubuwan da Gubbins suka gano sun tabbatar da RDP4 (Tables 4 da 5). Idan aka yi la'akari da duk saitin bayanan, an tabbatar da abubuwan sake haɗuwa guda uku, biyu sun haɗa da nodes na ciki da ɗayan da ke tattare da kwayar halitta guda ɗaya a cikin reshen tasha, wanda ba za a iya sanya ɗakunan clonal ba (Tables 4 da 5). Gano abubuwan da suka faru a rassan tasha na iya nuna cewa har yanzu ana ci gaba da haɗawa a cikin nau'ikan M. bovis na zamani ko kuma sakamakon ya ɓace70. A cikin wannan yanki na sake hadewa, kusan 20% na matsayi suna da nucleotides da ba a bayyana ba (N), don haka yana rinjayar siginar sake haɗuwa (Ƙarin Hoto 2). Bugu da kari, wannan yanki yana shafar kwayar halittar rrs, yana sanya 16S ribosomal RNA da ake tsammanin za a kiyaye shi sosai, don haka wannan siginar sake haɗawa na iya zama sakamakon kurakuran da aka tsara ko rashin daidaituwa. Sa'an nan dukan genome alignment tsakanin Mb0003 da Mycobacterium bovis AF2122/97 da aka yi, da kuma wanzuwar undefined nucleotides da SNPs aka tabbatar, don haka da yiwuwar matsaloli alaka da ba daidai ba jeri ba saboda nazarin halittu bayanai aiwatar a cikin wannan aikin Ya bayyana bayan koyon shirin.
Ba a sami raguwa ko nucleotides da ba a bayyana ba a cikin yankunan sake hadewa na nodes na ciki (Figures 4 da 5). Game da waɗannan abubuwan da suka faru, ɗayan ya ƙunshi nau'in halittar Eu2 kawai kuma yana shafar kwayar pks12, wanda ke ɓoye yuwuwar polyketide synthase; yayin da ɗayan ke rajista a cikin Eu1 genome kuma yana shafar ƙwayar narX wanda ke ɓoye yiwuwar nitrate reductase (Table 4). Gabaɗaya, ƙididdigar sake haɗawa ya nuna cewa akwai ƙayyadaddun adadin rarrabuwa na sake haɗawa tare da tallafin ƙididdiga, kuma alamomin da aka ƙididdige sun nuna cewa sake haɗawa yana da ƙarancin tasiri akan layin M. bovis. Ana sa ran siginar sake haɗawa ya zama ƙasa, amma yana da mahimmanci don bambanta siginar juyin halitta na gaskiya daga hayaniyar baya, wanda shine aiki mai wuyar gaske. Don rage siginar amo da aka gabatar ta hanyar haɗin kai na tushen tunani da matsalolin rashin daidaituwa na 70, 71, duk sauran sai dai cikakken genome an tattara su daga karce, kuma an duba ingancin taro kuma an tabbatar da ingancin bututun QUAST (Ƙarin Teburin 1) . Bugu da ƙari, an gudanar da jerin ƙarin bincike don samar da ƙarfi da daidaito na binciken gabaɗaya. Don haka, an kimanta ingancin jerin kwayoyin halittar narX da pks12 ta hanyar karanta taswira akan Mycobacterium bovis AF2122/97. Matsayin SNP da aka ba da shawarar a cikin yankin sake haɗawa an tabbatar da shi ta hanyar yin amfani da ka'idodin da aka ambata a cikin sashin hanyar (akalla karatun 20 da 0.9 sauyin canji). An tabbatar da polymorphism na kwayar halittar narX a cikin kwayoyin halitta guda biyu (Mb1792361 da Mb7240415; 2.3%) da kwayoyin halittar pks12: Mb0891, Mb1711, Mb1789, Mb1870, Mb17046, Mb1756, da Mb1256. Duk da haka, don genome Mb2043, shida daga cikin matsayi takwas ba su hadu da ma'aunin zurfin karantawa ba saboda SNP yana goyan bayan matsakaicin 17 karantawa, wanda ke ƙasa da ƙimar yankewar da aka kafa na 20. Saboda haka, ana iya tabbatar da sake haɗuwa da kwayoyin halitta guda shida (8.6%) a wannan rukunin kwayoyin halitta (Figures 4 da 5).
Cikakkun gani na daidaitawar yanki na sake haɗawa na Mycobacterium bovis dataset yana rinjayar kwayar narX wanda ke ɓoye yiwuwar nitrate reductase. Ba a sami gibi ko nucleotides da ba a bayyana ba a cikin yankin sake haɗawa na nodes na ciki. An yi rajistar wannan taron musamman a cikin genome na Eu1. An kimanta ingancin jeri na narX ta hanyar tsara karatun Mycobacterium bovis AF2122/97. Tabbatar da shawarar SNP da aka ba da shawarar a cikin yankin sake haɗawa ta hanyar yin amfani da ma'auni da aka ambata a cikin sashin hanyar (akalla 20 karatu da 0.9 sauyi sau). Polymorphism na kwayar halittar narX an tabbatar da shi sosai a cikin kwayoyin halittar Mb1792361 da Mb7240415 (2.3%).
Cikakken hangen nesa na daidaitawar yanki na sake hadewa na Mycoplasma bovis dataset wanda ya shafi kwayar pks12. Ba a sami gibi ko nucleotides da ba a bayyana ba a cikin yankin sake haɗawa na nodes na ciki. Dangane da lamarin da ya shafi kwayar pks12 da ke sanya yuwuwar polyketide synthase, ya ƙunshi kwayar halittar Eu2 kawai. An kimanta ingancin jerin pks12 ta hanyar karanta taswirar Mycobacterium bovis AF2122/97. Tabbatar da shawarar SNP da aka ba da shawarar a cikin yankin sake haɗawa ta hanyar yin amfani da ma'auni da aka ambata a cikin sashin hanyar (akalla 20 karatu da 0.9 sauyi sau). An tabbatar da cikakkiyar ma'anar halittar halittar halittar Mb0891, Mb1711, Mb1789, Mb1870, Mb1758, Mb2043, da Mb1960.
Kwayoyin PE da PPE suna da yankuna masu maimaitawa waɗanda ke da sauƙin karantawa ta hanyar Illumina sequencing da rashin taswira, don haka yawanci ana share su daga aikin bioinformatics na membobin tarin fuka kawai lokacin amfani da dabarun taswira-zuwa-jeri. Abubuwan da aka yi amfani da su na sake haɗawa a cikin wannan aikin sun dogara ne akan taron de novo ba tare da tace PE/PPE ba. Mun yi imanin cewa ta hanyar aiwatar da hanyoyi daban-daban na haɗin gwiwa da algorithms ta hanyar SplitsTree, Gubbins bututun da software na RDP4, dabarun da aka yi amfani da su suna da ƙarfi don sarrafawa da tace yankunan da aka sake tsarawa ta hanyar siginar kuskure. Duk da haka, don kawar da tsangwama na PE / PPE gene a kan Gubbins da software na RDP4 don gano gungun SNP, sabili da haka ganewar yankunan sake hadewa sun ba da shawarar yin tasiri ga kwayoyin narX da pks12, an bincika unguwar wadannan kwayoyin halitta (Ƙarin Fig. 3-5). A cikin M. bovis AF2122/97, an raba kwayoyin halittar narX ta narK2 da Mb1764c, yayin da pks12 ke kewaye da Mb2075c e Mb2073c (Ƙarin Hoto 3-5). Taswirar da aka ƙirƙira ta amfani da taswirar synline na MAUVE na cikakkiyar kwayar halitta tana ba da bayanai game da kiyaye jerin halittu da sake tsarawa, yana nuna tubalan collinear guda huɗu, kuma babu alamun juyawar kwayoyin halitta ko juyewa. Bugu da kari, binciken da aka yi tare da jerin amino acid ya tabbatar da homology a cikin dukkan cikakkun kwayoyin halittar jiki, kuma ba a sami PE/PPE a cikin yankunan da ke kusa da narX ko pks12 ba. Don narX, kwayar halitta guda ɗaya (Mb0030) tana da ƙarancin ma'auni na ma'ana saboda an gano kwayar halittar narX a matsayin gutsutsaye guda biyu (gutsuwa 1891 da 1890). Don pks12, saboda kamanceceniya, Mb0030 da Mb003 sun baje kolin ƙananan ma'aunin daidaitawa, yayin da aka gano pks12 a cikin guda biyu da uku, bi da bi, wakiltar yankuna daban-daban na furotin (Ƙarin Hoto 3-5). Yin la'akari da wannan bayanin, kuma Gubbins da software na RDP4 duka suna yin bincike, duba ainihin madaidaicin madaidaicin matsakaicin 500 bp a cikin taga, mun tabbatar da cewa PE/PPE gene ba zai tsoma baki tare da siginar sake haɗawa da ke shafar narX da pks12 ba.
Ko da yake ana iya la'akari da siginar sake haɗuwa da aka gano a cikin wannan saitin bayanai a matsayin saura, gaskiya ne cewa sake haɗuwa a cikin M. bovis ba za a iya kawar da shi ba, don haka ya kamata a ci gaba da kasancewa batun ƙarin bincike, wanda dukkanin kwayoyin halitta daga cututtuka daban-daban na annoba suna jera su zuwa mahimmanci.
Kwatanta bishiyoyin phylogenetic na ML da aka samu kafin da kuma bayan gyaran gyare-gyare (Figure 2A, B) bai haifar da canje-canje masu mahimmanci a cikin dangantakar da ke da alaƙa ba, kuma an haɗa nau'in M. bovis a cikin rukuni ɗaya.
Bayan taswira 42 sabon jerin M. bovis ya karanta tare da ma'anar kwayar halittar M. bovis AF2122/97, an sami daidaitawar SNP mai ɗauke da matsayi na 1816 polymorphic. Yawancin SNPs (87.1%) suna cikin yanki na coding, kuma kwayoyin da aka shafa ana nuna su bisa ga nau'ikan aikin da aka nuna a cikin Bovilist (Hoto 6A, B). Yin la'akari da yawan adadin kwayoyin halitta a cikin kowane nau'i na aiki, kwayoyin halitta a cikin nau'in "lipid metabolism" sun nuna karin SNPs, sannan "bangon tantanin halitta da tsarin tantanin halitta" da "tsakanin metabolism da numfashi", suna nuna cewa suna cikin juyin halitta na M. bovis.
Ƙididdigar matsayi na bayanan M. bovis daga Portugal (n = 42). Jimlar adadin SNPs masu rijista da kwayoyin halitta da abin ya shafa don kowane nau'in aiki (A). Jimlar adadin canje-canje masu kama da juna da mara ma'ana da aka yiwa rajista ta nau'in aiki (B).
A kan sikelin duniya, matsakaicin dN/dS ya fi 1.5, wanda ke nuna cewa matsin juyin juyin halitta na duniya shine kawar da jihar kakanni kuma yana wakiltar tabbataccen yanayin zaɓin zaɓin tsarkakewa. A cikin nau'o'in "virulence, detoxification, adaptation", "jerin shigarwa da phages", da "proteins na tsari", fiye da kashi biyu bisa uku na SNPs ba su da alaƙa (Hoto 6B).
A cikin dukkan nau'o'in, akwai kwayoyin halitta tare da SNPs masu yawa, wanda ya haifar da matsakaicin matsakaicin maye gurbin (wato, matsakaicin SNP a kowace jinsi) fiye da 1 (Hoto 6A). Pks12 (Mb2074c) tare da 15 SNPs da fas (Mb2553c) tare da 8 SNPs suna da ƙimar maye gurbi. Duk waɗannan kwayoyin halitta suna da hannu a cikin metabolism na fatty acid. Halin pks yana ɓoye polyketide synthase (PKS), wanda shine multifunctional enzyme wanda ke da hannu a cikin biosynthesis na bangon cell mycobacterial na lipid biosynthesis74,75. Wannan kwayar halitta tana ɓoye polypeptide multifunctional wanda ke da hannu a cikin haɗin mycoketides74,76. Halin fas yana shiga cikin haɗin mycolic acid. Duk waɗannan kwayoyin halitta suna taka muhimmiyar rawa a cikin biosynthesis na bangon tantanin halitta a cikin hulɗa da mai gida.
Don ci gaba da nazarin juyin halittar Mycobacterium bovis, an yi nazarin nau'i biyu na takamaiman kwayoyin halitta. Ayyukan da aka buga a baya ta amfani da tsarin tsari da hanyoyin phylogenetic sun gano kwayoyin halittar da kakannin MTBC suka samu ta hanyar HGT kafin rarrabuwa37,38. Wadannan kwayoyin halitta an jera su a cikin Ƙarin Table 2. An bincika rarraba SNP na jimillar kwayoyin 77 da ke da alaƙa da HGT, kuma an gano wuraren 26 polymorphic, wanda a mafi yawan lokuta (78%) ya haifar da canje-canje marasa daidaituwa (NS) (Ƙarin Table 2). Ayyukan da suka gabata akan kwayoyin halittar MTBC sun nuna cewa yankin HGT mai sakawa yana nuna mafi girman rabo na NS SNP idan aka kwatanta da sauran kwayoyin halitta. Idan mutum yana tunanin cewa waɗannan yankuna na sake haɗawa sun samo asali ne ta kakanni na MTBC, sabili da haka, suna wakiltar tsohuwar polymorphisms, to ana sa ran adadin sauye-sauye masu kama da juna zai kasance mafi girma, saboda ana sa ran za a kawar da maye gurbin NS ta hanyar zaɓi mara kyau saboda canje-canjen amino acid na iya canza aikin furotin. Sabili da haka, sakamakonmu yana nuna cewa sakamakon aiki na iya samo asali daga maye gurbin kwayoyin halitta irin na HGT, wanda ke nuna mahimmancin su ga bambancin kwayoyin halitta masu dacewa.
A layi daya da wannan bincike, kwayoyin halittar da ke ɓoye sassan tsarin 3R (gyaran DNA, maimaitawa, da sake haɗawa) an bincika su sosai daidai da jerin abubuwan da dos Vultos and collaborators suka buga a baya (2008)39. Ba za a iya lura da musayar ɓangarorin DNA iri ɗaya ba kai tsaye, kodayake yana iya zama tsari akai-akai lokacin da ƙwayoyin cuta masu alaƙa suka shiga, kamar yanayin wannan saitin bayanan; Bugu da ƙari, wannan tsari na iya zama mabuɗin hanyoyin gyaran DNA72, don haka Yi rawar jiki a cikin sake hadewa. An gano jimlar 26 polymorphic matsayi da aka rarraba ta hanyar 54 genes (Ƙarin Teburin 3). A cikin wannan saitin kwayoyin halitta, canje-canjen NS sun kai kusan kashi 65% na sakamakon, wanda ya yi daidai da rahotannin da suka gabata game da nau'ikan tarin fuka na Mycobacterium.
Lokacin aikawa: Oktoba-21-2021