Ua tsaug rau koj tuaj saib Nature. Qhov browser version koj siv tau txwv kev txhawb nqa rau CSS. Rau qhov kev paub zoo tshaj plaws, peb pom zoo kom koj siv qhov browser tshiab (lossis kaw qhov kev sib raug zoo hauv Internet Explorer). Nyob rau tib lub sijhawm, txhawm rau ua kom muaj kev txhawb nqa txuas ntxiv, peb yuav tso saib cov chaw tsis muaj cov qauv thiab JavaScript.
Genome sequencing tau txhawb nqa kev tshawb fawb txog kab mob sib kis, nthuav tawm cov kab mob sib kis, kab mob, kev sib cuam tshuam ntawm tus tswv tsev, thiab cov txheej txheem evolutionary yuam rau cov kab mob. Lub Mycobacterium tuberculosis complex (MTBC) suav nrog Mycobacterium bovis yog ib qho ntawm nws cov tsiaj hloov pauv uas ua rau mob ntsws (TB) hauv cov tsiaj txhu hauv av, thiab yog ib qho qauv ntawm cov kab mob evolution. Ib yam li lwm cov tswv cuab MTBC, Mycobacterium bovis yog suav tias yog ib qho nruj nruj, maj mam hloov cov kab mob, thiab tsis muaj qhov pom tseeb ntawm kev rov ua dua lossis hloov cov noob kab rov tav. Hauv txoj haujlwm no, peb siv cov kev sib piv genomics rau tag nrho genome sequence (WGS) dataset muaj li ntawm 70 nyuj M. los ntawm txawv pedigrees (Tebchaws Europe thiab Africa) kom nkag siab txog cov noob caj noob ces ntau haiv neeg nyuj M. Evolutionary hwj chim. Peb txoj kev sib txawv yog siv los kwv yees cov cim qhia ntawm kev tsim kho dua tshiab. Thoob plaws ntiaj teb, qee qhov kev sib koom ua ke me me tau raug txheeb xyuas thiab lees paub los ntawm ob txoj kev ywj pheej nrog kev txhawb nqa. Txawm li cas los xij, piv nrog kev hloov pauv, recombination muaj qhov cuam tshuam tsis zoo rau kev sib txawv ntawm M. bovis (tag nrho r / m = 0.037). Qhov sib txawv r / m nruab nrab tau txais nyob rau hauv lub clonal complex ntawm Mycobacterium bovis nyob rau hauv peb cov ntaub ntawv teev yog raws li lub tswvyim dav dav hais tias cov degree ntawm recombination yuav txawv heev ntawm cov kab uas muab rau tib hom taxonomic. Raws li txoj haujlwm no, kev rov ua dua hauv Mycobacterium bovis tsis tuaj yeem txiav tawm, yog li nws yuav tsum yog qhov kev siv zog ntxiv rau yav tom ntej kev tshawb fawb genomics piv, uas WGS ntawm cov ntaub ntawv loj los ntawm cov xwm txheej sib kis thoob ntiaj teb yog qhov tseem ceeb. Ib qho kev soj ntsuam ntxiv tau ua tiav ntawm cov ntaub ntawv me me ntawm Mycobacterium bovis cov ntaub ntawv teev tseg (n = 42) los ntawm ntau tus tswv tsev TB, thiab ntau dua 1,800 loci tau txheeb xyuas, ntawm qhov tsawg kawg yog ib hom kab mob pom ib zaug nucleotide polymorphism (SNP). Feem ntau (87.1%) yog nyob rau hauv cheeb tsam coding, thiab lub ntiaj teb no piv ntawm non-synonymous kev hloov (dN/dS) ntawm synonymous hloov ntau tshaj 1.5, qhia tau hais tias zoo xaiv yog ib qho tseem ceeb evolutionary quab yuam exerted ntawm M. bovis. Ib qho kev faib ua feem ntau ntawm SNPs tau kuaj pom nyob rau hauv cov noob nplua nuj nyob rau hauv kev ua haujlwm ntawm "lipid metabolism", "cell phab ntsa thiab cellular processes", thiab "intermediate metabolism thiab respiration", qhia lawv lub peev xwm hauv biology thiab evolution ntawm Mycobacterium bovis tseem ceeb. Kev saib ze dua ntawm cov noob hauv MTBC cov poj koob yawm txwv uas nquag hloov cov noob kab rov tav thiab suav nrog hauv 3R (DNA kho, rov ua dua, thiab rov ua dua) qhov system qhia txog lub ntiaj teb qhov nruab nrab qhov tsis zoo ntawm Taijima's D nruab nrab qhov kev sim, uas qhia tau hais tias yav dhau los xaiv scanning Lub bottleneck tsis ntev los no tom qab cov pej xeem nthuav dav tseem yog lub ntsiab evolution ntawm cov kab mob hauv lub ntiaj teb. tus tswv tsev.
Mycobacterium tuberculosis complex (MTBC) yog ib qho kev vam meej tshaj plaws ntawm cov kab mob kab mob thiab ib qho xwm txheej ntawm cov kab mob evolution. Nws cov tswvcuab qhia tau hais tias muaj qhov tsis txaus ntseeg siab ntawm nucleotide tus kheej ntawm qib genomic (> 99%) 1,2 . MTBC ecotypes sib txawv tuaj yeem ua rau tuberculosis (TB), uas yog kab mob granulomatous, nyob rau hauv ntau hom hom tsiaj los ntawm micro-mammals rau tib neeg3,4,5. Tam sim no, lub complex suav nrog tib neeg [M. Tuberculosis (Mtb), Mycobacterium africanum] thiab tsiaj-adapted pathogens (Mycobacterium bovis, Mycobacterium capitum, Mycobacterium pinnipedum, Mycobacterium microtobacter, Mycobacterium mongee, Mycobacterium miysani, Mycobacterium surika, "Bacillus "5" 6. M. canettii (tseem hu ua "Nodobacter glabrata") Qhov nruab nrab nucleotide tus kheej nrog cov mycobacteria tau hais los saum no yog 98%, thiab kev sib piv genomics ua haujlwm tau pom tias M. canettii thiab lwm tus ntawm MTBC tsis ntev los no tau sib txawv los ntawm cov poj koob yawm txwv.7 Xav txog lub tswv yim no, qee tus sau ntawv hu ua M. canetti.
MTBC tau piav qhia raws li ib qho kev nruj nruj, thiab nws cov qauv pej xeem tau tswj hwm kom meej los ntawm kev txo qis ntau haiv neeg, lub cev tsis muaj zog, kev xaiv scanning, thiab caj ces drift9,10. Piv txwv li txoj kev nruj nruj clonal evolution, xws li ploj lawm polymorphisms tsis tuaj yeem rov qab los ntawm recombination. Raws li qhov kev pom zoo no, cov xwm txheej sib law liag ntawm genomic deletion ntawm thaj chaw sib txawv (RD) thiab TbD1 (Mtb tshwj xeeb tshem tawm 1 cheeb tsam) tau raug npaj ua cov cim molecular ntawm MTBC evolution2,5,11. Comparative genomics thiab tag nrho genome sequencing (WGS) ua hauj lwm txhawb kev faib cov tib neeg-hloov cov tswv cuab rau hauv cuaj kab (Mycobacterium tuberculosis L1 txog L4, L7, thiab L8; thiab Mycobacterium africanum L5, L6, thiab L9), kab L2 mus rau L4 sib koom tshem tawm thaj tsam TbD1.12, 111. Tsis tas li ntawd, cov tswv cuab ntawm cov tsiaj yoog tau raug thov kom sib koom cov poj koob yawm txwv, uas yog txhais los ntawm cov kev tshem tawm tshwj xeeb hauv RD7, RD8, RD9 thiab RD102, 5, thiab 14.
Kab rov tav hloov pauv cov noob (HGT) thiab cov xwm txheej sib xyaw ua ke yog suav tias tsis tshua muaj thiab tshwm sim hauv cov poj koob yawm txwv ntawm MTBC, tsis yog cov keeb kwm sib txawv ntawm tag nrho MTBC cov tswv cuab15,16,17. Ob tsab ntawv ceeb toom thaum ntxov los ntawm Hughes thiab cov neeg koom tes (2002) thiab Gutacker thiab cov neeg koom tes (2006) tau qhia tias cov xwm txheej sib xyaw ua ke tuaj yeem pab tsim cov polymorphisms uas cim tshwj xeeb loci hauv M. tuberculosis strains18,19. Cov laj thawj rau qhov pom tseeb tsis muaj kev sib koom ua ke hauv MTBC yog: (1) cov txheej txheem kev siv tshuab thiab tsis muaj peev xwm ntawm HGT; (2) qhov tsis tshua muaj ntawm HGT cov xwm txheej; (3) tsis muaj caij nyoog ntawm kev sib koom ua ke hauv MTBC niche14,17. Tsis ntev los no, qee qhov kev tshawb fawb tag nrho-genome sequencing (WGS) tau siv rau MTBC strain 20 thiab Mycobacterium bovis 21 tau muab pov thawj ntawm kev sib xyaw ua ke, thawj zaug qhia tias MTBC hom kab mob feem ntau pauv cov DNA me me, tab sis vim muaj qee qhov sib txawv nucleotide sib txawv, cov xwm txheej no tseem tsis tau pom.
Mycobacterium bovis yog MTBC cov tswv cuab zoo tshaj plaws los ntawm cov tsiaj nyeg (feem ntau yog nyuj), txawm hais tias nws tseem tuaj yeem raug cais tawm ntawm cov tsiaj qus dawb thiab fenced4,22,23,24. M. bovis evolved mus rau tsib loj clonal complexes [European 1 (Eu1), European 2 (Eu2), European 3 (Eu3), African 1 (Af1) thiab Africa 2 (Af2)], raws li lub spoligotyping profile, tshwj xeeb deletions Thiab ib leeg nucleotide polymorphisms (SNP2, 2, 2, 2, 5, 2, noob. Cov clonal complexes qhia txog cov qauv sib txawv ntawm Mycobacterium bovis cov pej xeem thiab nws cov koom haum nrog thaj chaw. Tsis tas li ntawd, kev ua haujlwm WGS tsis ntev los no tau ua los ntawm Zimpel thiab cov neeg koom tes (2020) tau tsim cov phylogeny raws li SNP ntawm Mycobacterium bovis, nrog ntau dua 1,900 genomes, qhia tias muaj tsawg kawg yog plaub kab sib txawv (lub npe Lb1 txog Lb1 rau Lb4), lawv tsis yog tag nrho raws li qhov kev txiav txim siab yav dhau los. Cov kws sau ntawv no tau ua qhov kev soj ntsuam sib txawv ntawm phylogeny thiab molecular dating, tab sis tsis tau kawm recombination30.
Kev ua haujlwm dhau los uas siv cov txheej txheem sib txawv, xws li spoligotyping, MIRU-VNTR (mycobacterial interspersed repeat unit-variable tandem repeat number), thiab SNP typing tsis ntev los no tau qhia txog qee theem ntawm kev sib txawv ntawm caj ces ntawm M. bovis strains 31,32,33, 34,35. Kev sib txawv ntawm kev sib txawv ntawm caj ces tau dhau los ua ib qho tseem ceeb hauv kev tshawb fawb txog kab mob sib kis, uas yog pab tau rau kev nkag siab tob txog cov kab mob, kab mob thiab kis kab mob. Qhov tshwm sim ntawm WGS txoj kev muab qhov ua tau los nthuav tawm cov kev hloov pauv ntawm kev tsav tsheb los ntawm Mycobacterium bovis genome nyob rau hauv cov txheej txheem ntawm kev hloov kho thiab ua kom tsis tu ncua rau cov tswv tsev sib txawv thiab cov xwm txheej sib kis.
Hauv kev ua haujlwm no, peb siv qhov sib piv genomic tsom xam ntawm ntau yam Mycoplasma bovis datasets (n = 70), suav nrog kev sib cais los ntawm cov chaw sib txawv clonal, kom nkag siab txog cov txheej txheem evolutionary ntawm Mycoplasma bovis, tshwj xeeb tshaj yog los daws kev sib raug zoo ntawm phylogenetic kev sib raug zoo thiab kev sib koom ua ke. Raws li ib qho ntxiv rau qhov kev tshuaj ntsuam no, cov ntaub ntawv me me ntawm M. bovis isolates (n = 42) tau txais los ntawm thaj chaw zoo ntawm ntau tus tswv tsev tuberculosis hauv Portugal 31,36 tau tshawb nrhiav ntxiv rau infer tsis muaj tus kheej Qhov sib npaug ntawm qhov sib npaug ntawm qhov kev nkag siab (dN) rau synonymous (dS) nucleotide kev hloov pauv ntawm qhov tshwj xeeb, raws li qhov tshwj xeeb ntawm cov kev hloov pauv ntawm nucleotide. cov ntaub ntawv, lawv yog 37,38 tau los ntawm MTBC cov poj koob yawm txwv los ntawm HGT, thiab encode 3R (DNA kho, replication, thiab recombination) system gene Cheebtsam 39. Xaiv cov noob tau los ntawm HGT vim hais tias lawv yuav sawv cev rau thaum ub polymorphisms, yog li xav tias lawv yuav muaj feem ntau ntawm cov kev hloov pauv. Cov noob uas muaj nyob rau hauv 3R system tau raug xaiv vim tias yav dhau los ua haujlwm ntawm M. tuberculosis strains qhia cov kev xaiv tsis zoo/purification uas ua haujlwm ntawm cov noob no, thiab lawv tuaj yeem ua lub luag haujlwm tseem ceeb hauv evolution 39. Lwm lub hom phiaj ntawm txoj haujlwm no yog txhawm rau txheeb xyuas qhov tshwm sim ntawm kev tsim kho tshiab. Vim li no, xav tias peb dataset los ntawm Portugal tsuas muaj cov genomes ntawm European clone complex 2 thiab cov hom uas tsis tau muab lub clone complex, peb tau txiav txim siab los suav cov ntaub ntawv genome uas muaj rau pej xeem kom thaum kawg tau txais ib tus neeg sawv cev ntawm tag nrho cov clone complexes, thiab txhim kho robustness thiab dav ntawm cov txiaj ntsig.
42 cov kab mob tshiab tshiab Mycoplasma bovis genomes los ntawm Portuguese endemic multi-host tuberculosis scene (cov ntsiab lus hauv qab no), yav dhau los tus cwj pwm los ntawm kev sib kis kab mob 36, yog qhov chaw ntawm txoj haujlwm no. Xav txog tias cov ntaub ntawv teev tseg los ntawm Portugal tsuas yog muaj cov neeg sawv cev ntawm European 2 clone complexes thiab strains yam tsis tau xaiv complexes, pej xeem muaj tag nrho cov genome sequencing cov ntaub ntawv tau ntxiv los nthuav cov ntaub ntawv teev uas muaj xws li tag nrho cov neeg sawv cev ntawm lub M. bovis clone complexes. Yog li ntawd, peb cov ntaub ntawv genome sequencing tag nrho tau siv rau hauv txoj haujlwm no: ua kom tiav / cov qauv genome sib dhos, mus txog 10 scaffolds khaws cia hauv NCBI (National Center for Biotechnology Information) (n = 15 cais); khaws cia rau hauv SRA (Cov ntaub ntawv Illumina fastq ntawm cov kab ke nyeem archive) sawv cev rau ntau haiv neeg ntawm M. bovis clones (n = 12 cais tawm) 30; thiab 42 tshiab genomes los ntawm Portugal. Mycobacterium bovis BCG (Bacille Calmette-Guerin) raug tshem tawm los ntawm NCBI tshawb nrhiav. M. bovis AF2122/97 feem ntau yog siv los ua ib qho kev siv genome kom muab tso rau hauv cov ntaub ntawv. Vim tias pej xeem tsis muaj peev xwm ntawm tag nrho cov genome ib ntus sawv cev los ntawm African 1 cloning complex, thiab tsawg tus genomes los ntawm cov neeg sawv cev ntawm Af2 thiab Eu1, cov ntaub ntawv thawj zaug muab los ntawm SRA tau siv rau hauv cov xwm txheej no. Kev ua haujlwm ntawm Zimpel thiab nws cov neeg koom tes (2020) tau pab txheeb xyuas cov genome los ntawm cov lus hais txog cloning complex thiab pab xaiv Mycobacterium bovis rau suav nrog hauv cov ntaub ntawv teev. Rau Eu3, tsuas yog ib hom genome tau piav qhia (Branger li al., 2020), yog li cov genome peb suav nrog yog tus sawv cev sib cais ntawm Eu3 complex.
Thoob ntiaj teb, cov ntaub ntawv no suav nrog 70 tus nyuj M. bovis cais los ntawm 8 hom hom, faib hauv 12 lub teb chaws los ntawm 1985 txog 2016. 36 hom raug xaiv los ua Eu2, 7 hom yog Eu1, 1 hom yog Eu3, 3 hom yog Af1, 4 hom yog Af2 thiab 19 tsis muaj nyob hauv qab no). Cov ncauj lus kom ntxaws (nrog rau tus lej nkag) ntawm Mycobacterium bovis siv hauv txoj kev tshawb no yog qhia hauv Table 1 thiab Cov Lus Ntxiv 1.
42 tshiab genomes ntawm Mycobacterium bovis los ntawm Portugal cov tsiaj tuberculosis hotspots thiab muab faib rau ntau tshaj 12 xyoo yog qhov chaw ntawm txoj kev tshawb no, raws li tej zaum cov tsiaj qus-tsiaj kab mob systems tau saib xyuas tsis tu ncua 31,36 (Ntxiv daim duab 1). Raws li cov txheej txheem tom ntej, cov kab no raug cais tawm ntawm nyuj (n = 14), mos lwj liab (n = 16) thiab npua qus (n = 12) los ntawm 2003 txog 2015: sau thiab tuav cov tsiaj raws li cov txheej txheem pom zoo Cov qauv ntaub so ntswg muaj nyob rau hauv OIE Terrestrial Tsiaj Phau Ntawv thiab yog pysteinenvateJ. medium thiab kua nruab nrab. Cov kab lis kev cai yog incubated ntawm 37 ° C thiab kev loj hlob raug kuaj ib zaug ib lub lim tiam rau tsawg kawg 12 lub lis piam. Cov colony yog khaws cia ncaj qha rau hauv cov tshuaj glycerol ntawm -80ºC. Nyob rau hauv Mycobacterium xaiv nruab nrab (Middlebrook 7H9, BD Diagnostics), thawj cov qauv archived tau dhau los ntawm ib qho hauv vitro passage hauv vitro kom tau txais DNA ntawm WGS qhov kev pab cuam. Rau qhov no, cov kab lis kev cai khov khov kho tau ntxiv nrog 5% sodium pyruvate thiab 10% ADS (50 g albumin, 20 g qabzib, 8.5 g sodium chloride hauv 1 L dej) ntawm Middlebrook 7H9 ntawm 37 ° C Retrain. Tom qab 4 lub lis piam ntawm kev loj hlob, qhov nruab nrab tau rov ua dua tshiab thiab cov kab lis kev cai tau saib xyuas tsis tu ncua kom txog thaum pom kev loj hlob. Cov hlwb raug sau los ntawm centrifugation, cov pellet tau rov ua dua hauv 500 µL phosphate buffered saline (PBS), rhuab ntawm 99 ° C rau 30 feeb, centrifuged, thiab supernatant tau khaws cia ntawm -20 ° C kom txog thaum WGS. Tag nrho cov txheej txheem yog ua nyob rau hauv qib 3 biosafety chaw.
Lub tsev qiv ntawv WGS ua ke-kawg genome tau npaj siv qhov ntsuas tshwj xeeb ntawm txhua tus qauv DNA, thiab siv Illumina MiSeq (2 × 250 pb) (40 cov qauv) thiab HiSeq (2 × 150 pb) (ob cais tawm) thev naus laus zis (Eurofins Genomics, Lub teb chaws Yelemees) rau kev sib txuas. Raws li cov chaw tsim tshuaj paus cov lus qhia, siv Illumina Genome Analyzer nrog ob-kawg module txuas mus ua raws cov genomic DNA, thiab siv Nextera XT DNA Library Prep Kit los ntawm Illumina los tsim lub tsev qiv ntawv.
Muab rau hauv tus account cov ntaub ntawv rov qab los ntawm SRA (n = 12), kev txheeb xyuas ntawm clone complex yuav siv tau raws li cov metadata ntawm cov ntaub ntawv sib xws 30, 41, 43. Thaum xav txog cov genome tiav, tshwj tsis yog rau Mycobacterium bovis AF2122/97 thiab Mycobacterium bovis yog 3601 cov tswv cuab ntawm clone complex. 25, 29, ntsig txog, nws yog tib yam li tag nrho genome ntawm Mycobacterium tuberculosis H37Rv (NCBI accession naj npawb NC_000962.3). Genome alignment yog ua los ntawm MAFFT (ntau qhov kev pab cuam ntawm cov amino acid lossis nucleotide sib lawv liag, version 7.458) thiab parameter -addfragments48. Tom qab ntawd, tshawb nrhiav qhov tsis muaj qhov sib txawv clonal complexes thiab/lossis muaj SNP nta.
Qhov tshiab tau ua ntu zus Mycobacterium bovis (n = 42) thiab cov ntawv nyeem thawj ntawm cov ntawv sau genome (n = 3) dlhos cov complex nrog cov ntaub ntawv siv genome Mycobacterium tuberculosis H37Rv los ntawm vSNP pipeline thiab muaj kev tshem tawm thiab / lossis SNP nta ntawm cov clones sib txawv Kev tshawb nrhiav tau ua.
Sau cov ntaub ntawv los ntawm qhov tsis muaj cov yam ntxwv thiab / lossis qhov muaj / tsis muaj SNP thiab spoligotyping profiles los muab cov ntaub ntawv genomic rau qhov sib txuam clonal complex. Rau plaub qhov kev sib sau ua ke, spoligotyping profile tsis tuaj yeem suav tias yog, yog li lawv suav nrog "tsis muaj qhov nyuaj".
Kev ua haujlwm bioinformatics ua raws li txoj haujlwm no pib los ntawm de novo sib dhos thiab ua daim ntawv qhia rau lub tswv yim siv, nrog rau lub hom phiaj ntawm kev tshawb nrhiav cov xwm txheej recombination thiab tshwj xeeb genome polymorphisms. Daim duab 1 muab ib daim ntawv qhia txog cov kauj ruam ua raws. Rau kev txheeb xyuas rov ua dua, tag nrho cov genomes yog siv los ua kom muaj zog ntawm kev txiav txim siab thiab lwm yam ntsuas.
Txhawm rau txo cov kev ua yuam kev hauv tiam ntawm genome kev pom zoo ua ntu zus, peb thawj zaug tau txais lub rooj sib txoos de novo, thiab tom qab ntawd tau txais ntau yam kev sib dhos. Lub raj xa dej Unicycler tam sim no muaj nyob ntawm https://github.com/rrwick/Unicycler49 thiab yog siv los ua de novo sib sau ua ke ntawm 54 genomes sequenced (42 tshiab sequenced thiab 12 fastq cov ntaub ntawv rov qab los ntawm SRA). Hauv luv luv, ua ntej sib sau ua ke los ntawm kos, nyeem cov kev soj ntsuam zoo tau ua nyob rau hauv FastQC version 0.11.7 (https://github.com/s-andrews/FastQC), thiab Trimmomatic version 0.36 (xaiv "txiav Adapters thiab lwm yam teeb pom kev zoo los ntawm kev nyeem" thiab "Txiav cov hauv paus los ntawm qhov kawg ntawm qhov zoo tshaj 2th) reshes (http://www.usadellab.org/cms/?page= trimmomatic) 50. Tom qab ntawd, SPAdes optimiser49 tau siv rau kev sib dhos genome, thiab Pilon version 1.1851 tau siv rau kev ua kom zoo tom qab sib dhos. Ib hom kev sib txuas txuas txuas tau raug xaiv kom tsis txhob muaj kev sib dhos tsis raug, thiab k-mer loj tau tshawb nrhiav thiab xaiv ntawm 20% thiab 95% ntawm qhov ntev nyeem. Ua raws li SPAdes cov lus qhia thiab txiav txim siab qhov loj me, tshem tawm cov kab mob me dua 300 bp, thiab tsim kom muaj 20 nyeem qhov tob qhov kev pab cuam txiav tawm ntawm 52. Nyob rau hauv lub de novo lub tswv yim sib dhos, genomic cheeb tsam xws li repetitive proline-glutamate (PE) thiab proline-proline glutamate (PPE) paralogs raug tshem tawm.
Qhov zoo ntawm lub rooj sib txoos de novo raug soj ntsuam los ntawm QUAST pipeline (http://quast.sourceforge.net/quast.html), uas pab txhawb kev rov ua dua tshiab ntawm contig thiab M. bovis AF2122/97 reference genome (NCBI accession number LT708304.1) Mapping (saib 1 parameter ntxiv).
Nrog kev pab los ntawm vSNP pipeline (https://github.com/USDA-VS/vSNP), FASTQ cov ntaub ntawv ntawm cov tshiab sequenced M. bovis los ntawm Illumina sequencing yog muab piv nrog cov M. bovis AF2122/97 siv genome (LT708304.1)). Raws li cov lus pom zoo ntawm kev coj ua zoo tshaj plaws ntawm Genome Analysis Toolkit (GATK) 53, 54, 55 siv cov qauv lim dej lossis cov qhab nia sib txawv rau kev rov ua dua. Cov txiaj ntsig tau raug lim siv qhov qis tshaj SAMtools huab hwm coj qhab nia ntawm 150 thiab AC = 2. Kuj tseem siv Kraken (http://ccb.jhu.edu/software/kraken/) los xyuas cov ntawv nyeem kom tsis txhob muaj kab mob. Cov kav dej vSNP tau siv los qhia txog cov tswv yim sib txuas hauv peb txoj haujlwm tshuaj xyuas ntau yam ntawm SNPs thiab cov hom phiaj, thiab tseem tsis suav nrog cov xwm txheej sib xyaw. Qhov kev nyeem ntawv genome tau zoo dua li 99% (Cov Lus Qhia Ntxiv 1).
Txhawm rau zam kev ua yuam kev thiab SNPs tsis raug, lim tawm qhov sib txawv hauv cov xwm txheej hauv qab no: (1) nws tau txais kev txhawb los ntawm tsawg dua 20 nyeem, (2) nws pom muaj qhov zaus tsawg dua 0.9, (3) nws nyob hauv tsawg kawg ib hom kab mob, tab sis tsawg kawg muaj qhov sib txawv ntawm lwm hom. Kev sib koom ua ke genomics saib (IGV) version 2.4.19 (http://software.broadinstitute.org/software/igv/)56 tau siv los txheeb xyuas qhov muag pom SNPs thiab txoj haujlwm nrog teeb meem kev teeb tsa lossis kev sib dhos. Txij li thaum cov noob caj noob ces proline-glutamate (PE) thiab proline-proline glutamate (PPE) cov noob muaj ntau dua thiab yog ib feem ntawm ntau tsev neeg, lawv tau nkag siab yooj yim los ntawm Illumina sequencing thiab mismapping, yog li lawv nyiam Cov mycobacterial bioinformatics workflow tshem tawm cov tswv cuab ntawm tuberculosis complex thaum siv cov tswv yim sib luag. Yog li ntawd, peb lim tawm PE / PPE noob thiab indels los ntawm kev tshuaj xyuas.
Raws li Bovilist (http://genolist.pasteur.fr/BoviList/), tag nrho SNPs tau muab faib ua pawg ua haujlwm. Lub raj xa dej SnpEff (https://pcingola.github.io/SnpEff/) yog siv los txiav txim SNP qhov tshwm sim (kev hloov pauv lossis tsis sib xws). Tsim cov ntaub ntawv tshiab ntawm Mycobacterium bovis AF2122/97 genome (LT708304.1).
Cov tub ntxhais genome ntau qhov sib dhos tau ua tiav siv Parsnp v1.2, tam sim no muaj nyob ntawm https://github.com/marbl/parsnp57, siv 69 tiav genomes/draft cov rooj sib txoos (nrog kev xaiv -c) thiab M. bovis AF2122/97 (LT708304.1) yog siv los ua ib qho kev siv. Plaub qhov kev sib tw ntau yam tau ua tiav: tsuas yog cov tswv cuab ntawm Eu2 cloning complex (n = 37), suav nrog tag nrho cov tswv cuab ntawm European cloning complex (n = 44), suav nrog qhov sib txuas ntawm European thiab African cloning complex (n = 51), thiab suav nrog tag nrho Mycobacterium bovis hauv txoj kev tshawb no (n = 70).
Cov kev sib raug zoo tseem ceeb tsim los ntawm Parsnp yog siv los ua qhov siab tshaj plaws (ML) phylogenetic tsob ntoo siv CIPRES Science Gateway v3.3 (http://www.phylo.org/)58 siv RAxML, thiab ua 1000 coj rov ua dua.
Peb qhov sib txawv algorithms thiab bioinformatics cov cuab yeej siv los kuaj xyuas qhov muaj cov xwm txheej rov ua dua nyob rau hauv tib lub sijhawm: SplitsTree4 software, Gubbins (kev tsis sib haum xeeb los ntawm kev sib koom ua ke hauv nucleotide sequences) pipeline, thiab RDP4 (recombination detection program, version beta 4.101) software.
Txoj kev sib cais decomposition tau siv nyob rau hauv SplitsTree4 v4.15.1 (http://www.splitstree.org/)59 yog siv los xam cov rootless phylogenetic network, siv Phi test rau kev txheeb xyuas qhov tseeb, thiab qhov tseem ceeb pib yog p = 0.05. Qhov tseem ceeb ntawm ntau qhov kev tsom xam ntawm Parsnp yog siv los ua cov tswv yim, thiab kev sib cais ntawm kev sib cais raws li tus qauv network tau paub.
Gubbins pipeline v2.3.1 (https://github.com/sanger-pathogens/gubbins60 khiav nrog default parameters raws li lwm txoj hauv kev los ntsuam xyuas qhov cuam tshuam ntawm recombination ntawm Mycobacterium bovis. Cov algorithm siv nyob rau hauv lub raj xa dej reconstructs cov ntaub ntawv hais txog clone lineage Qhov ua tiav genome/databovis teeb tsa thiab kev sib dhos ntawm peb AF2122/97, LT708304.1) yog sib koom ua ke; thiab luam theej duab txoj hauj lwm ntawm SNP ntawm txhua ceg ntawm tsob ntoo kom pom cov SNP pawg sawv cev rau qhov kev tshwm sim recombination Qhov xoom ntawm ceg Xav tias tsis muaj qhov kev tshwm sim recombination, uas txhais tau hais tias SNPs uas tshwm sim ntawm ceg ntoo yuav tsum tau muab faib ua ntau qhov sib npaug ntawm ML ML siv raws li cov ntaub ntawv nkag.
Thaum kawg, txhawm rau txhawm rau paub meej qhov kev tsim kho tshiab tau pom zoo los ntawm Gubbins pipeline, rau algorithms tau siv hauv RDP467 (RDP61, GENECONV62, Bootscan63, Maxchi64, Chimaera65 thiab SiScan66) tau siv rau Parsnp's core ntau qhov kev sib dhos hauv qab qhov teeb tsa. Peb tau txiav txim siab tias yam tsawg kawg peb ntawm cov algorithms tau siv hauv RDP4 yuav tsum ua kom pom cov teeb liab tseem ceeb los xyuas txhua qhov kev sib koom ua ke.
Kev txiav txim siab tias ob qho tib si Gubbins thiab RDP software saib rau cov teeb liab sib txuas los ntawm kev txheeb xyuas cov tub ntxhais ntau qhov kev sib dhos hauv lub qhov rais txog li 500 bp, thiab lees paub tias kev suav nrog PE / PPE noob thaum lub sij hawm de novo los ua ke yuav tsis cuam tshuam nrog cov teeb liab recombination pom, ntxiv kev tshuaj ntsuam xyuas yog ua los ntawm homolinearity txheeb xyuas cov kab mob sib kis. Daim ntawv qhia synlinear siv cov genome ua tiav tau tsim los siv MAUVE-multi-genome alignment (http://darlinglab.org/mauve/mauve.html) kom tsis suav cov genome translocations lossis inversions hauv zos. Tsis tas li ntawd, tag nrho cov genome tau siv los ua homolinearity tsom xam ntawm cov amino acid ib ntus los ntawm SyntTax web server (https://archaea.i2bc.paris-saclay.fr/SyntTax/).
Ib qho kev soj ntsuam ntau ntxiv ntawm cov ntaub ntawv genome tau txais los ntawm Portuguese multi-host tuberculosis system yog los xyuas cov polymorphism ntawm cov noob tau hais hauv cov ntaub ntawv. Cov noob no yog 37,38 thiab cov noob encoding 3R tau los ntawm MTBC cov poj koob yawm txwv los ntawm HGT (DNA kho, rov ua dua thiab rov ua dua) cov txheej txheem 39. Siv ClustalX v2.1 (http://www.clustal.org/clustal2/) thiab siv DnaSP v6.12.03 (http://www. nucleotide diversity (π) thiab Tajima's D neutral test parameter input.
Qhov siab tshaj plaws (ML) phylogenetic ntoo raws li 69 Mycoplasma bovis cais thiab siv cov genomes tau txais (Daim duab 2A). Muab piv nrog cov ntoo ib leeg-raws li cov ntoo lossis ntau qhov chaw-raws li cov ntoo, lub tswv yim no tso cai rau cov ntoo muaj zog ntau dua uas tsis tuaj yeem ntes qhov sib txawv ntawm tag nrho cov genome thiab yog li ntawd muaj peev xwm txo qis kev ntxub ntxaug ntawm hom 68,69. Cov qauv topological ntawm tsob ntoo ML feem ntau yog ua raws li kev faib tawm ntawm cov clones. Lub genome ntawm Eu2 yog pawg nyob rau hauv ib ceg, thiab lub genome ntawm Af1 kuj yog pawg ua ke (Daim duab 2A). Qhov tshwm sim kuj zoo ib yam nrog kev paub txog kev sib raug zoo ntawm Mycobacterium bovis, uas yog, muaj qhov sib txawv loj ntawm cov tswv cuab Eu1 thiab cov pab pawg uas muaj tag nrho lwm cov clonal complexes thiab genomes, tab sis cov clonal complex 30 tsis tau teev tseg. Qhov me me tsis sib xws ntawm clonal complex thiab kev sib raug zoo ntawm tsob ntoo phylogenetic tuaj yeem piav qhia los ntawm qhov tseeb tias clonal complex tau piav qhia raws li cov cheeb tsam genomic, thaum tsob ntoo phylogenetic yog nyob ntawm ntau qhov sib txawv ntawm cov tub ntxhais genomes sawv cev rau tag nrho cov genome.
Qhov siab tshaj plaws phylogenetic tsob ntoo (GTR) yog tsim los ntawm cov genome alignment ntawm Mycobacterium bovis genome ua ntej (A) thiab tom qab (B) tshem tawm ntawm qhov chaw recombination. Cov ceg xim sawv cev rau Mycobacterium bovis clone complex: Europe 1 yog ntshav, Europe 2 yog liab, Europe 3 yog xiav, Africa 1 yog txiv kab ntxwv, thiab Africa 2 yog ntsuab. Tsob ntoo yog cag thiab kos rau nplai, thiab ceg ntev yog ntsuas raws li kev hloov pauv rau txhua qhov chaw.
Lub Mycobacterium tuberculosis complex tau piav raws li kev hloov pauv hloov pauv, thiab feem ntau ntawm cov pov thawj tau sau ntau xyoo txhawb lub tswv yim tias HGT txuas ntxiv thiab cov xwm txheej rov ua dua yuav tsis tshwm sim ntawm qib kuaj pom ntawm MTBC15,17,18.
Cov hauj lwm yav dhau los tau qhia tias yuav muaj kev txwv tsis pub rov ua dua ntawm MTBC strains20,21, thaum lwm tus tsis tuaj yeem txheeb xyuas cov xwm txheej uas ntsuas tau rov ua dua tshiab 70,71. Rov sib tham txog qhov teeb meem no nrog rau kev tsom mus rau Mycobacterium bovis, uas txawv ntawm kev ua haujlwm dhau los uas tsuas yog suav tias yog Mycobacterium tuberculosis 70,71; los yog xav txog MTBC tag nrho, yuav luag tsis muaj M. bovis sawv cev 20; los yog tsuas yog xav txog kev txwv cov nyuj feem. Lub mycobacteria dataset, nyob rau hauv txoj hauj lwm no, muaj tag nrho ntawm 70 hom, sawv cev rau tag nrho cov clonal complexes, siv los tshuaj ntsuam rau recombination. Cov ntaub ntawv teev yog ntsuas raws li plaub theem: (1) Eu2 cov tswv cuab, (2) tag nrho cov European clone complex cov tswv cuab (ie Europe), (3) European thiab African clone complex (Eu + Af) thiab (4) tag nrho cov ntaub ntawv Collections (xws li genomes uas tsis suav nrog nyob rau hauv tej cloning complexes twb tau piav qhia).
Txhawm rau kawm ntxiv txog qhov kev xav no, kev sib cais-decomposition network tau ua los ntsuas qhov tsis muaj cov xwm txheej rov ua dua ntawm cov genomes, vim tias txoj kev no tuaj yeem pom cov poj koob yawm txwv kev sib raug zoo ntawm cov tib neeg thiab pom cov teeb meem tsis sib haum xeeb. Tag nrho plaub cov ntaub ntawv teev tseg hauv kev txheeb xyuas tau lees paub qhov muaj nyob ntawm lub voj voog hauv lub network (uas yog, thaj chaw uas tsis sib sau ua ke rau hauv ib tsob ntoo), tab sis Phi kev sim tsis muaj kev txhawb nqa (Eu2, p = 0.0956; Europe, p = 0.1637; Eu + Af p = 0.2774; tag nrho cov ntaub ntawv teev cov pov thawj tsis zoo p = 0.2) (Daim duab 3A-D).
Hauv Tebchaws Europe 2 genomes (n = 37) (A), European genomes (n = 44) (B), European thiab African genomes (n = 51) (C) thiab tag nrho cov ntaub ntawv teev (n = 70) (D).
Tom qab qhov kev tshuaj ntsuam no, thiab coj mus rau hauv tus account lub cyclic kev soj ntsuam nyob rau hauv tag nrho cov tes hauj lwm, lub reconstruction algorithm siv nyob rau hauv lub Gubbins pipeline yog siv los reconstruct lub clonal kab thiab ntxiv rau kev kwv yees ntawm cov nyhuv ntawm recombination ntawm lub M. bovis genome. Infer tus naj npawb ntawm recombination txheej xwm, feem ntau tshwm sim nyob rau hauv lub davhlau ya nyob twg ceg (uas yog, nyob rau hauv ib tug tib genome) (Table 2). Cov ntsuas no qhia qhov sib xws ntawm tag nrho cov ntaub ntawv teeb tsa thiab qhia tias qhov zaus ntawm cov txheej xwm rov ua dua yog 200 txog 300 npaug ntawm kev hloov pauv. Thaum tus rho/theta parameter sawv cev rau tus txheeb ze tus nqi ntawm recombination thiab taw tes hloov pauv ntawm ceg zoo li nyob nruab nrab ntawm 0.0037 thiab 0.0056 (Table 3). Tsis ntev los no, kev tshaj tawm ua haujlwm ntawm 38 M. bovis strain pom tau tias muaj ntau dua rho / theta tus nqi (rho / theta = 0.1) ntau dua li tau txais hauv cov ntaub ntawv no, tab sis kev ua haujlwm ntawm Patané thiab cov npoj yaig siv cov ntaub ntawv siv los ua ke rau infer recombination tsis, Cov txheej txheem nthuav dav, vim yog cov txheej txheem sib dhos, tau cuam tshuam nrog cov txheej txheem rov ua dua. ceg.
Tom ntej no, qhov ntsuas r / m sawv cev rau qhov sib txawv ntawm qhov sib txawv ntawm kev sib txuam thiab kev hloov pauv, thiab nws qhov nruab nrab tus nqi yog nyob nruab nrab ntawm 0.025 thiab 0.037, qhia tias piv nrog kev hloov pauv, kev sib xyaw ua ke muaj feem cuam tshuam rau tag nrho cov caj ces ntawm M. bovis (Table 3)). Rau kev sib piv dav dav, ib txoj kev zoo sib xws tau siv los kwv yees qhov ntsuas r / m rau MTBC cov ntaub ntawv tsim los ntawm 23 genomes, qhia qhov nruab nrab tus nqi ntawm 0.48620, thaum rau Patané thiab cov npoj yaig '38 M. bovis dataset, nws ua pov thawj tus nqi nruab nrab yog 0.98. Nyob rau hauv thawj txoj kev tshawb no, tsuas yog ob ntawm 23 genomes suav nrog hauv kev ua haujlwm ntawm M. bovis (M. bovis BCG thiab cov ntaub ntawv siv hom), yog li tus nqi tau txais yuav tsis ncaj ncees vim yog overexpression ntawm M. tuberculosis genome. Hauv tsab ntawv tshaj tawm thib ob, cov tshuaj ntsuam xyuas Mycobacterium bovis cov pej xeem feem ntau tau rov qab los ntawm Tebchaws Meskas thiab cov tsiaj txhu. Nyob rau hauv sib piv, nyob rau hauv peb cov ntaub ntawv teev, ntau thaj chaw thiab host hom yog sawv cev, thiab genomes pawg nyob rau hauv sib txawv clonal complexes nrog txawv pej xeem cov yam ntxwv ntawm caj ces kuj siv, yog li ua tiav ib tug tob thiab dav pej xeem paub. Qhov sib txawv r / m nruab nrab tus nqi tau txais nrog peb cov ntaub ntawv teev yog ua raws li lub tswv yim hais tias qhov degree ntawm recombination txawv heev ntawm cov kab uas muab rau tib hom taxonomic, yog li cov txiaj ntsig no qhia tau hais tias M. bovis clone complex tuaj yeem pom qhov sib txawv ntawm qhov cuam tshuam kuj yog raws li tau hais los ntawm Didelot & Maiden72. Txawm li cas los xij, nthuav dav cov ntaub ntawv no los ntawm kev suav nrog ntau tus lej ntawm M. bovis genomes yuav tso cai rau kev nthuav dav ntxiv ntawm cov ntsiab lus no. Ob qho tib si r / m thiab rho / theta tsis pom muaj qhov sib txawv ntawm cov ceg, thiab qhov txiaj ntsig no yog raws li cov lus ceeb toom ntawm lwm hom kab mob 72,73.
Thaum kawg, txhawm rau kom paub meej txog cov xwm txheej rov tsim dua tshiab uas tau txheeb xyuas los ntawm Gubbins pipeline, rau qhov sib txawv algorithms tau siv rau hauv RDP4 software los ntawm nws tus kheej ntsuas cov tub ntxhais sib txawv ntau qhov sib piv. Thoob plaws ntiaj teb, tsawg dua ib nrab ntawm cov xwm txheej txheeb xyuas los ntawm Gubbins tau lees paub los ntawm RDP4 (Tables 4 thiab 5). Xav txog tag nrho cov ntaub ntawv teeb tsa, peb qhov kev sib koom ua ke tau lees paub, ob qho cuam tshuam nrog cov nodes thiab lwm qhov cuam tshuam nrog ib qho genome hauv ib ceg davhlau ya nyob twg, uas cov clonal complexes tsis tuaj yeem muab (Table 4 thiab 5). Kev txheeb xyuas cov xwm txheej hauv cov ceg dav hlau yuav qhia tau tias qhov kev sib txuas ua ke tseem nyob hauv kev kawm M. bovis strains lossis qhov tshwm sim yog misplaced70. Hauv cheeb tsam no hypothetical recombination, kwv yees li 20% ntawm txoj haujlwm muaj cov nucleotides uas tsis tau teev tseg (N), yog li cuam tshuam rau cov teeb liab recombination (Cov duab ntxiv 2). Tsis tas li ntawd, thaj av no cuam tshuam rau rrs gene, encoding 16S ribosomal RNA uas xav tias yuav tau txais kev cawmdim heev, yog li qhov teeb meem kev sib koom ua ke no tuaj yeem yog qhov tshwm sim ntawm kev ua yuam kev los yog misalignment. Tom qab ntawd tag nrho cov genome alignment ntawm Mb0003 thiab Mycobacterium bovis AF2122/97 tau ua tiav, thiab lub hav zoov ntawm cov nucleotides thiab SNPs tsis tau raug lees paub, yog li cov teeb meem muaj feem cuam tshuam txog kev sib raug zoo tsis yog vim cov ntaub ntawv lom neeg siv hauv txoj haujlwm no tshwm sim tom qab kawm txog qhov kev zov me nyuam.
Tsis muaj qhov sib txawv lossis tsis tau txhais cov nucleotides tau pom nyob rau hauv cov cheeb tsam recombination ntawm cov nodes sab hauv (Daim duab 4 thiab 5). Hais txog cov xwm txheej no, ib qho tsuas muaj Eu2 genome thiab cuam tshuam rau pks12 noob, uas encodes ib qho polyketide synthase; thaum lwm tus tau sau npe hauv Eu1 genome thiab cuam tshuam rau narX noob uas encodes ib qho nitrate reductase tau (Table 4). Nyob rau hauv dav dav, qhov recombination tsom xam qhia tau hais tias muaj ib tug tsawg tus naj npawb ntawm recombination fragments nrog kev txhawb nqa, thiab cov inferred indicators, As qhia hais tias cov recombination muaj ib tug tsis tshua muaj feem xyuam rau M. bovis kab. Lub teeb liab recombination yuav tsum tau qis, tab sis nws yog ib qho tseem ceeb kom paub qhov txawv ntawm qhov tseeb evolution teeb liab los ntawm lub suab nrov tom qab, uas yog ib txoj hauj lwm nyuaj. Txhawm rau txo cov suab nrov teeb liab qhia los ntawm kev siv raws li kev sib dhos thiab cov teeb meem tsis sib haum 70, 71, tag nrho cov seem tsuas yog ua tiav cov genome tau sib sau ua ke los ntawm kos, thiab cov khoom sib dhos tau raug kuaj xyuas thiab xyuas kom meej los ntawm QUAST pipeline tsom xam (Cov Lus Qhia Ntxiv 1). Tsis tas li ntawd, ib qho kev ntsuam xyuas ntxiv tau ua los muab qhov kev ua tau zoo thiab qhov tseeb ntawm kev soj ntsuam tag nrho. Yog li, qhov ua tau zoo ntawm narX thiab pks12 noob tau soj ntsuam los ntawm kev nyeem daim ntawv qhia tawm tsam Mycobacterium bovis AF2122/97. Qhov pom zoo SNP txoj hauj lwm nyob rau hauv cheeb tsam recombination tau lees paub los ntawm kev siv cov txheej txheem hais nyob rau hauv txoj kev seem (tsawg kawg yog 20 nyeem thiab 0.9 hloov zaus). Lub polymorphism ntawm narX noob tau lees paub tag nrho hauv ob lub genomes (Mb1792361 thiab Mb7240415; 2.3%) thiab cov genomes ntawm pks12 genome: Mb0891, Mb1711, Mb1789, Mb1870, Mb16, thiab Mb17. Txawm li cas los xij, rau genome Mb2043, rau ntawm yim txoj haujlwm tsis ua raws li qhov kev ntsuas qhov tob tob vim tias SNP tau txais kev txhawb nqa los ntawm qhov siab tshaj plaws ntawm 17 nyeem, uas yog qis dua tus nqi txiav tawm ntawm 20. Yog li ntawd, kev sib txuas ntawm rau genomes (8.6%) ntawm qhov chaw genome no tuaj yeem lees paub (daim duab 4 thiab 5).
Cov ncauj lus kom ntxaws pom ntawm thaj tsam recombination kev sib haum xeeb ntawm Mycobacterium bovis dataset cuam tshuam rau narX noob uas encodes ib qho tau nitrate reductase. Tsis muaj qhov sib txawv los yog tsis muaj cov nucleotides tau pom nyob rau hauv thaj tsam recombination ntawm cov nodes sab hauv. Qhov kev tshwm sim tshwj xeeb no tau sau npe hauv Eu1 genome. Qhov ua tau zoo ntawm cov noob narX tau soj ntsuam los ntawm kev npaj cov ntawv nyeem ntawm Mycobacterium bovis AF2122/97. Txheeb xyuas qhov pom zoo SNP qhov chaw nyob hauv thaj chaw recombination los ntawm kev siv cov txheej txheem hais hauv tshooj txheej txheem (tsawg kawg 20 nyeem ntawv thiab 0.9 hloov zaus). Lub polymorphism ntawm narX noob tau lees paub tag nrho hauv cov genomes ntawm Mb1792361 thiab Mb7240415 (2.3%).
Cov ncauj lus kom ntxaws pom ntawm thaj tsam recombination kev sib haum xeeb ntawm Mycoplasma bovis dataset cuam tshuam rau cov noob pks12. Tsis muaj qhov sib txawv los yog tsis muaj cov nucleotides tau pom nyob rau hauv thaj tsam recombination ntawm cov nodes sab hauv. Hais txog qhov xwm txheej cuam tshuam rau pks12 noob encoding tau polyketide synthase, nws tsuas muaj Eu2 genome. Qhov ua tau zoo ntawm pks12 tau soj ntsuam los ntawm kev nyeem daim ntawv qhia ntawm Mycobacterium bovis AF2122/97. Txheeb xyuas qhov pom zoo SNP qhov chaw nyob hauv thaj chaw recombination los ntawm kev siv cov txheej txheem hais hauv tshooj txheej txheem (tsawg kawg 20 nyeem ntawv thiab 0.9 hloov zaus). Cov polymorphisms ntawm genomes Mb0891, Mb1711, Mb1789, Mb1870, Mb1758, Mb2043, thiab Mb1960 tau lees paub tag nrho.
PE thiab PPE noob muaj cov cheeb tsam rov ua dua uas tau yooj yim tsis raug los ntawm Illumina sequencing thiab mismapping, yog li lawv feem ntau raug tshem tawm los ntawm bioinformatics workflow ntawm M. tuberculosis cov tswv cuab tsuas yog thaum siv cov phiaj xwm ua ke. Qhov kev txiav txim siab ntawm cov txheej xwm rov ua dua tau siv rau hauv txoj haujlwm no yog ua raws li de novo sib dhos yam tsis tau lim tawm PE / PPE. Peb ntseeg hais tias los ntawm kev siv peb txoj kev sib txawv thiab cov txheej txheem sib txawv los ntawm SplitsTree, Gubbins pipeline thiab RDP4 software, cov tswv yim siv tau zoo rau kev ua haujlwm thiab lim cov cheeb tsam rov tsim dua los ntawm cov teeb meem yuam kev. Txawm li cas los xij, txhawm rau kom tsis suav nrog kev cuam tshuam ntawm PE / PPE noob ntawm Gubbins thiab RDP4 software txhawm rau txheeb xyuas SNP pawg, thiab yog li kev txheeb xyuas cov cheeb tsam recombination tau npaj los cuam tshuam rau cov noob narX thiab pks12, cov neeg nyob ib puag ncig ntawm cov noob no tau kuaj xyuas (Ntxiv daim duab 3-5). Hauv M. bovis AF2122/97, narX noob cais los ntawm narK2 thiab Mb1764c, thaum pks12 yog nyob ib puag ncig los ntawm Mb2075c e Mb2073c (Cov duab ntxiv 3-5). Daim ntawv qhia tsim los siv MAUVE synline daim ntawv qhia ntawm cov genome ua tiav muab cov ntaub ntawv hais txog cov noob caj noob ces kev txuag thiab kev kho dua tshiab, uas qhia txog plaub qhov sib txuam sib luag, thiab tsis muaj cov cim qhia ntawm genome translocation lossis inversion. Tsis tas li ntawd, kev soj ntsuam ntxiv nrog cov amino acid ib ntus tau ua pov thawj homology nyob rau hauv tag nrho cov genomes tiav, thiab tsis muaj PE / PPE tau pom nyob rau hauv cov cheeb tsam uas nyob ib sab ntawm narX lossis pks12. Rau narX, ib qho genome (Mb0030) muaj cov qhab nia qis dua vim tias cov noob narX tau txheeb xyuas tias yog ob ntu (fragments 1891 thiab 1890). Rau pks12, vim qhov zoo sib xws, Mb0030 thiab Mb003 tau nthuav tawm cov qhab nia qis dua synlinearity, thaum pks12 tau txheeb xyuas hauv ob thiab peb ntu, raws li, sawv cev rau ntau qhov sib txawv ntawm cov protein (Cov duab ntxiv 3-5). Muab rau hauv tus account cov ntaub ntawv no, thiab Gubbins thiab RDP4 software ob qho tib si ua kev soj ntsuam, xyuas cov tub ntxhais ntau qhov kev sib dhos ntawm qhov siab tshaj 500 bp hauv lub qhov rais, peb tau lees paub tias PE / PPE noob yuav tsis cuam tshuam nrog cov teeb liab recombination uas cuam tshuam rau narX thiab pks12.
Txawm hais tias recombination signals pom nyob rau hauv cov ntaub ntawv no tej zaum yuav suav hais tias residual, nws yog tseeb hais tias recombination nyob rau hauv M. bovis tsis tuaj yeem txiav tawm, yog li nws yuav tsum txuas ntxiv mus rau qhov kev soj ntsuam ntxiv, nyob rau hauv uas tag nrho cov genomes los ntawm cov xwm txheej sib txawv ntawm cov kab mob sib kis tau ua rau tseem ceeb.
Sib piv cov ntoo ML phylogenetic tau ua ntej thiab tom qab kev kho dua tshiab (Daim duab 2A, B) tsis ua rau muaj kev hloov pauv tseem ceeb hauv kev sib raug zoo phylogenetic, thiab M. bovis hom kab mob tau muab faib ua pawg tib yam.
Tom qab daim ntawv qhia 42 tshiab tau ua tiav M. bovis nyeem nrog cov ntaub ntawv siv genome ntawm M. bovis AF2122/97, SNP kev sib raug zoo uas muaj 1816 polymorphic txoj haujlwm tau txais. Feem ntau SNPs (87.1%) yog nyob rau hauv cheeb tsam coding, thiab cov noob cuam tshuam yog cov yam ntxwv raws li cov kev ua haujlwm tau pom hauv Bovilist (Daim duab 6A, B). Muab rau hauv tus account tag nrho cov noob nyob rau hauv txhua yam kev ua haujlwm, cov noob hauv "lipid metabolism" qeb pom ntau SNPs, ua raws li "cell phab ntsa thiab cell txheej txheem" thiab "intermediate metabolism thiab ua pa", qhia tias lawv nyob rau hauv M. bovis evolution.
Hierarchical tsom xam ntawm M. bovis dataset los ntawm Portugal (n = 42). Tag nrho cov naj npawb ntawm cov npe SNPs thiab cov noob cuam tshuam rau txhua pawg ua haujlwm (A). Tag nrho tus naj npawb ntawm cov kev hloov pauv tsis zoo thiab tsis sib xws tau sau npe los ntawm qeb ua haujlwm (B).
Nyob rau hauv lub ntiaj teb no, qhov nruab nrab dN / dS piv yog zoo dua 1.5, uas qhia tau hais tias lub ntiaj teb no evolutionary siab yog kom tshem tawm ntawm cov poj koob yawm txwv lub xeev thiab sawv cev rau ib tug zoo (ntau yam los yog qhia) thiab / los yog so kom huv si purification scenario. Nyob rau hauv pawg "virulence, detoxification, adaptation", "insertion sequences and phages", thiab "regulatory proteins", ntau tshaj li ob feem peb ntawm SNPs yog non-synonymous (Daim duab 6B).
Hauv txhua pawg, muaj cov noob uas muaj ntau yam SNPs, uas ua rau qhov kev hloov pauv nruab nrab (uas yog, qhov nruab nrab SNP ib gene) ntau dua 1 (Daim duab 6A). Pks12 (Mb2074c) nrog 15 SNPs thiab fas (Mb2553c) nrog 8 SNPs muaj kev hloov pauv ntau dua. Ob leeg ntawm cov noob no koom nrog hauv fatty acid metabolism. Lub pks noob encodes polyketide synthase (PKS), uas yog ib tug multifunctional enzyme koom tes nyob rau hauv mycobacterial cell phab ntsa lipid biosynthesis74,75. Cov noob no encodes multifunctional polypeptide uas koom nrog hauv kev sib txuas ntawm mycoketides74,76. Cov noob fas koom nrog hauv kev sib txuas ntawm mycolic acid. Ob leeg ntawm cov noob no ua lub luag haujlwm tseem ceeb hauv biosynthesis ntawm phab ntsa ntawm tes hauv kev sib cuag nrog tus tswv tsev.
Txhawm rau kawm ntxiv txog kev hloov pauv ntawm Mycobacterium bovis, ob pawg ntawm cov noob tshwj xeeb tau txheeb xyuas. Yav dhau los luam tawm ua haujlwm siv cov kab ke sib xyaw thiab cov txheej txheem phylogenetic txheeb xyuas cov noob uas tau txais los ntawm MTBC cov poj koob yawm txwv los ntawm HGT ua ntej muaj kev sib txawv37,38. Cov noob no tau teev nyob rau hauv Cov Lus Ntxiv 2. SNP kev faib tawm ntawm tag nrho 77 cov noob caj noob ces uas yuav cuam tshuam nrog HGT tau txheeb xyuas, thiab 26 qhov chaw polymorphic tau txheeb xyuas, uas feem ntau (78%) ua rau tsis muaj kev hloov pauv (NS) (Sab Ntxiv Table 2). Kev ua haujlwm yav dhau los ntawm MTBC genome tau pom tias thaj av putative HGT nthuav tawm ntau dua NS SNP piv piv rau lwm cov genome. Yog hais tias ib tug xav tias cov recombination cheeb tsam no tau txais los ntawm MTBC cov poj koob yawm txwv, thiab yog li ntawd, lawv overrepresent ancient polymorphisms, ces qhov kev faib ua feem ntawm synonymous kev hloov yuav tsum siab dua, vim hais tias NS hloov yuav tsum raug tshem tawm los ntawm kev xaiv tsis zoo vim hais tias ntawm cov amino acid hloov tej zaum yuav hloov cov kev ua ntawm cov protein. Yog li ntawd, peb cov txiaj ntsig qhia tau hais tias cov txiaj ntsig ua haujlwm tuaj yeem tshwm sim los ntawm kev hloov cov noob zoo li HGT, uas qhia txog lawv qhov tseem ceeb rau kev hloov pauv caj ces muaj txiaj ntsig.
Nyob rau tib lub sijhawm nrog qhov kev tshuaj ntsuam no, cov noob encoding cov khoom ntawm 3R (DNA kho, rov ua dua, thiab rov ua dua) tau raug tshuaj xyuas kom zoo raws li cov npe yav dhau los luam tawm los ntawm dos Vultos thiab cov neeg koom tes (2008)39. Kev sib pauv ntawm cov DNA zoo ib yam tsis tuaj yeem pom ncaj qha, txawm hais tias nws yuav yog cov txheej txheem nquag thaum cov kab mob cuam tshuam nrog, xws li hauv cov ntaub ntawv no; Ntxiv rau, cov txheej txheem no yuav yog tus yuam sij rau DNA kho txoj kev72, yog li Ua lub luag haujlwm hauv homologous recombination. Tag nrho ntawm 26 txoj haujlwm polymorphic faib los ntawm 54 cov noob tau txheeb xyuas (Cov Lus Ntxiv 3). Hauv cov txheej txheem no, NS hloov pauv suav txog li 65% ntawm qhov tshwm sim, uas zoo ib yam nrog cov ntaub ntawv dhau los ntawm Mycobacterium tuberculosis hom.
Post lub sij hawm: Oct-21-2021