I-Specific-Locus Amplified Fragment Sequencing (SLAF-Seq)
Ukuhamba komsebenzi
Inkonzo inoyilo oluyilwe kwangaphambili kwi-silico ukuqinisekisa ukhetho lwe-enzyme ekulungisweni kwethala leencwadi.
Inkqubo yobuGcisa
Iimpawu zeNkonzo
● Ukulandelelanisa kwi-NovaSeq nge-PE150.
● Ukulungiswa kwethala leencwadi elineekhowudi ezimbini, okwenza ukuba kudityaniswe iisampuli ezingaphezu kwe-1000.
● Ukuzimela genome yesalathiso:
Ngokubhekiselele kwi-genome: i-SNP kunye nokufunyanwa kwe-InDel
Ngaphandle kwegenome yesalathiso: iisampulu yokuhlanganiswa kunye nokufunyanwa kwe-SNP
● Kwikwi-silicoinqanaba loyilo lwangaphambili imidibaniso ye-enzyme yothintelo oluninzi iyajongwa ukufumana ezo zivelisa ukuhanjiswa okufanayo kweethegi zeSLAF ecaleni kwegenome.
● Ngexesha lovavanyo lwangaphambili, indibaniselwano ye-enzyme emithathu ivavanywa kwiisampulu ezi-3 ukuvelisa amathala eencwadi angama-9 e-SLAF, kwaye olu lwazi lusetyenziselwa ukukhetha indibaniselwano ye-enzyme yesithintelo esilungileyo kwiprojekthi.
Iinkonzo eziluncedo
●UFundo oluPhezulu lweMakishi yemfuza: Sidibanisa i-high-throughput double barcode system evumela ulandelelwano ngaxeshanye lwabantu abaninzi, kunye ne-locus-specific amplification ephucula ukusebenza kakuhle, ukuqinisekisa ukuba amanani ethegi ahlangabezana neemfuno ezahlukeneyo zemibuzo yophando eyahlukeneyo.
● Ukuxhomekeka okuPhantsi kwiGenome: Ingasetyenziswa kwiintlobo ngeentlobo okanye ngaphandle kwe-genome yesalathiso.
●Uyilo lweSkimu esiguquguqukayo: I-enzyme enye, i-enzyme ephindwe kabini, i-digestion ye-enzyme eninzi, kunye neentlobo ezahlukeneyo ze-enzyme zinokukhethwa ukuba zihlangabezane neenjongo ezahlukeneyo zophando okanye iintlobo.
● Ukusebenza okuphezulu kwi-Enzymatic Digestion: Ukuqhuba kwekwi-silicoUyilo lwangaphambili kunye novavanyo lwangaphambili luqinisekisa uyilo olulolona lufanelekileyo kunye nokusasazwa kweethegi ze-SLAF kwikhromozomu (ithegi ye-1 SLAF / 4Kb) kunye nokunciphisa ukuphindaphinda ukulandelelana (<5%).
●UbuGcisa obuBanzi: Sizisa ubutyebi bamava kwiprojekthi nganye, kunye nerekhodi yokuvala iiprojekthi ze-SLAF-Seq ezingaphezulu kwama-5000 kumakhulu eentlobo zezilwanyana, kuquka izityalo, izilwanyana ezanyisayo, iintaka, izinambuzane, kunye nezinto eziphilayo zasemanzini.
● Ukuziphuhlisa kwe-Bioinformatic Workflow: Siphuhlise i-bioinformatic workflow edibeneyo ye-SLAF-Seq ukuqinisekisa ukuthembeka kunye nokuchaneka kwesiphumo sokugqibela.
IiNgcaciso zeNkonzo
| Uhlobo lokuhlalutya | Umlinganiselo wabemi ocetyiswayo | Isicwangciso sokulandelelana | |
| Ubunzulu bolandelelwano lwethegi | Inombolo yethegi | ||
| Iimephu zemfuzo | Abazali aba-2 kunye ne-150 yabantwana | Abazali: 20x WGS Inzala: 10x | Ubungakanani beGenome: <400 Mb: I-WGS iyacetyiswa <1Gb: 100K iithegi I-1-2Gb:: Iithegi ze-200K > 2Gb: 300K iithegi Ubuninzi bethegi ezingama-500k |
| Izifundo zeGenome-Wide Association (GWAS) | ≥200 iisampulu | 10x | |
| I-Genetic Evolution | ≥30 iisampulu, kunye> 10 iisampulu kwiqela elincinane ngalinye | 10x | |
IiMfuno zeNkonzo
Ugxininiso ≥ 5 ng/µL
Imali iyonke ≥ 80 ng
Nanodrop OD260/280=1.6-2.5
Ijeli yeAgarose: akukho okanye ukuthotywa okulinganiselweyo okanye ungcoliseko
Unikezelo lweSampuli eCetyisiweyo
Isikhongozeli: 2 ml centrifuge tube
(Kuninzi lweesampulu, sincoma ukuba ungayigcini kwi-ethanol)
Iisampulu zokufakwa ileyibhile: Iisampulu kufuneka zibhalwe ngokucacileyo kwaye zifane nefom yesampulu yolwazi engenisiweyo.
Ukuthumela: Umkhenkce owomileyo: Iisampulu kufuneka zipakishwe kwiingxowa kuqala kwaye zingcwatywe kwindawo eyomileyo.
Ukuhanjiswa kwenkonzo
Isampulu ye-QC
Ulingo lokulinga
SLAF-uvavanyo
ULungiselelo lweThala leeNcwadi
Ukulandelelanisa
Uhlalutyo lwedatha
Iinkonzo zasemva kokuthengiswa
Uhlalutyo lwethu lwe-bioinformatical lubandakanya:
Idatha ye-QC kunye nokunciphisa idatha ukususa ukufundwa kwe-N-rich, ukufundwa kwe-adapter okanye ukufundwa komgangatho ophantsi.
Ukulawulwa komgangatho wesibini wokufunda okucocekileyo ukujonga ukuhanjiswa kwesiseko, umgangatho wokulandelelana kunye novavanyo lwedatha, kodwa kwakhona ukujonga ukusebenza kakuhle kokugaya kunye nokufakwa okufunyenweyo.
Nje ukuba ufundo luhlolwe, kukho iindlela ezimbini zokukhetha:
- Imephu yereferensi yegenome
- Ngaphandle kwegenome yesalathiso: ukuhlanganisana
Emva kwayo, uhlalutyo lweethegi ze-SLAF zisetyenziselwa ukwenza ukufowuna okwahlukileyo ukunceda ukufunyanwa kophawu: SNP, InDel, SNV, CV ukufowuna kunye nochazo.
Ukuhanjiswa kweethegi zeSLAF kwiichromosomes:
Ukusasazwa kwee-SNPs kwiichromosomes:
Inkcazo ye-SNP
Jiang S, Li S, Luo J, Wang X and Shi C (2023) QTL mapping kunye nohlahlelo lwetranscriptome yesiqulatho seswekile ngexesha lokuvuthwa kweziqhamo.I-Pyrus pyrifolia.Ngaphambili. Isayensi yezityalo.14:1137104. doi: 10.3389/fpls.2023.1137104
Li, J., Zhang, Y., Ma, R., Huang, W., Hou, J., Fang, C., & Sun, L. (2022). Ukuchongwa kwe-st1 kutyhila ukhetho olubandakanya ukukhweliswa kwembewu kunye nomxholo we-oyile ngexesha lokufuywa kweembotyi zesoya.Ijenali yeBiyoteknoloji yeZityalo, 20(6), 1110-1121. https://doi.org/10.1111/pbi.13791
Xu, P., Zhang, X., Wang, X.okqhubekayo.Ulandelelwano lweGenome kunye nokwahluka kofuzo kwi-carp eqhelekileyo,Cyprinus carpio.UNat Genet 46, 1212-1219 (2014). https://doi.org/10.1038/ng.3098
Zhuang, W., Chen, H., Yang, M.okqhubekayo.I-genome ye-peanut elinywayo inika ulwazi malunga ne-karyotypes ye-legume, i-polyploid evolution kunye nokugcinwa kwezityalo.UNat Genet 51, 865–876 (2019). https://doi.org/10.1038/s41588-019-0402-2
| Unyaka | Ijenali | IF | Isihloko | Usetyenziso |
| 2022 | Unxibelelwano lwendalo | 17.694 | Isiseko se-genomic se-giga-chromosomes kunye ne-giga-genome ye-peony yomthi I-Paeonia ostii | I-SLAF-GWAS |
| 2015 | Ingcali yePhytologist entsha | 7.433 | Imizila yeenyawo zasekhaya ixhasa imimandla ye-genomic ebalulekileyo kwi-agronomic iimbotyi zesoya | I-SLAF-GWAS |
| 2022 | Ijenali yoPhando oluPhezulu | 12.822 | Genome-wide-wide artificial introgressions of Gossypium barbadense into G. hirsutum bonisa i-loci ephezulu yokuphucula ngaxeshanye umgangatho we-cotton fiber kunye nesivuno iimpawu | I-SLAF-Evolutionary genetics |
| 2019 | Isityalo seemolekyuli | 10.81 | Uhlalutyo lweGenomic yabemi kunye neNdibano yaseDe Novo ityhila iMvelaphi yeWeedy Irayisi njengoMdlalo wokuZiphendukela kwemvelo | I-SLAF-Evolutionary genetics |
| 2019 | Imfuzo yendalo | 31.616 | Ukulandelelana kweGenome kunye neentlobo ezahlukeneyo zemfuza ye-carp eqhelekileyo, i-Cyprinus carpio | Imephu ye-SLAF-Linkage |
| 2014 | Imfuzo yendalo | 25.455 | I-genome ye-peanut elinywayo inika ukuqonda kwi-karyotypes ye-legume, i-polyploid indaleko kunye nokufuywa kwezityalo. | Imephu ye-SLAF-Linkage |
| 2022 | Ijenali yeBiyoteknoloji yeZityalo | 9.803 | Ukuchongwa kwe-ST1 kutyhila ukhetho olubandakanya ukuhamba ngeenyawo kwimophology yembewu kunye nomxholo we-oyile ngexesha lokufuywa kweembotyi zesoya | Uphuhliso lwe-SLAF-Marker |
| 2022 | Ijenali yaMazwe ngaMazwe yeNzululwazi yeMolekyuli | 6.208 | Ukuchongwa kunye noPhuhliso lweMakishi yeDNA yeWheat-Leymus mollis 2Ns (2D) Ukutshintshwa kweChromosome yeDisomic | Uphuhliso lwe-SLAF-Marker |
| Unyaka | Ijenali | IF | Isihloko | Usetyenziso |
| 2023 | Imida kwinzululwazi yezityalo | 6.735 | Imephu ye-QTL kunye nohlalutyo lwe-transcriptome yesiqulatho seswekile ngexesha lokuvuthwa kweziqhamo zePyrus pyrifolia | Imephu yemfuzo |
| 2022 | Ijenali yeBiyoteknoloji yeZityalo | 8.154 | Ukuchongwa kwe-ST1 kutyhila ukhetho olubandakanya ukuhambahamba kwembewu kunye nomxholo we-oyile ngexesha lokufuywa kweembotyi zesoya.
| Ukufowuna kwe-SNP |
| 2022 | Imida kwinzululwazi yezityalo | 6.623 | Genome-Wide Association Mapping of Hulless Barely Phenotypes kwindawo yembalela.
| I-GWAS |
