Ukulandelana Kwezingcezu Ezihlanganisiwe Zendawo Ethile (SLAF-Seq)
Ukuhamba komsebenzi
Le nsizakalo inomklamo wangaphambilini ku-silico ukuqinisekisa ukukhethwa okuhle kakhulu kwama-enzyme ekulungiseleleni umtapo wolwazi.
Uhlelo Lobuchwepheshe
Izici Zesevisi
● Ukulandelana kwe-NovaSeq nge-PE150.
● Ukulungiswa komtapo wolwazi nge-barcode ephindwe kabili, okuvumela ukuqoqa amasampula angaphezu kwe-1000.
● Kungakhathaliseki ukuthi i-genome ibhekisela kuphi:
Ngokuphathelene ne-genome: Ukutholwa kwe-SNP kanye ne-InDel
Ngaphandle kwe-genome yokubhekisela: ukuhlanganiswa kwesampula kanye nokutholakala kwe-SNP
● Ku-i-in-silicoIzigaba zangaphambi kokuklama zihlolwe ukuze kutholakale lezo ezikhiqiza ukusatshalaliswa okufanayo kwamathegi e-SLAF ku-genome.
● Ngesikhathi sokuhlolwa kwangaphambi kokuhlolwa, kuhlolwa izinhlanganisela ezintathu zama-enzyme kumasampula amathathu ukuze kukhiqizwe imitapo yolwazi engu-9 ye-SLAF, futhi lolu lwazi lusetshenziselwa ukukhetha inhlanganisela ye-enzyme yokuvimbela efanele kakhulu yephrojekthi.
Izinzuzo Zesevisi
●Ukutholakala Kwezimpawu Eziphezulu Zezakhi Zofuzo: Sihlanganisa uhlelo lwebhakhodi ephindwe kabili olusebenzisa amandla aphezulu oluvumela ukulandelana kwenani elikhulu labantu ngesikhathi esisodwa, kanye nokukhulisa okuqondile kwendawo okuthuthukisa ukusebenza kahle, ukuqinisekisa ukuthi izinombolo zamathegi ziyahlangabezana nezidingo ezahlukahlukene zemibuzo eyahlukahlukene yocwaningo.
● Ukuthembela Okuphansi ku-Genome: Ingasetshenziswa ezinhlotsheni ezine-genome ebhekisele kuzo noma ezingenayo.
●Umklamo Wesikimu Oguquguqukayo: Ukugaya ukudla okune-enzyme eyodwa, i-enzyme ekabili, i-enzyme eningi, kanye nezinhlobo ezahlukene zama-enzyme kungakhethwa ukuze kuhlangatshezwane nezinhloso noma izinhlobo ezahlukene zocwaningo.
● Ukusebenza Kahle Kakhulu Ekugayweni Kwe-Enzymatic: Ukuqhutshwa kwe-i-in-silicoukwakheka kwangaphambilini kanye nokuhlolwa kwangaphambilini kuqinisekisa ukwakheka okuhle kakhulu ngokusatshalaliswa okulinganayo kwamathegi e-SLAF ku-chromosome (ithegi eli-1 le-SLAF/4Kb) kanye nokulandelana okuphindaphindwayo okuncishisiwe (<5%).
●Ubuchwepheshe Obubanzi: Siletha ulwazi oluningi kuyo yonke iphrojekthi, ngomlando wokuvala amaphrojekthi e-SLAF-Seq angaphezu kuka-5000 ezinhlobonhlobo zezilwane, okuhlanganisa izitshalo, izilwane ezincelisayo, izinyoni, izinambuzane, kanye nezinto eziphilayo zasemanzini.
● Ukuhamba Komsebenzi Ozithuthukisayo We-Bioinformatic: Sakhe umsebenzi ohlanganisiwe we-bioinformatic we-SLAF-Seq ukuqinisekisa ukuthembeka nokunemba komkhiqizo wokugcina.
Imininingwane Yesevisi
| Uhlobo lokuhlaziya | Isikali sabantu esinconywayo | Isu lokulandelana kwezigaba | |
| Ukujula kokulandelana kwamathegi | Inombolo yethegi | ||
| Amamephu Ezakhi Zofuzo | Abazali ababili kanye nezingane ezingaphezu kuka-150 | Abazali: 20x WGS Ukunciphisa: 10x | Usayizi we-genome: <400 Mb: Kunconywa i-WGS <1Gb: Amathegi angu-100K Amathegi angu-1-2Gb:: angu-200K >2Gb: Amathegi angu-300K Amathegi aphezulu angu-500k |
| Izifundo Zokuhlangana Kwama-Genome-Wide (i-GWAS) | Amasampula angu-≥200 | 10x | |
| Ukuziphendukela Kwezakhi Zofuzo | Amasampula angu-≥30, kanye namasampula angu->10 avela eqenjini ngalinye elincane | 10x | |
Izidingo Zesevisi
Ukugxila ≥ 5 ng/µL
Inani eliphelele ≥ 80 ng
I-Nanodrop OD260/280=1.6-2.5
Ijeli le-Agarose: akukho noma ukuwohloka okulinganiselwe noma ukungcola
Ukulethwa Kwesampula Okunconyiwe
Isitsha: ishubhu le-centrifuge elingu-2 ml
(Ngeningi lamasampula, sincoma ukuthi ungagcini ku-ethanol)
Ukulebula amasampula: Amasampula kudingeka abhalwe ngokucacile futhi afane nefomu lolwazi lesampula elithunyelwe.
Ukuthunyelwa: Iqhwa elomile: Amasampula kudingeka apakishwe ezikhwameni kuqala bese egqitshwa eqhweni elomile.
Ukuhamba Komsebenzi Wesevisi
Isampula ye-QC
Ukuhlolwa kwe-pilot
Ukuhlolwa kwe-SLAF
Ukulungiselela Umtapo Wolwazi
Ukulandelana
Ukuhlaziywa Kwedatha
Izinsizakalo Zangemva Kokuthengisa
Ukuhlaziywa kwethu kwe-bioinformatic kuhlanganisa:
I-Data QC kanye nokunciphisa idatha ukuze kususwe ukufundwa kwe-N-rich, ukufundwa kwe-adaptha noma ukufundwa kwekhwalithi ephansi.
Ukulawulwa kwekhwalithi yesibili kokufundwa okuhlanzekile ukuhlola ukusatshalaliswa kwesisekelo, ikhwalithi yokulandelana kanye nokuhlolwa kwedatha, kodwa futhi nokuhlola ukusebenza kahle kokugaya kanye nokufakwa okutholiwe.
Uma ukufundwa sekuhloliwe, kunezinketho ezimbili:
- Ukuhlela imephu ukuze kubhekwe i-genome
- Ngaphandle kwe-genome ebhekisela kuyo: ukuqoqana
Ngemva kwalokho, ukuhlaziywa kwamathegi e-SLAF kusetshenziselwa ukwenza ukubiza okuhlukile ukusiza ekutholakaleni komaka: i-SNP, i-InDel, i-SNV, ukubiza i-CV kanye nesichasiselo
Ukusatshalaliswa kwamathegi e-SLAF kuma-chromosome:
Ukusatshalaliswa kwama-SNP kuma-chromosome:
Isichasiselo se-SNP
UJiang S, uLi S, uLuo J, uWang X noShi C (2023) Ukuhlaziywa kwemephu ye-QTL kanye nokuhlaziywa kwe-transcriptome kokuqukethwe kukashukela ngesikhathi sokuvuthwa kwezitheloI-Pyrus pyrifolia.Ngaphambili. Isayensi Yezitshalo.14:1137104. doi: 10.3389/fpls.2023.1137104
ULi, J., Zhang, Y., Ma, R., Huang, W., Hou, J., Fang, C., & Sun, L. (2022). Ukuhlonza i-st1 kwembula ukukhetha okubandakanya ukuhamba ngebhayisikili kwesimo sembewu kanye nokuqukethwe kwamafutha ngesikhathi sokufuywa kwesoya.Ijenali ye-Plant Biotechnology, 20(6), 1110-1121. https://doi.org/10.1111/pbi.13791
Xu, P., Zhang, X., Wang, X.nabanye.Ukulandelana kwezakhi zofuzo kanye nokuhlukahluka kwezakhi zofuzo ze-carp evamile,uCyprinus carpio.UNat Genet 46, 1212–1219 (2014). https://doi.org/10.1038/ng.3098
Zhuang, W., Chen, H., Yang, M.nabanye.I-genome yamantongomane atshaliwe inikeza ukuqonda ngezinhlobo ze-legume, ukuvela kwe-polyploid kanye nokukhula kwezitshalo.UNat Genet 51, 865–876 (2019). https://doi.org/10.1038/s41588-019-0402-2
| Unyaka | Ijenali | IF | Isihloko | Izicelo |
| 2022 | Ukuxhumana kwemvelo | 17.694 | Isisekelo se-genomic sama-giga-chromosomes kanye ne-giga-genome ye-peony yesihlahla I-Paeonia ostii | I-SLAF-GWAS |
| 2015 | Isazi Sezitshalo Esisha | 7.433 | Izinyathelo zokufuywa kwezindawo zasekhaya ziqinisa izindawo zofuzo ezibalulekile kwezolimo ubhontshisi | I-SLAF-GWAS |
| 2022 | Ijenali Yocwaningo Oluthuthukisiwe | 12.822 | Ukungena kokwenziwa okubanzi kwe-genome kwe-Gossypium barbadense ku-G. hirsutum kwembula indawo engcono kakhulu yokuthuthukisa ikhwalithi kanye nokukhiqiza kwefayibha kakotini ngasikhathi sinye izici | I-SLAF-Izakhi zofuzo eziziphendukela kwemvelo |
| 2019 | Isitshalo Se-Molecular | 10.81 | Ukuhlaziywa Kwezakhi Zofuzo Zabantu kanye Nokuhlanganiswa Kwe-De Novo Kwembula Umsuka We-Weedy Ilayisi njengomdlalo wokuziphendukela kwemvelo | I-SLAF-Izakhi zofuzo eziziphendukela kwemvelo |
| 2019 | Izakhi Zofuzo Zemvelo | 31.616 | Ukulandelana kwezakhi zofuzo kanye nokuhlukahluka kwezakhi zofuzo ze-carp evamile, i-Cyprinus carpio | Imephu ye-SLAF-Linkage |
| 2014 | Izakhi Zofuzo Zemvelo | 25.455 | I-genome yamantongomane atshaliwe inikeza ukuqonda ngezinhlobo ze-legume, i-polyploid ukuvela kwemvelo kanye nokufuywa kwezitshalo. | Imephu ye-SLAF-Linkage |
| 2022 | Ijenali ye-Plant Biotechnology | 9.803 | Ukuhlonza i-ST1 kwembula ukukhetha okubandakanya ukugibela i-lifthill kwesimo sembewu kanye nokuqukethwe kwamafutha ngesikhathi sokusetshenziswa kwesoya | Ukuthuthukiswa kwe-SLAF-Marker |
| 2022 | Ijenali Yomhlaba Wonke Yesayensi Yamamolekyuli | 6.208 | Ukuhlonza kanye Nokuthuthukiswa Kophawu Lwe-DNA Lwe-Wheat-Leymus mollis 2Ns (2D) Ukufakwa esikhundleni kwe-Disomic Chromosome | Ukuthuthukiswa kwe-SLAF-Marker |
| Unyaka | Ijenali | IF | Isihloko | Izicelo |
| 2023 | Imingcele kwisayensi yezitshalo | 6.735 | Ukuhlaziywa kwemephu ye-QTL kanye nokuhlaziywa kwe-transcriptome kokuqukethwe kukashukela ngesikhathi sokuvuthwa kwezithelo ze-Pyrus pyrifolia | Imephu Yezakhi Zofuzo |
| 2022 | Ijenali ye-Plant Biotechnology | 8.154 | Ukuhlonza i-ST1 kwembula ukukhetha okubandakanya ukugibela i-lifthill kwesimo sembewu kanye nokuqukethwe kwamafutha ngesikhathi sokusetshenziswa kwesoya
| Ukushaya kwe-SNP |
| 2022 | Imingcele kwisayensi yezitshalo | 6.623 | Ukumapha Kokuhlobana Okubanzi Kwezakhi Zofuzo Zemvelo Ezingenazo Izinto Eziningi Emvelweni Yesomiso.
| I-GWAS |
