Tatellano ea Likaroloana tse Entsoeng ka Sebaka se Ikhethang (SLAF-Seq)
Mokgwa wa mosebetsi
Tšebeletso ena e na le moralo o itseng esale pele ka silico ho netefatsa khetho e ntle ka ho fetisisa ea li-enzyme ha ho lokisoa laeborari.
Morero oa Tekheniki
Likarolo tsa Tšebeletso
● Ho latellana ha NovaSeq le PE150.
● Tokisetso ea laeborari ka ho sebelisa li-barcode tse peli, e nolofalletsang ho bokella lisampole tse fetang 1000.
● Ho sa tsotelehe genome ea litšupiso:
Ka genome e referense: Ho sibolloa ha SNP le InDel
Ntle le genome ea referense: ho bokellana ha sampole le ho sibolloa ha SNP
● Ka hare hoka har'a silikoMetswako ya dienzyme tse ngata tse thibelang di-restriction e hlahlojwa mohatong wa pele ho moralo ho fumana tse hlahisang kabo e tshwanang ya di-tag tsa SLAF ho pholletsa le genome.
● Nakong ea teko ea pele ho liteko, metsoako e meraro ea li-enzyme e lekoa lisampoleng tse 3 ho hlahisa lilaebrari tse 9 tsa SLAF, 'me tlhahisoleseling ena e sebelisoa ho khetha motsoako o motle oa li-enzyme tsa thibelo bakeng sa projeke.
Melemo ea Tšebeletso
●Ho sibolloa ha Letšoao le Phahameng la Liphatsa tsa Lefutso: Re kopanya sistimi ea barcode e habeli e nang le phallo e phahameng e lumellang tatellano ea baahi ba bangata ka nako e le 'ngoe, le keketseho e khethehileng ea sebaka se itseng e ntlafatsang bokhoni, ho netefatsa hore linomoro tsa tag li fihlela litlhoko tse fapaneng tsa lipotso tse fapaneng tsa lipatlisiso.
● Ho Itšetleha ho Tlase ho Genome: E ka sebelisoa ho mofuta o nang le genome e referense kapa o se nang eona.
●Moralo oa Morero o Tenyetsehang: Enzyme e le 'ngoe, enzyme e habeli, tšilo ea lijo ea enzyme e mengata, le mefuta e fapaneng ea lienzyme kaofela li ka khethoa ho khotsofatsa lipheo kapa mefuta e fapaneng ea lipatlisiso.
● Bokgoni bo Phahameng ba ho Sila Dintho ka Enzymatic: Ho tsamaisoa haka har'a silikoMoralo oa pele le teko ea pele li netefatsa moralo o motle ka kabo e lekanang ea li-tag tsa SLAF ho chromosome (tag e le 'ngoe ea SLAF/4Kb) le tatellano e fokotsehileng ea ho pheta-pheta (<5%).
●Bokgoni bo Pharaletseng: Re tlisa boiphihlelo bo bongata morerong o mong le o mong, ka rekoto ea ho koala merero e fetang 5000 ea SLAF-Seq mefuteng e makholo ea limela, ho kenyeletsoa limela, liphoofolo tse anyesang, linonyana, likokoanyana le lintho tse phelang metsing.
● Mokhoa oa Mosebetsi oa Bioinformatic o iketselitseng: Re ntlafalitse mokhoa o kopaneng oa ho sebetsa oa bioinformatic bakeng sa SLAF-Seq ho netefatsa botšepehi le ho nepahala ha tlhahiso ea ho qetela.
Litlhaloso tsa Tšebeletso
| Mofuta oa tlhahlobo | Sekala se kgothaletswang sa baahi | Leano la ho latellana ha tatellano | |
| Botebo ba tatellano ea li-tag | Nomoro ea tag | ||
| Limmapa tsa Liphatsa tsa Lefutso | Batsoali ba 2 le bana ba ka holimo ho 150 | Batsoali: 20x WGS Ho fokotsa: 10x | Boholo ba liphatsa tsa lefutso: <400 Mb: WGS e kgothaletswa <1Gb: Li-tag tse 100K 1-2Gb:: Li-tag tse 200K >2Gb: Li-tag tsa 300K Li-tag tse phahameng ka ho fetisisa tsa 500k |
| Lithuto tsa Mokhatlo oa Genome-Wide (GWAS) | ≥ disampole tse 200 | 10x | |
| Phetoho ea Liphatsa tsa Lefutso | disampole tse ≥30, ka disampole tse ka bang >10 ho tsoa sehlopheng se seng le se seng | 10x | |
Litlhoko tsa Tšebeletso
Ho tsepamisa maikutlo ≥ 5 ng/µL
Kakaretso ea chelete ≥ 80 ng
Nanodrop OD260/280=1.6-2.5
Jeli ea Agarose: ha e na kapa ha e na ho senyeha kapa ho silafala ho lekanyelitsoeng
Thomello e Khothaletsoang ea Sampole
Setshelo: 2 ml tube ea centrifuge
(Bakeng sa boholo ba disampole, re kgothaletsa hore di se ke tsa bolokwa ka hara ethanol)
Ho ngolwa ha mehlala: Disampole di lokela ho ngolwa ka ho hlaka le ho tshwana le foromo ya tlhahisoleseding ya sampole e rometsweng.
Thomello: Leqhoa le omileng: Disampole di hloka ho pakwa ka mekotleng pele mme di patwe ka hara leqhoa le omileng.
Mokhoa oa Tšebeletso
Mohlala oa QC
Teko ea sefofane
Teko ea SLAF
Litokisetso tsa Laeborari
Tatellano
Tlhahlobo ea Lintlha
Litšebeletso tsa Kamora thekiso
Tlhahlobo ea rona ea bioinformatics e kenyelletsa:
QC ea data le ho fokotsa data ho tlosa li-read tse nang le N-rich, li-read tsa adaptara kapa li-read tsa boleng bo tlaase.
Taolo ea bobeli ea boleng ba ho bala ho hloekileng ho hlahloba kabo ea motheo, boleng ba tatellano le tlhahlobo ea data, empa hape le ho hlahloba katleho ea tšilo ea lijo le lintho tse kentsoeng tse fumanoeng.
Hang ha lipalo li hlahlobiloe, ho na le likhetho tse peli:
- Ho etsa 'mapa ho ea ho li-genome tsa litšupiso
- Ntle le genome ea referense: ho bokellana ha lihlopha
Kamora eona, tlhahlobo ea li-tag tsa SLAF e sebelisoa ho etsa mehala e fapaneng ho thusa ka ho sibolla letšoao: SNP, InDel, SNV, CV call le tlhaloso.
Kabo ea li-tag tsa SLAF ho li-chromosome:
Kabo ea li-SNP ho li-chromosome:
Tlhaloso ea SNP
Jiang S, Li S, Luo J, Wang X le Shi C (2023) Tlhahlobo ea 'mapa oa QTL le transcriptome ea litaba tsa tsoekere nakong ea ho butsoa ha litholoanaPyrus pyrifolia.Ka pele. Saense ea Semela.14:1137104. doi: 10.3389/fpls.2023.1137104
Li, J., Zhang, Y., Ma, R., Huang, W., Hou, J., Fang, C., & Sun, L. (2022). Ho tsebahatsa st1 ho senola kgetho e kenyeletsang ho palama lipere tsa peo le oli nakong ya ho rua soya.Koranta ea Theknoloji ea Limela, 20(6), 1110-1121. https://doi.org/10.1111/pbi.13791
Xu, P., Zhang, X., Wang, X.le ba bang.Tatellano ea liphatsa tsa lefutso le mefuta-futa ea liphatsa tsa lefutso tsa carp e tloaelehileng,Cyprinus carpio.Nat Genet 46, 1212–1219 (2014). https://doi.org/10.1038/ng.3098
Zhuang, W., Chen, H., Yang, M.le ba bang.Jenome ea peanut e lengoang e fana ka temohisiso mabapi le mefuta ea linaoa, phetoho ea polyploid le ho rua lijalo lapeng.Nat Genet 51, 865–876 (2019). https://doi.org/10.1038/s41588-019-0402-2
| Selemo | Koranta | IF | Sehlooho | Likopo |
| 2022 | Puisano ea tlhaho | 17.694 | Motheo oa genome oa li-chromosome tsa giga le genome ea giga ea peony ea sefate Paeonia ostii | SLAF-GWAS |
| 2015 | Setsebi se Secha sa Litho tsa 'Mele | 7.433 | Mehato ea ho rua liphoofolo lapeng e tšehetsa libaka tsa genomic tsa bohlokoa ba temo ho linaoa tsa soya | SLAF-GWAS |
| 2022 | Koranta ea Lipatlisiso tse Tsoetseng Pele | 12.822 | Ho kenella ha maiketsetso ka genome ho Gossypium barbadense ho G. hirsutum senola sebaka se setle sa ntlafatso ea boleng le chai ea k'hothone ka nako e le 'ngoe litšobotsi | SLAF-Liphatsa tsa lefutso tsa ho iphetola ha lintho |
| 2019 | Semela sa limolek'hule | 10.81 | Tlhahlobo ea Genomic ea Baahi le Kopano ea De Novo li Senola Tšimoloho ea Weedy Raese e le Papali ea ho Iphetola ha Lintho | SLAF-Liphatsa tsa lefutso tsa ho iphetola ha lintho |
| 2019 | Liphatsa tsa lefutso tsa tlhaho | 31.616 | Tatellano ea liphatsa tsa lefutso le mefuta-futa ea liphatsa tsa lefutso tsa carp e tloaelehileng, Cyprinus carpio | 'Mapa oa SLAF-Linkage |
| 2014 | Liphatsa tsa lefutso tsa tlhaho | 25.455 | Genome ea peanut e lengoang e fana ka temohisiso mabapi le mefuta ea linaoa, polyploid ho iphetola ha lintho le ho rua lijalo lapeng. | 'Mapa oa SLAF-Linkage |
| 2022 | Koranta ea Theknoloji ea Limela | 9.803 | Ho hlwaya ST1 ho senola kgetho e kenyeletsang ho palama lifte tsa sebopeho sa peo le oli nakong ea ho rua soya lapeng | Nts'etsopele ea Letšoao la SLAF |
| 2022 | Koranta ea Machaba ea Saense ea Limolek'hule | 6.208 | Tlhahisoleseding le Ntlafatso ea Letšoao la DNA bakeng sa 2Ns (2D) ea Koro-Leymus mollis Ho Nkela Disomic Chromosome Sebakeng | Nts'etsopele ea Letšoao la SLAF |
| Selemo | Koranta | IF | Sehlooho | Likopo |
| 2023 | Meeli ea saense ea limela | 6.735 | Tlhahlobo ea 'mapa oa QTL le tlhahlobo ea transcriptome ea litaba tsa tsoekere nakong ea ho butsoa ha litholoana tsa Pyrus pyrifolia | 'Mapa oa Liphatsa tsa Lefutso |
| 2022 | Koranta ea Theknoloji ea Limela | 8.154 | Ho hlwaya ST1 ho senola kgetho e kenyeletsang ho palama lieta tsa peo le oli nakong ea ho lema soya lapeng.
| Ho letsetsa SNP |
| 2022 | Meeli ea saense ea limela | 6.623 | 'Mapa oa Mokhatlo oa Li-genome-Wide oa Li-phenotype tse se nang Li-Hullless Barely Tikolohong ea Komello.
| GWAS |
