Ukulandelanisa Kwezitshalo/Isilwane Se-De Novo Genome
I-De NovoUkulandelana kubhekisela ekwakhiweni kwe-genome yonke yohlobo kusetshenziswa ubuchwepheshe bokulandelana ngaphandle kwe-genome yokubhekisela. Ukwethulwa kanye nokwamukelwa kabanzi kokulandelana kwesizukulwane sesithathu, okuhlanganisa ukufunda okude, kuye kwathuthukisa kakhulu ukuhlanganiswa kwe-genome ngokwandisa ukuhlangana phakathi kokufundwa. Lokhu kuthuthukiswa kubaluleke kakhulu lapho kubhekene nama-genome ayinselele, njengalawo abonisa i-heterozygosity ephezulu, isilinganiso esiphezulu sezifunda eziphindaphindayo, ama-polyploid, kanye nezifunda ezinezici eziphindaphindayo, okuqukethwe kwe-GC okungavamile, noma ubunzima obukhulu obuvame ukuhlanganiswa kabi kusetshenziswa ukulandelana kokufunda okufushane kuphela.
Isixazululo sethu esisodwa sinikeza izinsizakalo zokulandelana ezihlanganisiwe kanye nokuhlaziywa kwe-bioinformatic okuletha i-genome ehlanganisiwe yekhwalithi ephezulu. Ucwaningo lokuqala lwe-genome ne-Illumina lunikeza izilinganiso zobukhulu be-genome kanye nobunzima, futhi lolu lwazi lusetshenziselwa ukuqondisa isinyathelo esilandelayo sokulandelana okufundwa isikhathi eside nge-PacBio HiFi, kulandelwe yide novoukuhlanganiswa kwama-contig. Ukusetshenziswa okulandelayo kwe-HiC assembly kwenza kube lula ukunamathela kwama-contig ku-genome, kutholakale i-chromosome-level assembly. Okokugcina, i-genome ichazwa ngokubikezela izakhi zofuzo nangokulandelanisa izakhi zofuzo eziveziwe, kusetshenziswe ama-transcriptome anokufundwa okufushane nokude.
Izici Zesevisi
● Ukuhlanganiswa kwezinsizakalo eziningi zokulandelana kanye ne-bioinformatic kusisombululo esisodwa:
Ucwaningo lwe-genome ne-Illumina ukuze kulinganiswe usayizi we-genome futhi kuqondiswe izinyathelo ezilandelayo;
Ukulandelana kokufunda isikhathi eside kwede novoukuhlanganiswa kwama-contigs;
Ukulandelana kwe-Hi-C kokuhlanganiswa kwe-chromosome;
ukulandelana kwe-mRNA ukuze kuchazwe izakhi zofuzo;
Ukuqinisekiswa kwenhlangano.
● Isevisi efanelekela ukwakha ama-genome amasha noma ukuthuthukiswa kwama-genome okubhekisela akhona ezinhlobo ezithakazelisayo.
Izinzuzo Zesevisi
Ukuthuthukiswa kwamapulatifomu okulandelana kanye ne-bioinformatics kude novoukuhlanganiswa kwe-genome
(Amarasinghe SL et al.,Ibhayoloji Yezakhi Zofuzo, 2020)
●Ubuchwepheshe Obubanzi kanye Nerekhodi Lokushicilela: I-BMKGene iqoqe ulwazi olukhulu ekuhlanganisweni kwe-genome esezingeni eliphezulu yezinhlobo ezahlukahlukene, kufaka phakathi ama-genome e-diploid kanye nama-genome ayinkimbinkimbi kakhulu ezinhlobo ze-polyploid kanye ne-allopolyploid. Kusukela ngo-2018, sibe negalelo elingaphezu kwalokho.Izincwadi ezingu-300 ezinomthelela omkhulu, kanti ezingu-20+ zazo zishicilelwe ku-Nature Genetics.
● Isixazululo Esihamba Kanye: indlela yethu ehlanganisiwe ihlanganisa ubuchwepheshe obuningi bokulandelana kanye nokuhlaziywa kwe-bioinformatic kube ukuhamba komsebenzi okuhlangene, okuletha i-genome ehlanganisiwe esezingeni eliphezulu.
●Kulungiselelwe Izidingo Zakho: Ukuhamba komsebenzi wethu wesevisi kungenziwa ngokwezifiso, okuvumela ukuzivumelanisa nezimo zama-genome anezici ezahlukahlukene kanye nezidingo ezithile zocwaningo. Lokhu kufaka phakathi ukwamukela ama-genome amakhulu, ama-genome e-polyploid, ama-genome angenawo ugesi kakhulu, nokuningi.
●Ithimba Le-Bioinformatics Nelebhu Elinekhono Eliphezulu: onolwazi olukhulu kokubili emkhakheni wokuhlola kanye nowe-bioinformatics wezinhlangano ze-genome eziyinkimbinkimbi kanye nochungechunge lwamalungelo obunikazi kanye namalungelo okushicilela esofthiwe.
●Usekelo Lwangemva Kokuthengisa:Ukuzibophezela kwethu kudlulela ngale kokuqedwa kwephrojekthi ngesikhathi senkonzo yezinyanga ezintathu ngemuva kokuthengisa. Phakathi nalesi sikhathi, sinikeza ukulandelwa kwephrojekthi, usizo lokuxazulula izinkinga, kanye nezikhathi ze-Q&A ukuze kuxazululwe noma yimiphi imibuzo ehlobene nemiphumela.
Imininingwane Yesevisi
| Ucwaningo lwe-genome | Ukuhlanganiswa kwe-genome | Izinga le-Chromosome | Isichasiselo se-Genome |
| I-50X Illumina NovaSeq PE150
| Ama-HiFi reads angu-30X e-PacBio CCS | I-Hi-C engu-100X | I-RNA-seq Illumina PE150 10 Gb + (ongakukhetha) I-RNA-seq ephelele i-PacBio engu-40 Gb noma I-Nanopore engu-12 Gb |
Izidingo Zesevisi
Ngocwaningo lwe-Genome, i-Genome Assembly kanye ne-Hi-C Assembly:
| Ama-nucleic acid noma izicubu ezikhishwe | Ucwaningo lwe-Genome | Ukuhlanganiswa kwe-Genome nge-PacBio | Umhlangano we-Hi-C |
| I-Animal Viscera | ≥ 0.2 g
| ≥ 1 g | ≥ 1 g |
| Imisipha Yezilwane | |||
| Igazi Lezilwane Ezincelisayo | ≥ 0.3 mL | ≥ 1 mL | ≥ 2 mL |
| Igazi Lenkukhu/Lenhlanzi | ≥ 0.05 mL | ≥ 0.1 mL | |
| Isitshalo - Iqabunga Elisha | ≥ 0.5 g | ≥ 2 g | ≥ 2 g |
| Amaseli Akhuliswe | ≥ 1x106 | ≥ 5x106 | ≥ 1x107 |
| Isinambuzane | ≥ 0.2 g | ≥ 1 g | ≥ 1 g |
| Izinto eziphilayo zasemanzini – Izicubu zemisipha | ≥ 0.3 g | ≥ 1.5 g | ≥ 1.0 g |
| Izinto eziphilayo zasemanzini – Ulwelwe | ≥ 0.5 g | ≥ 3.0 g | ≥ 2.0 g |
| I-DNA ekhishwe | Ukugxila: ≥1 ng/ µL Inani ≥ 30 ng Ukuwohloka noma ukungcola okulinganiselwe noma okungenakho | Ukugxila: ≥ 50 ng/µL Inani: 8 µg/iseli eligelezayo/isampula OD260/280=1.7-2.2 OD260/230=1.8-2.5 Ukuwohloka noma ukungcola okulinganiselwe noma okungenakho |
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Ukuze uthole isichasiselo se-Genome nge-transcriptomics:
| Ama-nucleic acid noma izicubu ezikhishwe | I-Transcriptome ye-Illumina | I-Transcriptome ye-PacBio | I-Nanopore Transcriptome |
| Isitshalo - Impande/Isiqu/Iqabunga | 450 mg | 600 mg | |
| Isitshalo - Iqabunga/Imbewu | 300 mg | 300 mg | |
| Isitshalo - Izithelo | 1.2 g | 1.2 g | |
| Inhliziyo/Amathumbu Esilwane | 300 mg | 300 mg | |
| I-Animal Viscera/Ubuchopho | 240 mg | 240 mg | |
| Imisipha Yezilwane | 450 mg | 450 mg | |
| Amathambo Ezilwane/Izinwele/Isikhumba | 1 g | 1 g | |
| I-Arthropod - Izinambuzane | 6 | 6 | |
| I-Arthropod - I-Crustacea | 300 mg | 300 mg | |
| Igazi eliphelele | Ishubhu eli-1 | Ishubhu eli-1 | |
| I-RNA ekhishwe | Ukugxila: ≥ 20 ng/µL Inani ≥ 0.3 µg OD260/280=1.7-2.5 OD260/230=0.5-2.5 I-RIN≥ 6 5≥28S/18S≥1 | Ukugxila: ≥ 100 ng/µL Inani ≥ 1 µg OD260/280=1.7-2.5 OD260/230=0.5-2.5 I-RIN≥ 8 5≥28S/18S≥1 | Ukugxila: ≥ 100 ng/µL Inani ≥ 1 µg OD260/280=1.7-2.5 OD260/230=0.5-2.5 I-RIN≥ 7.5 5≥28S/18S≥1 |
Ukulethwa Kwesampula Okunconyiwe
Isitsha: Ishubhu le-centrifuge elingu-2 ml (Akukhuthazwa ukusebenzisa i-tin foil)
(Ngeningi lamasampula, sincoma ukuthi ungagcini ku-ethanol.)
Ukulebula isampula: Amasampula kumele abhalwe ngokucacile futhi afane nefomu lolwazi lesampula elithunyelwe.
Ukuthunyelwa: Iqhwa elomile: Amasampula kudingeka apakishwe ezikhwameni kuqala bese egqitshwa eqhweni elomile.
Ukuhamba komsebenzi
Ukugeleza Komsebenzi Wesevisi
Umklamo wokuhlola
Ukulethwa kwesampula
Ukukhishwa kwe-DNA
Ukwakhiwa komtapo wolwazi
Ukulandelana
Ukuhlaziywa kwedatha
Izinsizakalo zangemuva kokuthengisa
Ukuhlaziywa okuphelele kwe-bioinformatic, okuhlukaniswe ngezinyathelo ezine:
1) Ucwaningo lwe-Genome, olusekelwe ekuhlaziyweni kwe-k-mer nge-NGS lufundeka kanje:
Ukuqagela usayizi we-genome
Isilinganiso se-heterozygosity
Isilinganiso sezindawo eziphindaphindayo
2) Ukuhlanganiswa kwe-Genome nge-PacBio HiFi:
De novoumhlangano
Ukuhlolwa kokuhlanganiswa: kufaka phakathi ukuhlaziywa kwe-BUSCO kokuphelela kwe-genome kanye nokuhlela ngemuva kwe-NGS kanye ne-PacBio HiFi reads
3) Ukuhlanganiswa kwe-Hi-C:
I-QC yelabhulali ye-Hi-C: ukulinganisa ukusebenzisana kwe-Hi-C okusebenzayo
Umhlangano we-Hi-C: ukuhlanganiswa kwama-contig ngamaqembu, kulandelwe ukuhleleka kwama-contig ngaphakathi kweqembu ngalinye kanye nokwabela ukuqondiswa kwama-contig
Ukuhlolwa kwe-Hi-C
4) Isichasiselo se-genome:
Ukubikezela kwe-RNA okungasebenzisi ikhodi
Ukuhlonza ukulandelana okuphindaphindiwe (ama-transposon kanye nokuphindaphinda kwe-tandem)
Ukubikezela kwezakhi zofuzo
§De novo: ama-algorithms okuqala
§ Kusekelwe ku-homology
§ Ngokusekelwe ku-transcriptome, ngokufundwa okude nokufushane: ukufundwa kuyafanade novokuhlanganiswe noma kufakwe kumephu ye-genome eklanyiwe
§ Isichasiselo sezakhi zofuzo ezibikezelwe ngemininingwane eminingi
1) Ucwaningo lwe-Genome- k-mer analysis
2) Ukuhlanganiswa kwe-Genome
2) Ukuhlanganiswa kwe-Genome – I-PacBio HiFi ifunda ukumepha ukuhlanganiswa kohlaka
2) Ukuhlanganiswa kwe-Hi-C – ukulinganiselwa kwama-pair okusebenzisana asebenzayo e-Hi-C
3) Ukuhlolwa kwe-Hi-C ngemuva kokuhlanganiswa
4) Isichasiselo se-Genome – ukuhlanganiswa kwezakhi zofuzo ezibikezelwe
4) Isichasiselo se-Genome – isichasiselo se-Gene esibikezelwe
Hlola intuthuko eyenziwe yizinsizakalo zokuhlanganisa i-de novo genome ze-BMKGene ngeqoqo lezincwadi ezikhethiwe:
ULi, C. et al. (2021) 'Ukulandelana kwezakhi zofuzo kwembula imizila yokusabalala komhlaba wonke futhi kusikisela ukuzivumelanisa kwezakhi zofuzo ezihambisanayo ekuziphendukeleni kwehhashi lasolwandle', Nature Communications, 12(1). doi: 10.1038/S41467-021-21379-X.
ULi, Y. nabanye. (2023) 'Izinguquko Ezinkulu Ze-Chromosomal Ziholela Ekushintsheni Kokubonakaliswa Kwezinga Le-Genome, Ukuzivumelanisa Nemvelo, kanye Nokukhethekile ku-Gayal (Bos frontalis)', i-Molecular Biology kanye Nokuziphendukela Kwemvelo, 40(1). doi: 10.1093/MOLBEV/MSAD006.
UTian, T. et al. (2023) 'Ukuhlanganiswa kwezakhi zofuzo kanye nokwahlukaniswa kwezakhi zofuzo ze-germplasm yommbila evelele engamelani nesomiso', i-Nature Genetics 2023 55:3, 55(3), amakhasi 496–506. doi: 10.1038/s41588-023-01297-y.
UZhang, F. nabanye. (2023) 'Ukwembula ukuvela kwe-tropane alkaloid biosynthesis ngokuhlaziya ama-genome amabili emndenini wakwaSolanaceae', Nature Communications 2023 14:1, 14(1), pp. 1–18. doi: 10.1038/s41467-023-37133-4.
Izifundo zamacala eziyinselele:
Ukuhlanganiswa kwe-Telomere kuya ku-telomere:UFu, A. et al. (2023) 'Ukuhlanganiswa kwe-genome ye-Telomere-kuya-telomere ye-bitter melon (Momordica charantia L. var. abbreviata Ser.) kwembula ukuthuthukiswa kwezithelo, ukwakheka kanye nezici zofuzo zokuvuthwa', i-Horticulture Research, 10(1). doi: 10.1093/HR/UHAC228.
Ukuhlanganiswa kwe-Haplotype:UHu, W. et al. (2021) 'I-genome echazwe yi-allele yembula ukuhlukaniswa kwe-biallelic ngesikhathi sokuvela kwe-cassava', i-Molecular Plant, 14(6), amakhasi 851–854. doi: 10.1016/j.molp.2021.04.009.
Ukuhlanganiswa kwe-genome enkulu:UYuan, J. nabanye. (2022) 'Isisekelo se-genomic sama-chromosome e-giga kanye ne-genome ye-giga ye-peony yesihlahla i-Paeonia ostii', i-Nature Communications 2022 13:1, 13(1), amakhasi 1–16. doi: 10.1038/s41467-022-35063-1.
Ukuhlanganiswa kwe-genome ye-polyploid:UZhang, Q. et al. (2022) 'Ukuqonda kwe-genomic ekunciphiseni kwe-chromosome kwakamuva kwe-autopolyploid sugarmoba Saccharum spontaneum', Nature Genetics 2022 54:6, 54(6), pp. 885–896. doi: 10.1038/s41588-022-01084-1.
