Isityalo/Isilwanyana saseDe Novo Genome Sequencing
I-De NovoUkulandelelana kubhekisa ekwakhiweni kwe-genome epheleleyo yohlobo kusetyenziswa ubuchwepheshe bokulandelelana xa kungekho genome yokubhekisa. Ukwaziswa kunye nokwamkelwa ngokubanzi kokulandelelana kwesizukulwana sesithathu, okubandakanya ukufunda okude, kuye kwaphucula kakhulu ukuhlanganiswa kwe-genome ngokwandisa ukudibana phakathi kokufundwa. Olu phuculo lubaluleke kakhulu xa kujongwana nee-genome ezintsonkothileyo, ezifana nezo zibonisa i-heterozygosity ephezulu, umlinganiselo ophezulu weendawo eziphindaphindayo, ii-polyploids, kunye nemimandla enezinto eziphindaphindayo, umxholo ongaqhelekanga we-GC, okanye ubunzima obukhulu obuhlala buhlanganiswa kakubi kusetyenziswa ulandelelwano olufutshane lodwa.
Isisombululo sethu esipheleleyo sinikezela ngeenkonzo zokulandelelanisa ezidibeneyo kunye nohlalutyo lwe-bioinformatic olunikezela nge-genome ehlanganisiweyo esemgangathweni ophezulu. Uphando lokuqala lwe-genome kunye ne-Illumina lubonelela ngoqikelelo lobungakanani be-genome kunye nobunzima, kwaye olu lwazi lusetyenziselwa ukukhokela inyathelo elilandelayo lokulandelelanisa okude nge-PacBio HiFi, kulandelede novoukuhlanganiswa kwee-contigs. Ukusetyenziswa okulandelayo kwe-HiC assembly kwenza ukuba ii-contigs zinamathele kwi-genome, zifumane i-chromosome-level assembly. Okokugqibela, i-genome ichazwa ngokuqikelela i-gene kunye nokulandelelanisa ii-genes eziveziweyo, kusetyenziswe ii-transcriptomes ezinee-reads ezimfutshane nezinde.
Iimpawu zeNkonzo
● Ukuhlanganiswa kweenkonzo ezininzi zokulandelelana kunye neenkonzo ze-bioinformatic kwisisombululo esinye:
Uphando lwe-genome kunye ne-Illumina ukuqikelela ubungakanani be-genome kunye nokukhokela amanyathelo alandelayo;
Ulandelelwano olude lokufunda lwede novoukuhlanganiswa kwee-contigs;
Ulandelelwano lwe-Hi-C lokubambelela kwi-chromosome;
ukulandelelana kwe-mRNA ukuze kuchazwe iijini;
Ukuqinisekiswa kwendibano.
● Inkonzo efanelekileyo yokwakha ii-genome ezintsha okanye ukuphuculwa kwee-genome ezikhoyo zeentlobo zezinto ezinomdla.
Iingenelo zeNkonzo
Uphuhliso lwamaqonga okulandelelanisa kunye ne-bioinformatics kwide novoindibano ye-genome
(Amarasinghe SL et al.,Ibhayoloji yeGenome, 2020)
●Irekhodi yoBugcisa oBubanzi kunye nokuPapasha: I-BMKGene ifumene amava amaninzi ekuhlanganiseni i-genome esemgangathweni ophezulu yeentlobo ezahlukeneyo, kuquka i-diploid genomes kunye ne-genomes ezintsonkothileyo kakhulu ze-polyploid kunye ne-allopolyploid species. Ukusukela ngo-2018, sibe negalelo kwi-overIimpapasho ezingama-300 ezinefuthe elikhulu, kwaye ezingama-20+ zazo zipapashwe kwiNature Genetics.
● Isisombululo esinye: indlela yethu edibeneyo idibanisa iiteknoloji ezininzi zokulandelelana kunye nohlalutyo lwe-bioinformatic kwi-workflow edibeneyo, inika i-genome ehlanganisiweyo esemgangathweni ophezulu.
●Yenzelwe Iimfuno Zakho: Indlela esisebenza ngayo inkonzo yethu iyakwazi ukwenziwa ngokwezifiso, ivumela ukulungelelaniswa kwee-genomes ezineempawu ezahlukeneyo kunye neemfuno ezithile zophando. Oku kuquka ukwamkela ii-genomes ezinkulu, ii-genomes ze-polyploid, ii-genomes ze-heterozygous eziphezulu, kunye nokunye.
●Iqela leBioinformatics kunye neLebhu elinobuGcisa obuPhezulu: unamava amahle kakhulu kwicandelo lovavanyo kunye nele-bioinformatics kwiindibano ze-genome ezintsonkothileyo kunye nothotho lweepatenti kunye namalungelo okushicilela esoftware.
●Inkxaso emva kokuthengisa:Ukuzibophelela kwethu kudlulela ngaphaya kokugqitywa kweprojekthi kunye nexesha leenkonzo emva kokuthengiswa kweenyanga ezi-3. Ngeli xesha, sinikezela ngokulandela iprojekthi, uncedo lokusombulula iingxaki, kunye neeseshoni ze-Q&A ukuphendula nayiphi na imibuzo enxulumene neziphumo.
Iinkcukacha zeNkonzo
| Uphando lwe-genome | Ukuhlanganiswa kwejini | Inqanaba le-Chromosome | Isaziso seGenome |
| I-50X Illumina NovaSeq PE150
| Ii-30X PacBio CCS HiFi reads | 100X Hi-C | I-RNA-seq Illumina PE150 10 Gb + (ukhetho) Ubude obupheleleyo be-RNA-seq PacBio 40 Gb okanye I-Nanopore 12 Gb |
Iimfuno zeNkonzo
Kwi-Genome Survey, i-Genome Assembly kunye ne-Hi-C Assembly:
| Ii-tissue okanye ii-nucleic acids ezikhutshiweyo | Uphando lweGenome | Ukuhlanganiswa kweGenome ngePacBio | Indibano yeHi-C |
| I-Viscera yezilwanyana | 0.5-1 g
| ≥ 3.5 g | ≥2 g |
| Izihlunu zezilwanyana | ≥ 5 g | ||
| Igazi lezilwanyana ezincelisayo | 1.5 mL
| ≥ 5 mL | ≥2 mL |
| Igazi leNkukhu/Intlanzi | ≥ 0.5 mL | ||
| Isityalo- Igqabi Elitsha | 1-2 g | ≥ 5 g | ≥ 4 g |
| Iiseli ezikhuliswe kakuhle |
| ≥ 1x108 | ≥ 1x107 |
| Izinambuzane | 0.5-1 g | ≥ 3 g | ≥ 2 g |
| I-DNA ekhutshiweyo | Uxinzelelo: ≥1 ng/ µL Isixa ≥ 30 ng Ukonakala okanye ukungcola okulinganiselweyo okanye okungekhoyo | Uxinzelelo: ≥ 50 ng/ µL Ubungakanani: 10 µg/iseli yokuhamba/isampulu OD260/280=1.7-2.2 OD260/230=1.8-2.5 Ukonakala okanye ukungcola okulinganiselweyo okanye okungekhoyo |
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Kwisichasiselo seGenome nge-transcriptomics:
| Ii-tissue okanye ii-nucleic acids ezikhutshiweyo | I-Illumina Transcriptome | I-PacBio Transcriptome | I-Nanopore Transcriptome |
| Ingcambu yesityalo/Isiqu/Ipetali | 450 mg | 600 mg | |
| Isityalo – Igqabi/Imbewu | 300 mg | 300 mg | |
| Isityalo - Isiqhamo | 1.2 g | 1.2 g | |
| Intliziyo yesilwanyana/Amathumbu | 300 mg | 300 mg | |
| I-Viscera yezilwanyana/Ubuchopho | 240 mg | 240 mg | |
| Izihlunu zezilwanyana | 450 mg | 450 mg | |
| Amathambo ezilwanyana/Iinwele/Ulusu | 1 g | 1 g | |
| I-Arthropod - Izinambuzane | 6 | 6 | |
| I-Arthropod - iCrustacea | 300 mg | 300 mg | |
| Igazi elipheleleyo | Ityhubhu e-1 | Ityhubhu e-1 | |
| I-RNA ekhutshiweyo | Uxinzelelo: ≥ 20 ng/ µL Ubungakanani ≥ 0.3 µg OD260/280=1.7-2.5 OD260/230=0.5-2.5 I-RIN≥ 6 5≥28S/18S≥1 | Ugxininiso: ≥ 100 ng/µL Ubungakanani ≥ 0.75 µg OD260/280=1.7-2.5 OD260/230=0.5-2.5 I-RIN≥ 8 5≥28S/18S≥1 | Ugxininiso: ≥ 100 ng/µL Ubungakanani ≥ 0.75 µg OD260/280=1.7-2.5 OD260/230=0.5-2.5 I-RIN≥ 7.5 5≥28S/18S≥1 |
Ukuhanjiswa kweSampuli okuCetyisiweyo
Isikhongozeli: Ityhubhu ye-centrifuge ye-2 ml (Akukhuthazwa ukusebenzisa i-tin foil)
(Kwiisampuli ezininzi, sicebisa ukuba zingagcinwa kwi-ethanol.)
Ukubhalwa kwesampuli: Iisampulu kufuneka zibhalwe ngokucacileyo kwaye zifane nefomu yolwazi lwesampuli engenisiweyo.
Ukuthunyelwa: Umkhenkce owomileyo: Iisampulu kufuneka zipakishwe kwiingxowa kuqala zize zingcwatywe kumkhenkce owomileyo.
Ukuhamba komsebenzi
Ukuhamba komsebenzi wenkonzo
Uyilo lovavanyo
Ukuhanjiswa kwesampulu
Ukukhupha i-DNA
Ukwakhiwa kweThala leeNcwadi
Ukulandelelana
Uhlalutyo lwedatha
Iinkonzo zasemva kokuthengisa
Uhlalutyo olupheleleyo lwe-bioinformatic, olwahlulwe ngamanyathelo ama-4:
1) Uphando lweGenome, olusekelwe kuhlalutyo lwe-k-mer olusebenzisa i-NGS lufundeka ngolu hlobo:
Uqikelelo lobungakanani be-genome
Uqikelelo lwe-heterozygosity
Uqikelelo lweendawo eziphindaphindayo
2) Ukuhlanganiswa kweGenome ngePacBio HiFi:
De novoindibano
Uvavanyo lokuhlanganisa: kuquka uhlalutyo lweBUSCO lokugqibelela kwe-genome kunye nokumakisha ngasemva kwe-NGS kunye ne-PacBio HiFi reads
3) Indibano ye-Hi-C:
I-QC yelayibrari ye-Hi-C: uqikelelo lonxibelelwano olusebenzayo lwe-Hi-C
Indibano ye-Hi-C: ukuhlanganiswa kwee-contigs ngamaqela, kulandele uku-odola ii-contig ngaphakathi kweqela ngalinye kunye nokwabela ulwalathiso lwe-contig
Uvavanyo lwe-Hi-C
4) Isichazi-magama se-genome:
Uqikelelo lwe-RNA olungabhalwanga kwikhowudi
Ukuchongwa kokulandelelana okuphindaphindwayo (ii-transposons kunye nokuphindaphinda kwe-tandem)
Uqikelelo lwe-gene
§De novo: ii-algorithms ze-ab initio
§ Isekelwe kwi-homology
§ Isekelwe kwi-transcriptome, enee-reads ezinde nezimfutshane: ii-reads zide novoidityaniswe okanye idweliswe kwi-genome eyilwayo
§ Inkcazo yezakhi zofuzo eziqikelelweyo ezineedathabheyisi ezininzi
1) Uhlalutyo lweGenome- k-mer
2) Ukuhlanganiswa kweGenome
2) I-Genome Assembly – I-PacBio HiFi ifunda imephu ukuya kwi-draft assembly
2) I-Hi-C Assembly – uqikelelo lwezibini zonxibelelwano ezisemthethweni ze-Hi-C
3) Uvavanyo lwe-Hi-C emva kokuhlanganiswa
4) Ingcaciso yeGenome – ukuhlanganiswa kwezakhi zofuzo eziqikelelweyo
4) Isichazi-magama sejini – isichazi-magama sejini esiqikelelweyo
Hlola inkqubela phambili eyenziwe ziinkonzo ze-BMKGene's de novo genome assembly ngokusebenzisa ingqokelela yeempapasho ezikhethiweyo:
ULi, C. et al. (2021) 'Ulandelelwano lwee-genome lubonisa iindlela zokusasazeka kwehlabathi kwaye lucebisa uhlengahlengiso lwe-genetic oluhambelanayo kwi-seahorse evolution', Nature Communications, 12(1). doi: 10.1038/S41467-021-21379-X.
ULi, Y. et al. (2023) 'Utshintsho olukhulu lweChromosomal lukhokelela ekuTshintsheni kweNqanaba leGenome, uLungiso lweNdalo, kunye neSpeciation kwiGayal (Bos frontalis)', iMolecular Biology kunye neEvolution, 40(1). doi: 10.1093/MOLBEV/MSAD006.
UTian, T. et al. (2023) 'Ukuhlanganiswa kwejini kunye nokwahlulwa kwejini kwe-germplasm yombona ebalaseleyo enganyangekiyo yimbalela', iNature Genetics 2023 55:3, 55(3), iphe. 496–506. doi: 10.1038/s41588-023-01297-y.
UZhang, F. et al. (2023) 'Ukutyhila uphuhliso lwe-tropane alkaloid biosynthesis ngokuhlalutya ii-genomes ezimbini kusapho lweSolanaceae', Nature Communications 2023 14:1, 14(1), iphe. 1–18. doi: 10.1038/s41467-023-37133-4.
Izifundo zetyala ezinzima:
Indibano yeTelomere-ukuya kwiTelomere:UFu, A. et al. (2023) 'Ukuhlanganiswa kwe-genome ye-Telomere-to-telomere ye-bitter melon (uMomordica charantia L. var. abbreviata Ser.) kutyhila uphuhliso lweziqhamo, ukwakheka kunye neempawu zemfuza ezivuthwayo', uPhando lwezoLimo, 10(1). doi: 10.1093/HR/UHAC228.
Indibano yeHaplotype:UHu, W. et al. (2021) 'I-genome echazwe yi-allele ityhila ukwahluka kwe-biallelic ngexesha lokuvela kwe-cassava', iMolecular Plant, 14(6), iphe. 851–854. doi: 10.1016/j.molp.2021.04.009.
Ukuhlanganiswa kwe-genome enkulu:UYuan, J. et al. (2022) 'Isiseko se-genomic se-giga-chromosomes kunye ne-giga-genome ye-tree peony Paeonia ostii', Nature Communications 2022 13:1, 13(1), iphe. 1–16. doi: 10.1038/s41467-022-35063-1.
Ukuhlanganiswa kwe-genome ye-polyploid:UZhang, Q. et al. (2022) 'Iingcamango ze-genomic malunga nokunciphisa kwe-chromosome yakutshanje ye-autopolyploid sugarcane Saccharum spontaneum', i-Nature Genetics 2022 54:6, 54(6), iphe. 885–896. doi: 10.1038/s41588-022-01084-1.
