Isityalo/Isilwanyana saseDe Novo Genome Sequencing
De Novoulandelelwano lubhekisa kulwakhiwo lwegenome yohlobo lonke kusetyenziswa ubugcisa bolandelelwano ngokungabikho kwegenome yesalathiso. Intshayelelo kunye nokwamkelwa okubanzi kolandelelwano lwesizukulwana sesithathu, olunofundo olude, luphucule kakhulu ukuhlanganiswa kwegenome ngokwandisa ukuhlangana phakathi kokufunda. Olu lwandiso lufanelekile ngakumbi xa kujongwana ne-genomes ecela umngeni, njengaleyo ibonisa i-heterozygosity ephezulu, umlinganiselo ophezulu wemimandla ephindaphindayo, i-polyploids, kunye nemimandla enezinto eziphindaphindayo, imixholo ye-GC engaqhelekanga, okanye ubunzima obuphezulu obuqhelekanga ngokungahambi kakuhle kusetyenziswa ukulandelelana kokufundwa okufutshane kuphela.
Isisombululo sethu sokumisa esinye sibonelela ngeenkonzo ezidibeneyo zolandelelwano kunye nohlalutyo lwe-bioinformatic oluhambisa igenome edityanisiweyo ye-de novo ephezulu. Uphando lokuqala lwe-genome kunye ne-Illumina lubonelela ngoqikelelo lobungakanani be-genome kunye nobunzima, kwaye olu lwazi lusetyenziselwa ukukhokela inyathelo elilandelayo lokulandelelana kwexesha elide kunye nePacBio HiFi, elandelwade novoukuhlanganiswa kwee-contigs. Ukusetyenziswa okulandelayo kwendibano ye-HiC kwenza ukuba i-anchoring ye-contigs kwi-genome, ifumane indibano yezinga le-chromosome. Okokugqibela, i-genome ichazwa ngokuqikelelwa kofuzo kunye nolandelelwano lwemfuza echaziweyo, ibhenela kwii-transcriptomes ezinokufundwa okufutshane kunye nokude.
Iimpawu zeNkonzo
● Ukudityaniswa kolandelelwano oluninzi kunye neenkonzo ze-bioinformatic kwisisombululo esisodwa:
Uphando lweGenome kunye ne-Illumina ukuqikelela ubungakanani be-genome kunye nokukhokela amanyathelo alandelayo;
Ukufunda ixesha elide ngokulandelelana kwede novoukuhlanganiswa kwee-contigs;
Ulandelelwano lwe-Hi-C lwe-anchoring yechromosome;
Ulandelelwano lwe-mRNA lwenkcazo yemfuza;
Ukuqinisekiswa kwendibano.
● Inkonzo elungele ukwakhiwa kwegenomes yenoveli okanye ukuphuculwa kweegenome zesalathiso ezikhoyo ukulungiselela iintlobo zomdla.
Iinkonzo eziluncedo
Ukuphuhliswa kwamaqonga okulandelelanisa kunye ne-bioinformatics kwide novoindibano yegenome
(Amarasinghe SL et al.,Ibhayoloji yeGenome, 2020)
●UbuGcisa obuBanzi kunye neRekhodi yoPapasho: I-BMKGene iqokelele amava amakhulu ekudibaneni kwegenome ekumgangatho ophezulu weentlobo ezahlukeneyo, kubandakanya igenomes yediploid kunye negenomes ezintsonkothileyo zepolyploid kunye neallopolyploid. Ukusukela ngo-2018, sibe negalelo ngaphayaUshicilelo lwe-300 olunempembelelo ephezulu, kwaye i-20 + kubo ipapashwe kwi-Nature Genetics.
● One-stop Solution: indlela yethu edibeneyo idibanisa itekhnoloji yolandelelwano oluninzi kunye nohlalutyo lwe-bioinformatic ibe yinkqubo edibeneyo yokusebenza, ukuhambisa i-genome edibeneyo ekumgangatho ophezulu.
●Yenzelwe Iimfuno Zakho: Umsebenzi wethu wenkonzo unokwenziwa ngokwezifiso, uvumela ukulungelelaniswa kweegenomes ezineempawu ezahlukeneyo kunye neemfuno ezithile zophando. Oku kubandakanya ukuhlalisa iigenomes ezinkulu, i-polyploid genomes, i-heterozygous genomes ephezulu, kunye nokunye.
●IQela leBioinformatics kunye neLabhoratri eneSakhono esiPhezulu: ngamava amnandi kuvavanyo kunye ne-bioinformatics phambi kweeassemblies ezintsonkothileyo zegenome kunye nothotho lwamalungelo awodwa omenzi wechiza kunye namalungelo okushicilela esoftware.
●Inkxaso yasemva kwentengiso:Ukuzinikela kwethu kudlulela ngaphaya kokugqitywa kweprojekthi kunye neenyanga ezi-3 emva kokuthengiswa kwenkonzo. Ngeli xesha, sinikezela ngokulandelelana kweprojekthi, uncedo lokusombulula ingxaki, kunye neeseshoni ze-Q&A ukujongana nayo nayiphi na imibuzo enxulumene neziphumo.
IiNgcaciso zeNkonzo
| Uphando lweGenome | Indibano yeGenome | Inqanaba leChromosome | I-Genome Annotation |
| 50X Illumina NovaSeq PE150
| 30X PacBio CCS HiFi ifundeka | 100X Hi-C | I-RNA-seq Illumina PE150 10 Gb + (ukhetho) Ubude obupheleleyo be-RNA-seq PacBio 40 Gb okanye Nanopore 12 Gb |
IiMfuno zeNkonzo
KuPhando lweGenome, iNdibano yeGenome kunye neNdibano ye-Hi-C:
| Izicubu okanye i-nucleic acids ekhutshiweyo | Uvavanyo lweGenome | INdibano yeGenome kunye nePacBio | INdibano ye-Hi-C |
| Animal Viscera | 0.5-1 g
| ≥ 3.5 g | ≥2 g |
| Animal Muscle | ≥ 5 g | ||
| Igazi leMammalian | 1.5 ml
| ≥ 5 ml | ≥2 ml |
| Iinkukhu / Igazi lentlanzi | ≥ 0.5 mL | ||
| Isityalo- Igqabi Elitsha | 1-2 g | ≥ 5 g | ≥ 4 g |
| Iiseli Cultured |
| ≥ 1x108 | ≥ 1x107 |
| isinambuzane | 0.5-1 g | ≥ 3 g | ≥ 2 g |
| I-DNA ekhutshiweyo | Ugxininiso: ≥1 ng/µL Imali ≥ 30 ng Umda okanye akukho ukuthotywa okanye ukosuleleka | Ugxininiso: ≥ 50 ng/µL Isixa: 10 µg/flow cell/isampulu OD260/280=1.7-2.2 OD260/230=1.8-2.5 Umda okanye akukho ukuthotywa okanye ukosuleleka |
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|
Kwinkcazo yeGenome ene-transcriptomics:
| Izicubu okanye i-nucleic acids ekhutshiweyo | Illumina Transcriptome | I-PacBio Transcriptome | Nanopore Transcriptome |
| Isityalo- Ingcambu/Isiqu/iPetal | 450 mg | 600 mg | |
| Isityalo – Igqabi/Imbewu | 300 mg | 300 mg | |
| Isityalo - Isiqhamo | 1.2 g | 1.2 g | |
| Intliziyo yezilwanyana / Amathumbu | 300 mg | 300 mg | |
| Animal Viscera / Brain | 240 mg | 240 mg | |
| Animal Muscle | 450 mg | 450 mg | |
| Amathambo Ezilwanyana / Iinwele / Ulusu | 1 g | 1 g | |
| I-Arthropod - isinambuzane | 6 | 6 | |
| Arthropod -Crustacea | 300 mg | 300 mg | |
| Igazi elipheleleyo | ityhubhu e-1 | ityhubhu e-1 | |
| I-RNA etsaliweyo | Ugxininiso: ≥ 20 ng/ µL Isixa ≥ 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 Isixa ≥ 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 Isixa ≥ 0.75 µg OD260/280=1.7-2.5 OD260/230=0.5-2.5 I-RIN≥ 7.5 5≥28S/18S≥1 |
Unikezelo lweSampuli eCetyisiweyo
Isikhongozeli: 2 ml centrifuge ityhubhu (Ifoyile yeTin ayikhuthazwa)
(Kuninzi lweesampulu, sincoma ukuba ungayigcini kwi-ethanol.)
Iisampulu zokufakwa ileyibhile: Iisampulu kufuneka zibhalwe ngokucacileyo kwaye zifane nefom yolwazi yesampulu engenisiweyo.
Ukuthumela: Umkhenkce owomileyo: Iisampulu kufuneka zipakishwe kwiingxowa kuqala kwaye zingcwatywe kwindawo eyomileyo.
Ukuhamba komsebenzi
Ukuhamba komsebenzi weNkonzo
Uyilo lovavanyo
Ukuhanjiswa kwesampuli
Ukutsalwa kwe-DNA
Ukwakhiwa kwethala leencwadi
Ukulandelelanisa
Uhlalutyo lwedatha
Iinkonzo emva kokuthengisa
Gqibezela uhlalutyo lwe-bioinformatic, lwahlulwe ngamanyathelo ama-4:
1) Uphando lweGenome, olusekwe kuhlalutyo lwe-k-mer kunye ne-NGS ifundeka ngolu hlobo:
Uqikelelo lobungakanani begenome
Uqikelelo lwe-heterozygosity
Uqikelelo lwemimandla ephindaphindwayo
2) INdibano yeGenome kunye nePacBio HiFi:
De novoindibano
Uvavanyo lweNdibano: kubandakanya uhlalutyo lwe-BUSCO lokugqibelela kwegenome kunye nemephu yasemva kwe-NGS kunye ne-PacBio HiFi ifundeka
3) Indibano ye-Hi-C:
Ithala leencwadi le-Hi-C le-QC: uqikelelo lonxibelelwano olusebenzayo lwe-Hi-C
Indibano ye-Hi-C: ukudityaniswa kwee-contig ngokwamaqela, kulandelwa yi-contig odolo phakathi kweqela ngalinye kunye nokwabela i-contig orientation
Uvavanyo lwe-Hi-C
4) Isihlomelo seGenome:
Uqikelelo lwe-RNA olungabhaliyo
Ukuchongwa kolandelelwano oluphinda-phindayo (iitransposons kunye nophindaphindo lwe-tandem)
Uqikelelo lwemfuza
§De novo: ab initio algorithms
§ Ngokusekelwe kwi-homology
§ Ngokusekwe kwi-transcriptome, kunye nofundo olude nolufutshane: ufundo lude novozidityanisiwe okanye zidweliswe kwidrafti yegenome
§ Isihlomelo semfuza eqikelelweyo enovimba weenkcukacha ezininzi
1) Genome Survey- k-mer uhlalutyo
2) INdibano yeGenome
2) INdibano yeGenome - iPacBio HiFi ifunda imephu ukuyila indibano
2) INdibano ye-Hi-C - ukuqikelelwa kwe-Hi-C izibini ezisebenzayo zokusebenzisana
3) Uvavanyo lwe-Hi-C Post-assembly
I-4) I-Genome Annotation - ukuhlanganiswa kweegenes eziqikelelweyo
I-4) Inkcazo ye-Genome - inkcazo yemfuza eqikelelweyo
Phonononga inkqubela phambili eququzelelwa yi-BMKGene yeenkonzo zendibano ye-de novo genome ngengqokelela egciniweyo yopapasho:
Li, C. et al. (2021) 'Ulandelelwano lweGenome lutyhila iindlela zokusasazeka kwehlabathi kwaye zicebisa ulungelelwaniso lwemfuzo oluguqukayo kwi-evolution ye-seahorse', uNxibelelwano lweNdalo, i-12(1). doi: 10.1038/S41467-021-21379-X.
Li, Y. et al. (2023) 'Iinguqu ezinkulu zeChromosomal ziKhokela kwiGenome-Level Expression Change, ukulungelelaniswa kweNdawo, kunye neNkcazo kwiGayal (Bos frontalis)', i-Molecular Biology kunye ne-Evolution, i-40 (1). doi: 10.1093/MOLBEV/MSAD006.
Tian, T. et al. (2023) 'Indibano ye-Genome kunye ne-genetic dissection ye-germplasm yombona eveleleyo kwimbalela', i-Nature Genetics 2023 55: 3, 55 (3), iphe. 496-506. doi: 10.1038/s41588-023-01297-y.
UZhang, F. et al. (2023) 'Ukutyhila ukuguquka kwe-tropane alkaloid biosynthesis ngokuhlalutya i-genomes ezimbini kwintsapho yaseSolanaceae', uNxibelelwano lweNdalo 2023 14: 1, 14 (1), iphe. 1-18. doi: 10.1038/s41467-023-37133-4.
Ucelomngeni lwezifundo:
Indibano yeTelomere-to-telomere:Fu, A. et al. (2023) 'i-Telomere-to-telomere genome assembly of bitter melon (Momordica charantia L. var. abbreviata Ser.) ibonisa uphuhliso lweziqhamo, ukubunjwa kunye nokuvuthwa kweempawu zemfuza', Uphando lweHorticulture, i-10 (1). doi: 10.1093/HR/UHAC228.
Indibano yeHaplotype:Hu, W. et al. (2021) 'I-allele-defined genome ibonisa ukuhlukana kwe-biallelic ngexesha le-cassava evolution', i-Molecular Plant, i-14 (6), iphe. 851-854. doi: 10.1016/j.molp.2021.04.009.
Indibano enkulu yegenome:Yuan, J. et al. (2022) 'Isiseko se-Genomic ye-giga-chromosomes kunye ne-giga-genome yomthi we-peony Paeonia ostii', i-Nature Communications 2022 13: 1, 13 (1), iphe. 1-16. doi: 10.1038/s41467-022-35063-1.
Indibano yegenome yePolyploid:Zhang, Q. et al. (2022) 'Iingcinga zeGenomic kwi-chromosome yokunciphisa i-autopolyploid iswekile ye-Saccharum spontaneum', i-Nature Genetics 2022 54: 6, 54 (6), iphe. 885-896. doi: 10.1038/s41588-022-01084-1.
