Zomera / Zinyama za De Novo Genome Sequencing
De Novokutsatizana kumatanthauza kupanga ma genome amtundu wonse pogwiritsa ntchito umisiri wotsatizana popanda ma genome. Kuyamba ndi kufalikira kwa kutsatizana kwa m'badwo wachitatu, wokhala ndi kuwerenga kwanthawi yayitali, kwathandizira kwambiri kuphatikiza kwa ma genome pakuwonjezera kuphatikizika pakati pa zowerenga. Kupititsa patsogolo kumeneku kumakhala koyenera makamaka polimbana ndi majeremusi ovuta, monga omwe amawonetsa heterozygosity yapamwamba, chiŵerengero chachikulu cha zigawo zobwerezabwereza, ma polyploids, ndi zigawo zomwe zimakhala ndi zinthu zobwerezabwereza, zomwe zili mu GC zosazolowereka, kapena zovuta kwambiri zomwe nthawi zambiri zimasonkhanitsidwa bwino pogwiritsa ntchito masanjidwe afupipafupi okha.
Yankho lathu loyimitsa limodzi limapereka ntchito zophatikizika zotsatizana komanso kusanthula kwa bioinformatic komwe kumapereka mtundu wapamwamba kwambiri wa de novo assembled genome. Kufufuza koyambirira kwa ma genome ndi Illumina kumapereka kuyerekezera kwa kukula ndi zovuta za genome, ndipo chidziwitsochi chimagwiritsidwa ntchito kutsogolera gawo lotsatira la kuwerengera kwanthawi yayitali ndi PacBio HiFi, kutsatiridwa ndindi novomsonkhano wa contigs. Kugwiritsiridwa ntchito kotsatira kwa msonkhano wa HiC kumathandizira kukhazikika kwa ma contigs ku genome, kupeza msonkhano wa chromosome. Pomaliza, ma genome amatsimikiziridwa ndi kulosera kwa jini komanso potsata ma jini owonetsedwa, kutengera ma transcriptomes okhala ndi zowerengera zazifupi komanso zazitali.
Mawonekedwe a Utumiki
● Kuphatikizika kwa njira zingapo zotsatirira ndi bioinformatics mu njira yoyimitsa kamodzi:
Kafukufuku wa genome ndi Illumina kuti ayese kukula kwa ma genome ndikuwongolera njira zotsatila;
Kuwerengera kwanthawi yayitali kwandi novokuphatikiza kwa contigs;
Kutsata kwa Hi-C kwa chromosome anchoring;
kutsatizana kwa mRNA kwa kafotokozedwe ka majini;
Kutsimikizika kwa msonkhano.
● Ntchito yoyenera kupanga ma genome atsopano kapena kukonza ma genome omwe alipo amitundu yosangalatsa.
Ubwino wa Utumiki
Kukula kwamapulatifomu otsatizana ndi bioinformatics mundi novomsonkhano wa genome
(Amarasinghe SL et al.,Genome Biology, 2020)
●Mbiri Yambiri Yaukatswiri ndi Kufalitsa: BMKGene yapeza zambiri pakupanga ma genome apamwamba kwambiri amitundu yosiyanasiyana, kuphatikiza ma diploid genomes ndi ma genome ovuta kwambiri a polyploid ndi allopolyploid. Kuyambira 2018, tathandizira kupitiliraZofalitsidwa zokwana 300, ndipo 20+ mwa izo zimasindikizidwa mu Nature Genetics.
● One-Stop Solution: Njira yathu yophatikizika imaphatikiza matekinoloje angapo otsatizana ndi kusanthula kwa bioinformatic kukhala mayendedwe ogwirizana, ndikupereka matupi amtundu wapamwamba kwambiri.
●Zogwirizana ndi Zosowa Zanu: Mayendedwe athu a ntchito amatha kusintha makonda, kulola kusinthika kwa ma genome okhala ndi mawonekedwe osiyanasiyana komanso zofunikira pakufufuza. Izi zikuphatikizapo kukhala ndi ma genome akuluakulu, ma polyploid genomes, heterozygous genomes, ndi zina.
●Luso Lapamwamba la Bioinformatics ndi Gulu Lasayansi: wodziwa zambiri pazoyeserera ndi bioinformatics kutsogolo kwa magulu ovuta a genome ndi mndandanda wa ma patent ndi kukopera kwa mapulogalamu.
●Thandizo Pambuyo Pakugulitsa:Kudzipereka kwathu kumapitilira kutha kwa projekiti ndi nthawi ya miyezi itatu mutagulitsa. Panthawiyi, timapereka zotsatila za polojekiti, chithandizo chazovuta, ndi magawo a Q&A kuti tiyankhe mafunso aliwonse okhudzana ndi zotsatira.
Mafotokozedwe a Utumiki
| Kafukufuku wa Genome | Msonkhano wa Genome | Chromosome-level | Zolemba za Genome |
| 50X Illumina NovaSeq PE150
| 30X PacBio CCS HiFi imawerenga | 100X Hi-C | RNA-seq Illumina PE150 10 Gb + (posankha) Utali wonse wa RNA-seq PacBio 40 Gb kapena Nanopore 12 Gb |
Zofunikira pa Utumiki
Kwa Genome Survey, Genome Assembly ndi Hi-C Assembly:
| Minofu kapena yotengedwa ma nucleic acid | Kafukufuku wa Genome | Genome Assembly ndi PacBio | Msonkhano wa Hi-C |
| Viscera Wanyama | 0.5-1 g
| ≥ 3.5 g | ≥2 g |
| Minofu Yanyama | ≥ 5 g | ||
| Mammalian Blood | 1.5 ml
| ≥ 5 ml | ≥2 ml |
| Nkhuku/Nsomba Magazi | ≥ 0.5 mL | ||
| Chomera - Tsamba Latsopano | 1-2 g pa | ≥ 5 g | ≥ 4 g |
| Maselo Okhazikika |
| ≥ 1x108 | ≥ 1x107 |
| Tizilombo | 0.5-1 g | ≥ 3 g | ≥ 2 g |
| DNA yotengedwa | Kukhazikika: ≥1 ng/µL Kuchuluka ≥ 30 ng Zochepa kapena zopanda kutsitsa kapena kuipitsidwa | Kukhazikika: ≥ 50 ng/µL Kuchuluka: 10 µg/flow cell/chitsanzo OD260/280=1.7-2.2 OD260/230=1.8-2.5 Zochepa kapena zopanda kutsitsa kapena kuipitsidwa |
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Pazofotokozera za Genome ndi transcriptomics:
| Minofu kapena yotengedwa ma nucleic acid | Illumina Transcriptome | PacBio Transcriptome | Nanopore Transcriptome |
| Chomera- Muzu/Stem/Petal | 450 mg | 600 mg | |
| Chomera – Masamba/Mbewu | 300 mg | 300 mg | |
| Chomera - Chipatso | 1.2g pa | 1.2g pa | |
| Mtima Wanyama/Matumbo | 300 mg | 300 mg | |
| Animal Viscera / Ubongo | 240 mg | 240 mg | |
| Minofu Yanyama | 450 mg | 450 mg | |
| Mafupa a Zinyama / Tsitsi / Khungu | 1 g pa | 1 g pa | |
| Arthropod - Tizilombo | 6 | 6 | |
| Arthropod - Crustacea | 300 mg | 300 mg | |
| Mwazi wonse | 1 chubu | 1 chubu | |
| RNA yotengedwa | Kukhazikika: ≥ 20 ng/µL Kuchuluka ≥ 0.3 µg OD260/280=1.7-2.5 OD260/230=0.5-2.5 RIN≥ 6 5≥28S/18S≥1 | Kukhazikika: ≥ 100 ng/µL Kuchuluka ≥ 0.75 µg OD260/280=1.7-2.5 OD260/230=0.5-2.5 RIN≥ 8 5≥28S/18S≥1 | Kukhazikika: ≥ 100 ng/µL Kuchuluka ≥ 0.75 µg OD260/280=1.7-2.5 OD260/230=0.5-2.5 RIN≥ 7.5 5≥28S/18S≥1 |
Kutumiza Kwachitsanzo Kovomerezeka
Chidebe: 2 ml centrifuge chubu (zojambula za malata ndizosavomerezeka)
(Pazitsanzo zambiri, timalimbikitsa kuti tisasunge ethanol.)
Zitsanzo zolembetsera: Zitsanzo ziyenera kulembedwa momveka bwino komanso zofanana ndi zomwe zatumizidwa.
Kutumiza: Owuma-Izi: Zitsanzo ziyenera kulongedzedwa m'matumba kaye ndikukwiriridwa mu ayezi wouma.
Kayendedwe kantchito
Kuyenda kwa Ntchito Yantchito
Kuyesera kupanga
Kupereka zitsanzo
Kutulutsa kwa DNA
Kumanga laibulale
Kutsata
Kusanthula deta
Pambuyo pogulitsa ntchito
Malizitsani kusanthula kwa bioinformatics, kugawa magawo 4:
1) Kafukufuku wa genome, kutengera kusanthula kwa k-mer ndi NGS amawerenga:
Kuyerekeza kukula kwa ma genome
Kuyerekeza kwa heterozygosity
Kuyerekeza kwa zigawo zobwerezabwereza
2) Msonkhano wa Genome wokhala ndi PacBio HiFi:
Pa novomsonkhano
Kuwunika kwa msonkhano: kuphatikiza kusanthula kwa BUSCO kwa kukwanira kwa ma genome ndikujambula kumbuyo kwa NGS ndi kuwerengedwa kwa PacBio HiFi
3) Msonkhano wa Hi-C:
Hi-C laibulale ya QC: kuyerekezera kovomerezeka kwa Hi-C
Msonkhano wa Hi-C: kuphatikiza kwa ma contig m'magulu, kutsatiridwa ndi kuyitanitsa ma contig mkati mwa gulu lililonse ndikugawa mawonekedwe a contig
Kuwunika kwa Hi-C
4) Ndemanga za Genome:
Kuneneratu kwa RNA kosalemba
Chizindikiritso chobwerezabwereza (ma transposon ndi kubwereza tandem)
Kuneneratu kwa Gene
§Pa novo: ab initio algorithms
§ Kutengera ndi Homology
§ Kutengera zolemba, zowerengera zazitali komanso zazifupi: zowerengera ndizondi novozosonkhanitsidwa kapena kujambulidwa ku ma genome
§ Kufotokozera za majini onenedweratu okhala ndi ma database angapo
1) Genome Survey- k-mer kusanthula
2) Msonkhano wa Genome
2) Genome Assembly - PacBio HiFi imawerenga mapu kuti ikonzekere msonkhano
2) Msonkhano wa Hi-C - kuyerekezera kwa mawiri awiri ogwirizana a Hi-C
3) Kuwunika kwa Hi-C Post-assembly
4) Genome Annotation - kuphatikiza kwa majini onenedweratu
4) Ndemanga za genome - zonenedweratu za majini
Onani kupita patsogolo komwe kumayendetsedwa ndi misonkhano ya BMKGene's de novo genome kudzera mgulu lazofalitsa:
Li, C. et al. (2021) 'Kutsatizana kwa ma genome kumawulula njira zobalalitsira padziko lonse lapansi ndikuwonetsa kusintha kwa majini mu seahorse evolution', Nature Communications, 12(1). doi: 10.1038/S41467-021-21379-X.
Li, Y. et al. (2023) 'Kusintha Kwakukulu Kwa Chromosomal Kumabweretsa Kusintha Kwa Mawonekedwe a Genome-Level, Kusintha Kwachilengedwe, ndi Kuzindikira mu Gayal (Bos frontalis)', Molecular Biology ndi Evolution, 40 (1). doi: 10.1093/MOLBEV/MSAD006.
Tian, T. et al. (2023) 'Genome assembly and genetic dissection ya majeremusi odziwika bwino olimbana ndi chilala', Nature Genetics 2023 55:3, 55(3), pp. 496-506. doi: 10.1038/s41588-023-01297-y.
Zhang, F. et al. (2023) 'Kuwulula kusinthika kwa tropane alkaloid biosynthesis mwa kusanthula ma genomes awiri mubanja la Solanaceae', Nature Communications 2023 14: 1, 14 (1), pp. 1-18. doi: 10.1038/s41467-023-37133-4.
Mayesero ovuta:
Msonkhano wa Telomere-to-telomere:Fu, A. et al. (2023) 'Telomere-to-telomere genome assembly of bitter melon (Momordica charantia L. var. abbreviata Ser.) imasonyeza kukula kwa zipatso, mapangidwe ndi kukhwima kwa majini', Horticulture Research, 10 (1). doi: 10.1093/HR/UHAC228.
Msonkhano wa Haplotype:Hu, W. et al. (2021) 'Allele-defined genome imasonyeza kusiyana kwa biallelic panthawi ya kusinthika kwa chinangwa', Molecular Plant, 14 (6), pp. 851-854. doi: 10.1016/j.molp.2021.04.009.
Msonkhano waukulu wa genome:Yuan, J. et al. (2022) 'Genomic maziko a giga-chromosomes ndi giga-genome ya mtengo peony Paeonia ostii', Nature Communications 2022 13: 1, 13 (1), pp. 1-16. doi: 10.1038/s41467-022-35063-1.
Msonkhano wa Polyploid genome:Zhang, Q. et al. (2022) 'Kuwunika kwa Genomic pakuchepetsa kwaposachedwa kwa chromosome kwa nzimbe ya autopolyploid Saccharum spontaneum', Nature Genetics 2022 54: 6, 54 (6), pp. 885-896. doi: 10.1038/s41588-022-01084-1.
