Dhirta/Xayawaanka De Novo Genome Sequencing
De Novoisku xigxiga waxaa loola jeedaa dhismaha noocyada 'genome' oo dhan iyadoo la adeegsanayo tignoolajiyada isku xigxiga ah maqnaanshaha genome tixraaceed. Horudhac iyo qaadashada baahsan ee taxanaha jiilka saddexaad, oo leh wax-akhris dheer, ayaa si weyn u xoojiyey isu-ururinta genome-ka iyada oo kordhinaysa isku-dhafka u dhexeeya akhrinta. Kobcintani waxay si gaar ah u khusaysaa marka lala tacaalayo genome-yada adag, sida kuwa muujinaya heterozygosity sare, saamiga sare ee gobollada soo noqnoqda, polyploids, iyo gobollada leh walxo soo noqnoqda, waxyaabaha GC-da aan caadiga ahayn, ama kakanaanta sare ee sida caadiga ah si liidata la isugu keeno iyadoo la isticmaalayo taxane akhris gaaban keligiis.
Xalkayaga halka joogsiga ah wuxuu bixiyaa adeegyo isku xigxig ah oo isku xiga iyo falanqaynta bioinformatic taasoo keenaysa genome-ga urursan ee de novo oo tayo sare leh. Sahanka genome-ga bilowga ah ee Illumina wuxuu bixiyaa qiyaasaha cabbirka genome-ga iyo kakanaanta, macluumaadkan waxaa loo isticmaalaa in lagu hago tillaabada xigta ee isku xigxiga muddada dheer ee akhriska ee PacBio HiFi, oo ay ku xigtode novoshirka contigs. Isticmaalka xiga ee isu-ururinta HiC waxa ay awood u siinaysaa ku xidhidhiyaha xididdada genome-ga, helitaanka isu-ururinta heerka koromosoomyada. Ugu dambayntii, genome-ka waxa lagu qeexaa saadaalinta hidda-wadaha iyo iyada oo la isku xig-xiro hiddo-sidaha la muujiyey, iyada oo la adeegsanayo qoraallo qoraallo ah oo akhrin gaaban iyo kuwo dheer leh.
Tilmaamaha Adeegga
● Isku darka adeegyo badan oo isku xigxig ah iyo bioinformatic ah oo lagu helo xal hal joogsi ah:
Sahanka genome ee leh Illumina si loo qiyaaso cabbirka genome-ga oo hago tillaabooyinka xiga;
Akhriska dheer ee taxanaha ahde novoururinta contigs;
Taxanaha Hi-C ee koromosoomyada soo xidhidhiyaha;
taxanaha mRNA ee sharraxaadda hiddo-wadaha;
Ansixinta golaha.
● Adeeg ku habboon dhisidda genome-ga cusub ama hagaajinta genome tixraaceedka jira ee noocyada xiisaha.
Faa'iidooyinka Adeegga
Horumarinta aaladaha isku xigxiga iyo bioinformatics inde novoururinta genome
(Amarasinghe SL iyo al.,Biology-ga Genome, 2020)
●Khibrad Balaadhan iyo Diiwaanka DaabacaaddaBMKGene waxay soo ururisay waayo-aragnimo ballaaran oo ku saabsan ururinta genome-ga tayada sare leh ee noocyada kala duwan, oo ay ku jiraan genomes diploid iyo genomes aadka u adag ee polyploid iyo noocyada allopolyploid. Ilaa 2018, waxaanu wax ku darsannay in ka badan300 oo daabacaadyo saamayn sare leh, iyo 20+ ka mid ah ayaa lagu daabacay Dabeecada Hiddaha.
● Xalka hal joogsi ah: Habkayaga isku dhafan wuxuu isku daraa teknoolojiyad isdaba-joog ah oo badan iyo falanqaynta bioinformatic-ka si loo helo shaqo isku-dhafan, oo keenaya genome la isku daray oo tayo sare leh.
●Ku Haboon Baahidaada: Socodka shaqadeenu waa mid la beddeli karo, taasoo u oggolaanaysa la qabsiga genome-yada leh astaamo kala duwan iyo baahiyo cilmi-baaris oo gaar ah. Tan waxaa ka mid ah in la waafajiyo genomes-ka weyn, genomes polyploid, genomes heterozygous aad u sarreeya, iyo in ka badan.
●Kooxda shaybaadhka iyo Bioinformatics oo aad u xirfad badan: khibrad weyn u leh labadaba tijaabada iyo bioinformatics ee hore ee shirarka genome-ga adag iyo taxane ah oo patents iyo xuquuq-dhaqaaleedka software.
●Taageerada Iibka Kadib:Ballanqaadkayaga waxa uu dhaafsiisan yahay dhamaystirka mashruuca oo leh 3 bilood oo adeega iibka kadib. Inta lagu jiro wakhtigan, waxaanu bixinaa dabagalka mashruuca, caawinta cilad-raadinta, iyo fadhiyada Q&A si wax looga qabto wixii su'aalo ah ee la xidhiidha natiijooyinka.
Tilmaamaha Adeegga
| sahanka genome | Isugeynta genome | Heerka-chromosome | Tilmaamaha Genome |
| 50X Illumina NovaSeq PE150
| 30X PacBio CCS HiFi ayaa akhriya | 100X Hi-C | RNA-seq Illumina PE150 10 Gb + (ikhtiyaar) Dhererka buuxa RNA-seq PacBio 40 Gb ama Nanopore 12 Gb |
Shuruudaha Adeegga
Sahanka Genome, Golaha Genome iyo Golaha Hi-C:
| Nudaha ama asiidhyada nucleic-ka ah ee la soo saaray | Sahanka Genome | Genome Assembly oo leh PacBio | Golaha Hi-C |
| Viscera xoolaha | 0.5-1 g
| ≥ 3.5 g | ≥2 g |
| Murqaha Xoolaha | ≥ 5 g | ||
| Dhiiga naasleyda | 1.5 ml
| ≥ 5 ml | ≥2 ml |
| Dhiiga digaaga/Kalluunka | ≥ 0.5 ml | ||
| Dhirta- Caleen Cusub | 1-2 g | ≥ 5 g | ≥ 4 g |
| Unugyada Dhaqanka |
| ≥ 1x108 | ≥ 1x107 |
| Cayayaanka | 0.5-1 g | ≥ 3 g | ≥ 2 g |
| DNA la soo saaray | Xooga saarida: ≥1 ng/µL Qadarka ≥ 30 ng Xaddidan ama aan hoos u dhicin ama faddarayn | Xooga saarida: ≥ 50 ng/µL Qadarka: 10 µg / unug qulqulaya / muunad OD260/280=1.7-2.2 OD260/230=1.8-2.5 Xaddidan ama aan hoos u dhicin ama faddarayn |
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Wixii faahfaahin ah ee Genome oo leh qoraal-qoraal:
| Nudaha ama asiidhyada nucleic-ka ah ee la soo saaray | Illumina Transcriptome | PacBio Transcriptome | Nanopore Transcriptome |
| Dhirta- Xidid/Sem/Beed | 450 mg | 600 mg | |
| Dhirta – Caleen/Iniinaha | 300 mg | 300 mg | |
| Dhirta - Miraha | 1.2 g | 1.2 g | |
| Wadnaha/mindhicirka xoolaha | 300 mg | 300 mg | |
| Animal Viscera / Maskaxda | 240 mg | 240 mg | |
| Murqaha Xoolaha | 450 mg | 450 mg | |
| Lafaha Xoolaha/Timaha/Maqaarka | 1 g | 1 g | |
| Arthropod - Cayayaanka | 6 | 6 | |
| Arthropod - Crustacea | 300 mg | 300 mg | |
| Dhiig dhan | 1 tube | 1 tube | |
| RNA la soo saaray | Xoog-saarista: ≥ 20 ng/µL Qadarka ≥ 0.3 µg OD260/280=1.7-2.5 OD260/230=0.5-2.5 RIN≥ 6 5≥28S/18S≥1 | Xoog-saarista: ≥ 100 ng/µL Qadarka ≥ 0.75 µg OD260/280=1.7-2.5 OD260/230=0.5-2.5 RIN≥ 8 5≥28S/18S≥1 | Xoog-saarista: ≥ 100 ng/µL Qadarka ≥ 0.75 µg OD260/280=1.7-2.5 OD260/230=0.5-2.5 RIN≥ 7.5 5≥28S/18S≥1 |
Tusaalaha keenista lagu taliyay
Weel: 2 ml oo tuubo sentrifuge ah (Faylada qasacadaysan laguma talinayo)
(Samballada intooda badan, waxaan kugula talineynaa inaan lagu keydin ethanol.)
Calaamadaynta Tusaalaha: Tusaalooyinka waa in si cad loo calaamadiyaa oo la mid ah foomka macluumaadka muunada ee la gudbiyay.
Shixnadda: Barafka qallalan: Muunadodu waxay u baahan yihiin in marka hore bac lagu shubo oo lagu aaso baraf qallalan.
Socodka shaqada
Socodka Shaqada Adeegga
Naqshadaynta tijaabada
Tusaalaha keenista
Soo saarista DNA
Dhismaha maktabadda
Isku xigxiga
Falanqaynta xogta
Adeegyada iibka kadib
Falanqaynta bioinformatic oo dhammaystiran, oo loo qaybiyay 4 tillaabo:
1) Sahanka Genome, oo ku salaysan falanqaynta k-mer ee NGS waxay akhridaa:
Qiyaasta cabbirka genome
Qiyaasta heterozygosity
Qiyaasta gobollada soo noqnoqda
2) Genom Assembly oo leh PacBio HiFi:
De novoshirka
Qiimaynta golaha: oo ay ku jirto falanqaynta BUSCO ee dhamaystirka genome-ga iyo khariidaynta dhabarka ee NGS iyo PacBio HiFi akhri
3) Kulanka Hi-C:
Maktabadda Hi-C QC: qiyaasta isdhexgalka Hi-C ee sax ah
Kulanka Hi-C: ururinta kontiig kooxeedyo, oo ay ku xigto amar-ku-gudbinta koox kasta iyo u xilsaarida hanuuninta contig
Qiimaynta Hi-C
4) Faahfaahinta Genome:
Saadaasha RNA aan codeeyn
Aqoonsiga taxanaha soo noqnoqda (transposons iyo tandem ku celcelinta)
Saadaasha hidda-socodka
§De novo: ab initio algorithms
§ Iyada oo ku saleysan homology
§ Iyada oo ku saleysan qoraalka qoraalka, oo leh akhrin dheer iyo mid gaaban: akhrintu waade novola ururiyey ama lagu sawiray genome qabyada
§ Sharaxaada hiddo-wadaha la saadaaliyay oo leh xog ururin badan
1) Baadhitaanka Genome- k-mer falanqaynta
2) Genom Assembly
2) Golaha Genome - PacBio HiFi wuxuu akhriyaa khariidad samaynta golaha qabyada
2) Golaha Hi-C - qiyaasida Hi-C lammaane is dhexgalka ansax ah
3) Qiimaynta Hi-C Golaha Kadib
4) Annotation Genome - isdhexgalka hiddo-wadaha la saadaaliyay
4) Sharaxaada genome - sharraxaadda hiddo-wadaha la saadaaliyay
Sahami horumarka ay fududaysay BMKGene's de novo genome Assembly adeegyada iyada oo loo marayo ururinta daabacaado la soo xulay:
Li, C. iyo al. (2021) 'Tixanaha genome-ga waxay muujinayaan dariiqyada kala firdhisan caalamiga ah waxayna soo jeedinayaan laqabsiga hidde-sidaha isku dhafan ee kobcinta badda', Isgaarsiinta Dabeecadda, 12(1). doi: 10.1038 / S41467-021-21379-X.
Li, Y. iyo al. (2023) 'Isbeddelada Weyn ee Chromosomal waxay u horseedaan Beddelka Muujinta Heerka Genome-Level, Laqabsiga Deegaanka, iyo Gaarka ee Gayal (Bos frontalis)', Biology Molecular and Evolution, 40(1). doi: 10.1093/MOLBEV/MSAD006.
Tian, T. iyo al. (2023) 'Geome-ga ururinta iyo kala qaybinta hidde-sidaha ee jeermiska galleyda abaarta u adkeysta ee caanka ah', Nature Genetics 2023 55:3, 55(3), bogga. 496-506. doi: 10.1038/s41588-023-01297-y.
Zhang, F. iyo al. (2023) 'Daah furka horumarka tropane alkaloid biosynthesis iyadoo la falanqeynayo laba genomes ee qoyska Solanaceae', Isgaarsiinta Dabeecadda 2023 14:1, 14(1), pp. 1-18. doi: 10.1038/s41467-023-37133-4.
Kiis-daraasad adag:
Isku-dhafka Telomere-ilaa-telomere:Fu, A. iyo al. (2023) 'Telomere-to-telomere genome genome shirkii qaraha qadhaadh (Momordica charantia L. var. abbreviata Ser.) waxay muujinaysaa horumarinta miraha, halabuurka iyo bisaylka sifooyinka hidaha', Cilmi-baarista Horticulture, 10 (1). doi: 10.1093/HR/UHAC228.
Haplotype shirka:Hu, W. iyo al. (2021) 'Allele-qeexay genome-ku wuxuu daaha ka qaadayaa kala duwanaanshaha biillelic inta lagu guda jiro koboca kasaafada', Plant Molecular, 14(6), bogga. 851-854. doi: 10.1016/j.molp.2021.04.009.
Isugeynta genome-ga weyn:Yuan, J. iyo al. (2022) 'Genomic base of giga-chromosomes iyo giga-genome of tree peony Paeonia ostii', Nature Communications 2022 13:1, 13(1), pp. 1-16. doi: 10.1038/s41467-022-35063-1.
Unugyada genome polyploid:Zhang, Q. iyo al. (2022) 'Aragtida genomic ee koromosoomyada dhowaan la dhimay ee sonkorowga autopolyploid Saccharum spontaneum', Nature Genetics 2022 54: 6, 54(6), pp. 885-896. doi: 10.1038/s41588-022-01084-1.
