Osisi/Anụmanụ De Novo Genome Sequencing
De Novousoro n'usoro na-ezo aka n'iwupụta genome nke ụdị dị iche iche site na iji teknụzụ usoro n'usoro na enweghị genome ntụaka. Iwebata na nnabata nke usoro ọgbọ nke atọ, nke na-egosi ogologo ọgụgụ, emeela ka mgbakọ genome dịkwuo mma site n'ịbawanye ndakọrịta n'etiti ọgụgụ. Nkwalite a dị mkpa karịsịa mgbe a na-emeso genomes ndị siri ike, dị ka ndị na-egosipụta heterozygosity dị elu, ọnụ ọgụgụ dị elu nke mpaghara ugboro ugboro, polyploids, na mpaghara nwere ihe ndị na-emegharị ugboro ugboro, ọdịnaya GC na-adịghị mma, ma ọ bụ mgbagwoju anya dị elu nke a na-ezukọta nke ọma site na iji usoro ọgụgụ dị mkpirikpi.
Ihe ngwọta otu nkwụsị anyị na-enye ọrụ nhazi usoro na nyocha bioinformatic nke na-ebuga genome dị elu nke de novo kpọkọtara. Nnyocha genome nke mbụ na Illumina na-enye atụmatụ nke nha genome na mgbagwoju anya, a na-ejikwa ozi a na-eduzi usoro ọzọ nke usoro ọgụgụ ogologo oge na PacBio HiFi, sochiri ya.de novomgbakọ nke contigs. Ojiji nke mgbakọ HiC na-esote na-enyere aka ịkwado contigs na genome, nweta mgbakọ ọkwa chromosome. N'ikpeazụ, a na-akọwa genome ahụ site na amụma mkpụrụ ndụ ihe nketa na site n'usoro usoro mkpụrụ ndụ ihe nketa egosipụtara, na-amalite na transcriptome nwere ọgụgụ dị mkpụmkpụ na ogologo.
Atụmatụ ọrụ
● Mmekọrịta nke ọtụtụ usoro na ọrụ bioinformatic n'otu ngwọta nkwụsịtụ:
Nyocha genome na Illumina iji tụọ nha genome na ntuziaka usoro ndị ọzọ;
Ogologo usoro ọgụgụ makade novomgbakọ nke contigs;
Usoro Hi-C maka anchoring chromosome;
usoro mRNA maka nkọwa nke mkpụrụ ndụ ihe nketa;
Nkwado nke mgbakọ ahụ.
● Ọrụ kwesịrị ekwesị maka iwulite genome ọhụrụ ma ọ bụ melite genome dị ugbu a maka ụdị mmasị.
Uru ọrụ
Mmepe nke usoro nhazi usoro na bioinformatics inde novomgbakọ genome
(Amarasinghe SL et al.,Genome Biology, 2020)
●Ọkachamara dị ukwuu na ndekọ mbipụta: BMKGene achịkọtala nnukwu ahụmahụ na mgbakọ genome dị elu nke ụdị dị iche iche, gụnyere diploid genomes na nnukwu mgbagwoju anya nke polyploid na allopolyploid ụdị. Kemgbe afọ 2018, anyị enyela aka n'ihe karịrịAkwụkwọ 300 nwere mmetụta dị elu, na 20+ n'ime ha ka ebipụtara na Nature Genetics.
● Ngwọta otu nkwụsị: ụzọ anyị jikọtara ọnụ na-ejikọta ọtụtụ teknụzụ usoro usoro na nyocha bioinformatic n'ime usoro ọrụ na-arụkọ ọrụ ọnụ, na-ebuga genome agbakọtara dị elu.
●Haziri maka mkpa gị: Ọrụ ọrụ anyị nwere ike ịhazi ya, na-enye ohere mgbanwe maka genomes nwere njirimara dị iche iche na mkpa nyocha kpọmkwem. Nke a na-agụnye nnabata nnukwu genomes, polyploid genomes, nnukwu heterozygous genomes, na ndị ọzọ.
●Ndị otu Bioinformatics na Laboratorial nwere nkà nke ukwuu: nwere ahụmahụ dị ukwuu na ma nnwale na bioinformatics n'ihu mgbakọ genome dị mgbagwoju anya na usoro patent na ikike nwebisiinka software.
●Nkwado mgbe ọrịre ahịa:Nkwenye anyị ga-agafe karịa mmecha ọrụ yana oge ọrụ ọnwa 3 gachara. N'ime oge a, anyị na-enye nlebanya ọrụ, enyemaka nchọpụta nsogbu, yana oge ajụjụ & A iji zaa ajụjụ ọ bụla metụtara nsonaazụ ya.
Nkọwapụta ọrụ
| Nyocha genome | Mgbakọ genome | Ọkwa chromosome | Nkọwa genome |
| 50X Illumina NovaSeq PE150
| 30X PacBio CCS HiFi na-agụ | 100X Hi-C | RNA-seq Illumina PE150 10 Gb + (nhọrọ) Ogologo zuru oke RNA-seq PacBio 40 Gb ma ọ bụ Nanopore 12 GB |
Ihe achọrọ ọrụ
Maka nyocha Genome, Mgbakọ Genome na Mgbakọ Hi-C:
| Anụ ahụ ma ọ bụ amịpụtara nucleic acid | Nyocha genome | Mgbakọ Genome na PacBio | Mgbakọ Hi-C |
| Viscera anụmanụ | 0,5-1 g
| ≥ 3.5 g | ≥2 g |
| Akwara anụ | ≥5 g | ||
| Ọbara Mammalian | 1.5 ml
| 5 ml nke mmiri ara ehi | 2 ml |
| Ọbara anụ ọkụkọ/Azụ | 0.5 ml nke mmiri ara ehi | ||
| Osisi- Akwụkwọ ọhụrụ | 1-2 g | ≥5 g | ≥4 g |
| Selụ mepere emepe |
| 1x108 | 1x107 |
| Ụmụ ahụhụ | 0,5-1 g | ≥3 g | ≥2 g |
| DNA ewepụtara | Ntinye uche: ≥1 ng/µL Ọnụ ego ≥ 30 ng Oke ma ọ bụ enweghị mmebi ma ọ bụ mmetọ | Ntinye uche: ≥ 50 ng/µL Ọnụ ego: 10 μg / cell flu / sample OD260/280=1.7-2.2 OD260/230=1.8-2.5 Oke ma ọ bụ enweghị mmebi ma ọ bụ mmetọ |
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|
Maka nkọwa Genome nwere transcriptomics:
| Anụ ahụ ma ọ bụ amịpụtara nucleic acid | Nkọwapụta nke Illumina | PacBio Transcriptome | Nanopore transcriptome |
| Osisi- Mgbọrọgwụ/Stem/Petal | 450 mg | 600 mg | |
| Osisi - Akwukwo/Mkpụrụ | 300 mg | 300 mg | |
| Osisi - Mkpụrụ osisi | 1.2 g | 1.2 g | |
| Obi/ eriri afọ anụmanụ | 300 mg | 300 mg | |
| Animal Viscera/ụbụrụ | 240 mg | 240 mg | |
| Akwara anụ | 450 mg | 450 mg | |
| Ọkpụkpụ anụmanụ/Ntutu/Akpụkpọ ahụ | 1 g | 1 g | |
| Arthropod - ahụhụ | 6 | 6 | |
| Arthropod - Crustacea | 300 mg | 300 mg | |
| Ọbara dum | 1 tube | 1 tube | |
| RNA ewepụtara | Ntinye uche: ≥ 20 ng/µL Ọnụ ego ≥ 0.3 µg OD260/280=1.7-2.5 OD260/230=0.5-2.5 RIN≥ 6 5≥28S/18S≥1 | Ntinye uche: ≥ 100 ng/µL Ọnụ ego ≥ 0.75 µg OD260/280=1.7-2.5 OD260/230=0.5-2.5 RIN≥ 8 5≥28S/18S≥1 | Ntinye uche: ≥ 100 ng/µL Ọnụ ego ≥ 0.75 µg OD260/280=1.7-2.5 OD260/230=0.5-2.5 RIN7.5 5≥28S/18S≥1 |
Nnyefe nlele akwadoro
Akpa: 2 ml centrifuge tube (Tin foil adịghị akwadoro)
(Maka ọtụtụ n'ime ihe atụ, anyị na-akwado ka ị ghara ichekwa na ethanol.)
Ịkpọ aha nlele: A ga-enwerịrị akara nke ọma ma yie ụdị ozi nlele etinyere.
Mbupu: ice-akọrọ: A ga-ebu ụzọ tinye ihe nlele n'ime akpa wee lie ya na ice-akọrọ.
Usoro ọrụ
Usoro ọrụ ọrụ
Nnwale imewe
Nbufe ihe atụ
Mwepụta DNA
Ịrụ ụlọ akwụkwọ
Usoro
Nyocha data
Mgbe-ire ọrụ
Nyocha bioinformatic zuru ezu, kewapụrụ na usoro anọ:
1) Nyocha Genome, dabere na nyocha k-mer na NGS na-agụ:
Atụmatụ nha genome
Atụmatụ heterozygosity
Atụmatụ mpaghara ugboro ugboro
2) Mgbakọ Genome na PacBio HiFi:
De novomgbakọ
Ntụle mgbakọ: gụnyere nyocha BUSCO maka izu oke genome na eserese azụ nke NGS na PacBio HiFi na-agụ.
3) Mgbakọ Hi-C:
Ọbá akwụkwọ Hi-C QC: atụmatụ nke mmekọrịta Hi-C bara uru
Mgbakọ Hi-C: nchịkọta nke contigs n'otu dị iche iche, na-esochi usoro nhazi n'ime otu ọ bụla ma na-ekenye nhazi contig.
Nyocha Hi-C
4) Nkọwa genome:
Amụma RNA na-abụghị koodu
Nchọpụta usoro ugboro ugboro (transposons na tandem ikwugharị)
Amụma mkpụrụ ndụ ihe nketa
§De novo: ab initio algọridim
§ Dabere na homology
§ Dabere na transcriptome, na ogologo na nkenke na-agụ: na-agụ bụde novokpọkọtara ma ọ bụ depụta ya na genome nke a gbara akwụkwọ
§ Nkọwa nke mkpụrụ ndụ ihe nketa buru amụma nwere ọtụtụ ọdụ data
1) Nyocha genome-k-mer nyocha
2) Mgbakọ Genome
2) Mgbakọ Genome - PacBio HiFi na-agụ nkewa ka ọ bụrụ mgbakọ
2) Mgbakọ Hi-C - atụmatụ Hi-C dị mma mmekọrịta abụọ
3) Hi-C Post-mgbakọ nlebanya
4) Nkọwapụta Genome - njikọ nke mkpụrụ ndụ ihe nketa buru amụma
4) Nkọwa genome - nkọwa mkpụrụ ndụ ihe nketa buru amụma
Nyochaa ọganihu nke BMKGene's de novo genome mgbakọ ọrụ kwadoro site na nchịkọta akwụkwọ ndị a chịkọtara:
Li, C. et al. (2021) 'usoro genome na-ekpughe ụzọ mgbasa ozi zuru ụwa ọnụ ma na-atụ aro mgbanwe mkpụrụ ndụ ihe nketa na evolushọn nke oké osimiri', Nature Communications, 12(1). doi: 10.1038/S41467-021-21379-X.
Li, Y. et al. (2023) 'Mgbanwe chromosomal buru ibu na-eduga na mgbanwe nkwupụta okwu Genome-Level, mgbanwe gburugburu ebe obibi, na nkọwapụta na Gayal (Bos frontalis)', Molecular Biology and Evolution, 40(1). doi: 10.1093/MOLBEV/MSAD006.
Tian, T. et al. (2023) 'Mgbakọ genome na mkpọsa mkpụrụ ndụ ihe nketa nke germplasm ọka a ma ama na-eguzogide ụkọ mmiri ozuzo', Nature Genetics 2023 55:3, 55(3), p. 496–506. doi: 10.1038/s41588-023-01297-y.
Zhang, F. et al. (2023) 'Ikpughe evolushọn nke tropane alkaloid biosynthesis site n'ịtụle genome abụọ n'ime ezinụlọ Solanaceae', Nature Communications 2023 14:1, 14(1), p. 1–18. doi: 10.1038/s41467-023-37133-4.
Ọmụmụ ikpe na-ama aka:
Mgbakọ Telomere-to-telomere:Fu, A. et al. (2023) 'Telomere-to-telomere genome Assembly of bitter melon (Momordica charantia L. var. abbreviata Ser.) na-ekpughe mmepe nke mkpụrụ osisi, ihe mejupụtara na njirimara mkpụrụ ndụ mkpụrụ osisi ', Horticulture Research, 10 (1). doi: 10.1093/HR/UHAC228.
Mgbakọ Haplotype:Hu, W. et al. (2021) 'Geneme-akọwapụtara Allele na-ekpughe ọdịiche bialelic n'oge evolushọn akpu', Osisi Molecular, 14(6), p. 851–854. doi: 10.1016/j.molp.2021.04.009.
Nnukwu genome mgbakọ:Yuan, J. et al. (2022) 'Genomic ndabere nke giga-chromosomes na giga-genome nke osisi peony Paeonia ostii', Nature Communications 2022 13:1, 13(1), p. 1–16. doi: 10.1038/s41467-022-35063-1.
Polyploid genome mgbakọ:Zhang, Q. et al. (2022) 'Nghọta Genomic n'ime mbelata chromosome na nso nso a nke autopolyploid sugarcane Saccharum spontaneum', Nature Genetics 2022 54:6, 54(6), p. 885–896. doi: 10.1038/s41588-022-01084-1.
