Semela / Phoofolo De Novo Genome Sequencing
Ho tloha Novotatellano e bolela kaho ea mofuta oohle oa lefutso ho sebelisoa mahlale a tatellano ha ho se na genome. Kenyelletso le kamohelo e pharalletseng ea tatellano ea moloko oa boraro, e nang le lipalo tse telele, e ntlafalitse haholo kopano ea genome ka ho eketsa ho fetana lipakeng tsa ho bala. Ntlafatso ena e bohlokoa haholo ha o sebetsana le liphatsa tsa lefutso tse phephetsang, tse kang tse bonts'ang heterozygosity e phahameng, karo-karolelano e phahameng ea libaka tse ipheta-phetang, li-polyploid, le libaka tse nang le likarolo tse pheta-phetoang, litaba tse sa tloaelehang tsa GC, kapa ho rarahana ho hoholo hoo hangata ho sa kopanngoeng hantle ho sebelisoa tatellano ea ho bala nako e khuts'oane. feela.
Tharollo ea rona ea ho emisa e le 'ngoe e fana ka lits'ebeletso tse kopaneng tsa tatellano le tlhahlobo ea bioinformatic e fanang ka genome ea boleng bo holimo e kopaneng ea de novo. Phuputso ea pele ea genome le Illumina e fana ka likhakanyo tsa boholo ba genome le ho rarahana, 'me boitsebiso bona bo sebelisetsoa ho tataisa mohato o latelang oa tatellano ea nako e telele le PacBio HiFi, e lateloa kede novokopano ea li-contigs. Tšebeliso e latelang ea kopano ea HiC e thusa ho tiisa li-contigs ho genome, ho fumana kopano ea boemo ba chromosome. Qetellong, genome e hlalosoa ka ho bolela esale pele ka liphatsa tsa lefutso le ka tatellano ea liphatsa tsa lefutso tse hlalositsoeng, ho sebelisa li-transcriptome tse nang le lipalo tse khutšoane le tse telele.
Likarolo tsa Tšebeletso
● Ho kopanngoa ha lits'ebeletso tse ngata tsa tatelano le bioinformatic ka tharollo ea sebaka se le seng:
Phuputso ea Genome le Illumina ho hakanya boholo ba genome le ho tataisa mehato e latelang;
Ho bala nako e telele tatellano bakeng sade novokopano ea li-contigs;
Tatelano ea Hi-C bakeng sa ho tiisa chromosome;
tatellano ea mRNA bakeng sa tlhaloso ea liphatsa tsa lefutso;
Netefatso ya kopano.
● Ts'ebeletso e loketseng ho aha liphatsa tsa lefutso tse ncha kapa ntlafatso ea liphatsa tsa lefutso tse teng bakeng sa mefuta e khahlisang.
Melemo ea Tšebeletso
Nts'etsopele ea li-platform tsa tatellano le bioinformatics hode novokopano ea genome
(Amarasinghe SL et al.,Genome Biology, 2020)
●Rekoto e Pharalletseng ea Boitsebi le Phatlalatso: BMKGene e bokelletse boiphihlelo bo boholo ba ho kopanya liphatsa tsa lefutso tsa boleng bo holimo tsa mefuta e fapaneng, ho kenyeletsoa liphatsa tsa lefutso tsa diploid le liphatsa tsa lefutso tse rarahaneng haholo tsa mefuta ea polyploid le allopolyploid. Ho tloha ka 2018, re kentse letsoho ho fetaLingoliloeng tse 300 tse nang le tšusumetso e kholo, 'me tse 20+ tsa tsona li hatisitsoe ho Nature Genetics.
● One-Stop Tharollo: mokhoa oa rona o kopantsoeng o kopanya mekhoa e mengata ea tatellano le litlhahlobo tsa bioinformatic hore e be mokhoa o kopanetsoeng oa ho sebetsa, ho fana ka genome ea boleng bo holimo e kopaneng.
●E Eketsehile ho ea ka Litlhoko Tsa Hao: Ts'ebetso ea rona ea ts'ebeletso e ka tloaeleha, e lumella ho ikamahanya le liphatsa tsa lefutso tse nang le likarolo tse fapaneng le litlhoko tse ikhethang tsa lipatlisiso. Sena se kenyelletsa ho amohela liphatsa tsa lefutso tse kholo, liphatsa tsa lefutso tsa polyploid, liphatsa tsa lefutso tsa heterozygous haholo, le tse ling.
●Sehlopha sa Bioinformatics le Laboratori ba nang le Tsebo e Phahameng: ea nang le boiphihlelo bo botle ho liteko le bioinformatics tse ka pele ho likopano tse rarahaneng tsa liphatsa tsa lefutso le letoto la litokelo tsa molao le litokelo tsa li-software.
●Tšehetso ea Ka mor'a Thekiso:Boitlamo ba rona bo fetela ka nģ'ane ho ho phethoa ha morero ka nako ea tšebeletso ea likhoeli tse 3 ka mor'a thekiso. Nakong ena, re fana ka tatellano ea projeke, thuso ea ho rarolla mathata, le linako tsa Q&A ho araba lipotso life kapa life tse amanang le sephetho.
Litlhaloso tsa Tšebeletso
| Phuputso ea Genome | Kopano ea Genome | Boemo ba chromosome | Tlhaloso ea Genome |
| 50X Illumina NovaSeq PE150
| 30X PacBio CCS HiFi ea bala | 100X Hi-C | RNA-seq Illumina PE150 10 Gb + (boikhethelo) Bolelele bo felletseng ba RNA-seq PacBio 40 Gb kapa Nanopore 12 Gb |
Litlhoko tsa Tšebeletso
Bakeng sa Patlisiso ea Genome, Kopano ea Genome le Kopano ea Hi-C:
| Lithane kapa li-nucleic acid tse nkiloeng | Patlisiso ea Genome | Kopano ea Genome le PacBio | Kopano ea Hi-C |
| Viscera ea liphoofolo | 0.5-1 g
| ≥ 3.5 g | ≥2 g |
| Mosifa oa Phoofolo | ≥ 5 g | ||
| Mali a Mammalian | 1.5 mL
| ≥ 5 ml | ≥2 ml |
| Mali a likhoho/Litlhapi | ≥ 0.5 mL | ||
| Semela- Lekhasi le Lecha | 1-2 g | ≥ 5 g | ≥ 4 g |
| Lisele tsa Cultured |
| ≥ 1x108 | ≥ 1x107 |
| Koanyana | 0.5-1 g | ≥3 g | ≥ 2 g |
| DNA e ntšitsoeng | Tsepamiso ea maikutlo: ≥1 ng/µL Chelete ≥ 30 ng Ho fokotsoa kapa ho se na seriti kapa tšilafalo | Tsepamiso ea maikutlo: ≥ 50 ng/µL Chelete: 10 µg/flow cell/sample OD260/280=1.7-2.2 OD260/230=1.8-2.5 Ho fokotsoa kapa ho se na seriti kapa tšilafalo |
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Bakeng sa tlhaloso ea Genome e nang le transcriptomics:
| Lithane kapa li-nucleic acid tse nkiloeng | Illumina Transcriptome | PacBio Transcriptome | Nanopore Transcriptome |
| Semela- Motso/Stem/Petal | 450 mg | 600 mg | |
| Semela – Lekhasi/Peo | 300 mg | 300 mg | |
| Semela - Litholoana | 1.2 g | 1.2 g | |
| Pelo ya Phoofolo/ Mala | 300 mg | 300 mg | |
| Phoofolo Viscera/Boko | 240 mg | 240 mg | |
| Mosifa oa Phoofolo | 450 mg | 450 mg | |
| Marapo a Phoofolo/Moriri/Letlalo | 1 g | 1 g | |
| Arthropod - Kokoane | 6 | 6 | |
| Arthropod - Crustacea | 300 mg | 300 mg | |
| Madi kaofela | 1 tube | 1 tube | |
| RNA e ntšitsoeng | Tsepamiso ea maikutlo: ≥ 20 ng/µL Chelete ≥ 0.3 µg OD260/280=1.7-2.5 OD260/230=0.5-2.5 RIN≥6 5≥28S/18S≥1 | Tsepamiso ea maikutlo: ≥ 100 ng/µL Chelete ≥ 0.75 µg OD260/280=1.7-2.5 OD260/230=0.5-2.5 RIN≥8 5≥28S/18S≥1 | Tsepamiso ea maikutlo: ≥ 100 ng/µL Chelete ≥ 0.75 µg OD260/280=1.7-2.5 OD260/230=0.5-2.5 RIN≥ 7.5 5≥28S/18S≥1 |
Tlhahiso ea Mohlala e khothalelitsoeng
Setshelo: 2 ml centrifuge tube (Tin foil ha e khothalletsoe)
(Bakeng sa disampole tse ngata, re khothaletsa ho se boloke ethanol.)
Mohlala oa ho ngola: Mehlala e tlameha ho ngoloa ka ho hlaka le ho tšoana le foromo ea tlhaiso-leseling e rometsoeng.
Thomello: Leqhoa le omileng: lisampole li hloka ho kenngoa ka mekotleng pele ebe li patoa ka leqhoa le omileng.
Mokhoa oa ho sebetsa
Phallo ea Mosebetsi oa Ts'ebeletso
Moqapi oa liteko
Mohlala oa thomello
Ho ntšoa ha DNA
Kaho ea laebrari
Tatelano
Tlhahlobo ea lintlha
Litšebeletso tsa ka mor'a thekiso
Tlhahlobo e felletseng ea bioinformatics, e arotsoe ka mehato e 4:
1) Tlhahlobo ea Genome, e ipapisitseng le tlhahlobo ea k-mer le NGS e baleha tjena:
Khakanyo ea boholo ba genome
Khakanyo ea heterozygosity
Khakanyo ea libaka tse iphetang
2) Kopano ea Genome e nang le PacBio HiFi:
De novokopano
Tlhahlobo ea Kopano: ho kenyelletsa tlhahlobo ea BUSCO bakeng sa botlalo ba genome le 'mapa oa morao oa NGS le PacBio HiFi e baloa.
3) Kopano ea Hi-C:
Hi-C laeborari ea QC: khakanyo ea litšebelisano tse nepahetseng tsa Hi-C
Kopano ea Hi-C: ho bokellana ha li-contig ka lihlopha, ho lateloa ke ho laela li-contig ka har'a sehlopha ka seng le ho abela li-contig
Tlhahlobo ea Hi-C
4) Tlhaloso ea Genome:
Bolelele ba RNA bo sa khouteng
Tsebiso ea tatellano e pheta-phetoang (li-transposons le pheta-pheto ea tandem)
Boprofeta ba Gene
§De novo: ab initio algorithms
§ E thehiloe ho homology
§ E ipapisitse le transcriptome, e nang le lipalo tse telele le tse khutšoane: tse baloang lide novoe bokelletsoe kapa e entsoe 'mapa oa sebopeho sa genome
§ Tlhaloso ea liphatsa tsa lefutso tse boletsoeng esale pele tse nang le database tse ngata
1) Tlhahlobo ea Genome- k-mer analysis
2) Kopano ea Genome
2) Kopano ea Genome - PacBio HiFi e bala 'mapa ho hlophisa kopano
2) Kopano ea Hi-C - khakanyo ea lipara tsa tšebelisano tse sebetsang tsa Hi-C
3) Tlhahlobo ea Hi-C Post-assembly
4) Tlhaloso ea Genome - kopanyo ea liphatsa tsa lefutso tse boletsoeng esale pele
4) Tlhaloso ea Genome - tlhaloso ea liphatsa tsa lefutso e boletsoeng esale pele
Lekola tsoelopele e tsamaisoang ke litšebeletso tsa kopano ea BMKGene's de novo genome ka pokello e hlophisitsoeng ea lingoliloeng:
Li, C. le ba bang. (2021) 'Tatelano ea liphatsa tsa lefutso e senola litsela tsa ho hasana lefatšeng ka bophara 'me e fana ka maikutlo a phetoho ea liphatsa tsa lefutso ho seahorse evolution', Nature Communications, 12(1). doi: 10.1038/S41467-021-21379-X.
Li, Y. et al. (2023) 'Liphetoho tse Khōlō tsa Chromosomal li Lebisa Liphetohong tsa Phatlalatso ea Boemo ba Genome, Ho Fetolana ha Tikoloho, le Tlhaloso ho Gayal (Bos frontalis)', Biology ea Molek'hule le Evolution, 40 (1). doi: 10.1093/MOLBEV/MSAD006.
Tian, T. et al. (2023) 'Genome assembly le genetic dissection ea poone e hlahelletseng e hananang le komello', Nature Genetics 2023 55:3, 55(3), maq. 496–506. doi: 10.1038/s41588-023-01297-y.
Zhang, F. et al. (2023) 'Ho senola ho iphetola ha lintho tsa tropane alkaloid biosynthesis ka ho hlahloba liphatsa tsa lefutso tse peli lelapeng la Solanaceae', Nature Communications 2023 14: 1, 14 (1), maq. 1-18. doi: 10.1038/s41467-023-37133-4.
Liphuputso tse phephetsang:
Kopano ea Telomere-to-telomere:Fu, A. et al. (2023) 'Telomere-to-telomere genome assembly of bitter melon (Momordica charantia L. var. abbreviata Ser.) e senola tsoelo-pele ea litholoana, sebopeho le liphatsa tsa lefutso tse butsoitseng', Horticulture Research, 10 (1). doi: 10.1093/HR/UHAC228.
Kopano ea Haplotype:Hu, W. et al. (2021) 'Allele-defined genome e senola phapang ea biallelic nakong ea ho iphetola ha cassava', Molecular Plant, 14 (6), maq. 851-854. doi: 10.1016/j.molp.2021.04.009.
Kopano e kholo ea genome:Yuan, J. et al. (2022) 'Genomic motheo oa giga-chromosomes le giga-genome ea sefate peony Paeonia ostii', Nature Communications 2022 13: 1, 13 (1), maq. 1-16. doi: 10.1038/s41467-022-35063-1.
Kopano ea genome ea Polyploid:Zhang, Q. et al. (2022) 'Litlhaloso tsa Genomic mabapi le phokotso ea morao-rao ea chromosome ea 'moba oa autopolyploid Saccharum spontaneum', Nature Genetics 2022 54: 6, 54 (6), maq. 885-896. doi: 10.1038/s41588-022-01084-1.
