La'au/Manu De Novo Genome Sequencing
De Novofa'asologa o lo'o fa'atatau i le fausiaina o se ituaiga 'genome atoa e fa'aaoga ai tekinolosi fa'asologa i le leai o se genome fa'asino. O le folasaga ma le lautele o le faʻaaogaina o le faʻasologa o augatupulaga lona tolu, e faʻaalia ai le umi o faitauga, ua matua faʻaleleia atili ai le genome faʻapotopotoga e ala i le faʻateleina o le faʻaogaina i le va o faitauga. O lenei faʻaleleia e sili ona taua pe a feagai ma genomes luʻitau, e pei oi latou o loʻo faʻaalia le maualuga o le heterozygosity, maualuga maualuga o itulagi toe fai, polyploids, ma itulagi o loʻo i ai elemene faifaipea, mea e le masani ai GC, poʻo le lavelave maualuga e masani ona le lelei le faʻapipiʻiina e faʻaaoga ai le faʻasologa o faitauga pupuu. toatasi.
O la matou fofo e tasi e tu'uina atu auaunaga tu'ufa'atasiga fa'asologa ma su'esu'ega o le bioinformatic lea e tu'uina atu ai se genome tu'ufa'atasia maualuga de novo. O se suʻesuʻega muamua o le genome ma Illumina e maua ai faʻatusatusaga o le genome tele ma le lavelave, ma o lenei faʻamatalaga e faʻaaogaina e taʻitaʻia ai le isi laasaga o le umi-faitau faʻasologa ma PacBio HiFi, sosoo ai ma leo le foupotopotoga o contigs. O le fa'aogaina mulimuli ane o le fa'apotopotoga o le HiC e mafai ai ona fa'amauina fa'atasiga i le genome, ma maua ai se fa'apotopotoga maualuga o le chromosome. Ma le mea mulimuli, o le genome e faʻamatalaina e ala i le vaʻaiga o kenera ma e ala i le faʻasologaina o kenera faʻaalia, faʻaoga i tusitusiga faʻatasi ma faitau pupuu ma umi.
Vaega Auaunaga
● Tu'ufa'atasiga o le tele o fa'asologa ma le bioinformatic 'au'aunaga i se vaifofo tasi-taofi:
Genome su'esu'ega ma Illumina e fa'atatau le tele o le genome ma ta'ita'ia laasaga mulimuli ane;
Fa'asologa umi faitau moo le foufaʻapotopotoga o contigs;
Hi-C sequencing mo chromosome taula;
mRNA fa'asologa mo fa'amatalaga o kenera;
Fa'amaoniga o le fa'apotopotoga.
● Auaunaga talafeagai mo le fausiaina o genomes fou poʻo le faʻaleleia o genomes o loʻo iai mo ituaiga o fiafia.
Tulaga Auaunaga
Atina'e o fa'asologa fa'asologa ma bioinformatics io le foufa'apotopotoga o genome
(Amarasinghe SL et al.,Genome Biology, 2020)
●Tomai Tele ma Faamaumauga Lomiga: Ua fa'aputuina e le BMKGene le tele o le poto masani i le fa'apotopotoina o genome maualuga o ituaiga eseese, e aofia ai genomes diploid ma genomes sili ona lavelave o ituaiga polyploid ma allopolyploid. Talu mai le 2018, ua matou saofagā i luga300 faʻasalalauga maualuga, ma 20+ o loʻo faʻasalalau i le Nature Genetics.
● Fofo e tasi le taofi: o la matou faiga tu'ufa'atasia e tu'ufa'atasia ai le tele o tekinolosi fa'asologa ma su'esu'ega o le bioinformatic i totonu o se fa'agaluega felagolagoma'i, e tu'uina atu ai se genome tu'ufa'atasia maualuga.
●Fa'atatau i ou Manaoga: O la matou galuega fa'au'u tautua e fa'apitoa, fa'ataga fetuutuuna'i mo genomes ma uiga 'ese'ese ma mana'oga fa'apitoa su'esu'ega. E aofia ai le faʻaogaina o genomes tetele, polyploid genomes, heterozygous genomes, ma isi mea.
●Tomai Maualuga Bioinformatics ma Laboratorial Team: fa'atasi ai ma le poto masani i fa'ata'ita'iga ma bioinformatics i luma o fa'apotopotoga fa'apitoa genome ma se faasologa o pateni ma puletaofia polokalama.
●Lagolago ina ua uma ona faatau atu:O la matou tautinoga e faʻalautele atu i tua atu o le faʻamaeʻaina o galuega faʻatasi ma le 3-masina pe a maeʻa le faʻatau atu vaitaimi auaunaga. I le taimi lea, matou te ofoina atu le tulitatao o poloketi, fesoasoani faʻafitauli, ma Q&A sauniga e faʻafeiloaʻi ai soʻo se fesili e fesoʻotaʻi ma iʻuga.
Fa'amatalaga Auaunaga
| Su'esu'ega Genome | Genome fono | Chromosome-tulaga | Genome Annotation |
| 50X Illumina NovaSeq PE150
| 30X PacBio CCS HiFi faitau | 100X Hi-C | RNA-seq Illumina PE150 10 Gb + (filifiliga) Le umi atoa RNA-seq PacBio 40 Gb poʻo Nanopore 12 Gb |
Manaoga Auaunaga
Mo Genome Survey, Genome Assembly ma Hi-C Assembly:
| Tisu po'o su'esu'e nucleic acids | Su'esu'ega Genome | Genome Assembly ma PacBio | Hi-C Fonotaga |
| Manu Viscera | 0.5-1 g
| ≥ 3.5 g | ≥2 g |
| Musele Manu | ≥ 5 g | ||
| Toto Mamalia | 1.5 mL
| ≥ 5 mL | ≥2 mL |
| Toto moa/I'a | ≥ 0.5 mL | ||
| La'au- Laulau fou | 1-2 g | ≥ 5 g | ≥ 4 g |
| Selula Aganuu |
| ≥ 1x108 | ≥ 1x107 |
| Iniseti | 0.5-1 g | ≥ 3 g | ≥ 2 g |
| DNA aumai | Fa'atonuga: ≥1 ng/ µL Aofaiga ≥ 30 ng Fa'atapula'a pe leai se fa'aleagaina po'o le fa'aleagaina | Fa'atonuga: ≥ 50 ng/ µL Aofa'i: 10 µg/fesosolo sela/fa'ata'ita'iga OD260/280=1.7-2.2 OD260/230=1.8-2.5 Fa'atapula'a pe leai se fa'aleagaina po'o le fa'aleagaina |
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Mo faʻamatalaga Genome faʻatasi ai ma tusitusiga:
| Tisu po'o su'esu'e nucleic acids | Illumina Transcriptome | PacBio Transcriptome | Nanopore Transcriptome |
| La'au- A'a/Stem/Petal | 450 mg | 600 mg | |
| La’au – Lau/Fua | 300 mg | 300 mg | |
| La'au - Fua | 1.2 g | 1.2 g | |
| Loto Manu/Anu | 300 mg | 300 mg | |
| Manu Viscera/ Faiai | 240 mg | 240 mg | |
| Musele Manu | 450 mg | 450 mg | |
| Ivi/Laulu/Pa'u | 1 g | 1 g | |
| Arthropod - Iniseti | 6 | 6 | |
| Arthropod -Crustacea | 300 mg | 300 mg | |
| Le toto atoa | 1 paipa | 1 paipa | |
| RNA aumai | Fa'atonuga: ≥ 20 ng/ µL Aofa'i ≥ 0.3 µg OD260/280=1.7-2.5 OD260/230=0.5-2.5 RIN≥ 6 5≥28S/18S≥1 | Fa'atonuga: ≥ 100 ng/ µL Aofa'i ≥ 0.75 µg OD260/280=1.7-2.5 OD260/230=0.5-2.5 RIN≥ 8 5≥28S/18S≥1 | Fa'atonuga: ≥ 100 ng/ µL Aofa'i ≥ 0.75 µg OD260/280=1.7-2.5 OD260/230=0.5-2.5 RIN≥ 7.5 5≥28S/18S≥1 |
Fautuaina Fa'ata'ita'iga Tu'uina atu
Containe: 2 ml centrifuge tube (E le fautuaina le pepa apa)
(Mo le tele o faʻataʻitaʻiga, matou te fautuaina e aua le teuina i le ethanol.)
Fa'ailoga fa'ata'ita'iga: E tatau ona fa'aigoa manino fa'ata'ita'iga ma tutusa ma le fomu fa'amatalaga fa'ata'ita'i ua tu'uina atu.
La'uina: Fa'amamago-aisa: Fa'ata'ita'iga e mana'omia ona teu muamua i taga ma tanu i le aisa-mago.
Fa'asologa o galuega
Auala o Galuega Au'aunaga
Fa'ata'ita'iga mamanu
Fa'ata'ita'iga tu'uina atu
Su'ega DNA
Fausia faletusi
Fa'asologa
Iloiloga fa'amaumauga
Au'aunaga pe a uma ona fa'atau
Fa'ato'a su'esu'ega o le bioinformatic, fa'avasega i la'asaga e 4:
1) Genome suʻesuʻega, faʻavae ile k-mer suʻesuʻega ma NGS faitau:
Fuafuaga o le tele o le genome
Fuafuaga o le heterozygosity
Fuafuaga o itulagi fai soo
2) Genome Assembly ma PacBio HiFi:
De novopotopotoga
Iloiloga o le Fonotaga: e aofia ai suʻesuʻega a le BUSCO mo le faʻamaeʻaina o genome ma faʻafanua tua o le NGS ma le PacBio HiFi faitau.
3) Fonotaga maualuga:
Hi-C faletusi QC: fa'atatau o feso'ota'iga Hi-C aoga
Hi-C faʻapotopotoga: faʻapipiʻiina o contigs i vaega, sosoo ai ma le contig ordering i totonu o vaega taʻitasi ma tofia contig orientation
Iloiloga maualuga
4) Fa'amatalaga Genome:
Fa'ailoga RNA e le fa'amaonia
Fa'ailoga fa'asologa faifaipea (transposons ma toe fai fa'atasi)
Va'aiga o kenera
§De novo: ab initio algorithms
§ Faʻavae i luga o le homology
§ Fa'avae i luga o le transcriptome, ma faitau umi ma pupuu: faitau eo le foufa'apotopoto pe fa'afanua i le genome ata
§ Fa'amatalaga o kenera vavalo ma le tele o fa'amaumauga
1) Genome Survey- su'esu'ega k-mer
2) Genome Assembly
2) Genome Assembly - PacBio HiFi faitau fa'afanua e tusi fa'apotopotoga
2) Fonotaga Hi-C - faʻatusatusaga o paga fefaʻatauaʻiga aoga a Hi-C
3) Hi-C Iloiloga Fa'auma-fa'apotopotoga
4) Genome Annotation - tu'ufa'atasiga o kenera vavalo
4) Fa'amatalaga Genome - fa'amatalaga o kenera vavalo
Su'esu'e le aga'iga'i i luma na fa'afaigofieina e le BMKGene's de novo genome assembly services e ala i le aoina o fa'asalalauga fa'apitoa:
Li, C. et al. (2021) 'O fa'asologa o le genome e fa'aalia ai ala ta'ape i le lalolagi atoa ma fa'ailoa mai ai suiga fa'atupu fa'atupu fa'atasi i le evolusione o le solofanua', Natura Feso'ota'iga, 12(1). doi: 10.1038/S41467-021-21379-X.
Li, Y. et al. (2023) 'Suiga Chromosomal Lapopo'a Lapopo'a Ta'ita'i atu i Suiga Fa'amatalaga Genome-Level, Fetuuna'iga Si'osi'omaga, ma Fa'amatalaga Fa'apitoa i le Gayal (Bos frontalis)', Molecular Biology and Evolution, 40(1). doi: 10.1093/MOLBEV/MSAD006.
Tian, T. et al. (2023) 'Genome fa'apotopotoga ma le fa'a'ese'esega fa'atupu o se fa'ama'i mata'utia maize germplasm', Nature Genetics 2023 55:3, 55(3), itulau 496–506. doi: 10.1038/s41588-023-01297-y.
Zhang, F. et al. (2023) 'Fa'aalia le fa'aleleia o le tropane alkaloid biosynthesis e ala i le su'esu'eina o genomes e lua i le aiga o Solanaceae', Natura Feso'ota'iga 2023 14:1, 14(1), itulau 1–18. doi: 10.1038/s41467-023-37133-4.
Su'esu'ega lu'itau:
Telomere-i-telomere fono:Fu, A. et al. (2023) 'Telomere-to-telomere genome assembly of melon oona (Momordica charantia L. var. abbreviata Ser.) faʻaalia le atinaʻeina o fualaʻau, tuʻufaʻatasia ma uiga faʻatupuina matua', Horticulture Research, 10 (1). doi: 10.1093/HR/UHAC228.
Fa'apotopotoga Haplotype:Hu, W. et al. (2021) 'Allele-defined genome fa'aalia le eseesega biallelic i le taimi o le evolusione manioka', Molecular Plant, 14(6), itulau 851–854. doi: 10.1016/j.molp.2021.04.009.
Fonotaga genome tele:Yuan, J. et al. (2022) 'Genomic faavae o giga-chromosomes ma giga-genome o laau peony Paeonia ostii', Nature Communications 2022 13:1, 13(1), itulau 1–16. doi: 10.1038/s41467-022-35063-1.
Polyploid genome fa'apotopotoga:Zhang, Q. et al. (2022) 'Genomic insights to the recent chromosome reduction of autopolyploid sugarcane Saccharum spontaneum', Nature Genetics 2022 54:6, 54(6), itulau 885–896. doi: 10.1038/s41588-022-01084-1.
