MRNA fa'asologa atoa -PacBio
Tulaga Auaunaga
● Faitau sa'o le mole mole cDNA atoa mai le 3'- pito i le 5'- pito
● Iso-form tulaga maualuga iugafono i le faatulagaga o le faasologa
● Faʻamaumauga e maualuga le saʻo ma le faʻamaoni
● E fetaui lelei ma ituaiga vaiours
● Tele fa'asologa fa'asologa ma 4 PacBio Sequel II fa'asologa fa'asologa fa'apipi'iina
● Maua le poto masani ile sili atu ile 700 Pacbio-based RNA sequencing projects
● BMKCloud fa'avae fa'ai'uga tu'uina atu: Fa'asinomaga fa'amaumauga e maua i luga ole tulaga.
● Au'aunaga ina ua mae'a fa'atauga aoga mo le 3 masina pe a mae'a galuega
Fa'amatalaga Auaunaga
Fa'avae: PacBio Sequel II
Faletusi fa'asologa: Poly A- fa'atamaoaigaina mRNA faletusi
Tuuina atu o faʻamatalaga fautuaina: 20 Gb / faʻataʻitaʻiga (Faʻalagolago i ituaiga)
FLNC（%）： ≥75%
*FLNC: Fa'aliliuga e le o chimeric umi atoa
Bioinformatics su'esu'ega
● Fa'asologa o fa'amatalaga mata'utia
● Faʻamatalaga faʻamatalaga
● Fa'asologa fa'asologa
● Fa'amatalaga Fa'atatau
● Fa'amatalaga Galuega
Fa'ata'ita'iga Mana'oga ma le Tu'uina atu
Fa'ata'ita'iga Mana'oga:
Nucleotides:
| Conc.(ng/μl) | Aofa'i (μg) | Mama | Amiotonu |
| ≥ 120 | ≥ 0.6 | OD260/280=1.7-2.5 OD260/230=0.5-2.5 Fa'atapula'a pe leai se polotini po'o le fa'aleagaina o DNA o lo'o fa'aalia ile gel. | Mo laau toto: RIN≥7.5; Mo manu: RIN≥8.0; 5.0≥ 28S/18S≥1.0; fa'atapula'a pe leai se maualuga maualuga |
Tisu: mamafa(mamago):≥1 g
*Mo mea e la'ititi ifo i le 5 mg, matou te fautua atu e lafo atu le moli fa'aaisa (i le nitrogen vai) fa'ata'ita'iga.
Taofi le sela:Faitauga sela = 3×106- 1×107
* Matou te fautua atu e lafo le lysate cell frozen.I le tulaga o lena sela e laʻititi ifo nai lo le 5x105, e fautuaina le moli fa'aaisa i le nitrogen vai, lea e sili ona lelei mo le fa'apipi'iina.
Su'ega toto:Volume≥1 mL
Microorganism:Massa ≥ 1 g
Fautuaina Fa'ata'ita'iga Tu'uina atu
pusa:
2 ml centrifuge paipa (E le fautuaina le pepa apa)
Fa'ailoga fa'ata'ita'iga: Vaega+fa'atusa eg A1, A2, A3;B1, B2, B3... ...
utaina:
1. Fa'amamago-aisa: Fa'ata'ita'iga e mana'omia ona fa'apipi'i i taga ma tanu i le aisa-mago.
2. RNAstable tubes: RNA samples e mafai ona fa'amago ile RNA stabilization tube(eg RNAstable®) ma lafo ile potu vevela.
Auala o Galuega Au'aunaga
Fa'ata'ita'iga mamanu
Fa'ata'ita'iga tu'uina atu
RNA su'ega
Fauga faletusi
Fa'asologa
Iloiloga fa'amaumauga
Au'aunaga pe a uma ona fa'atau
1.FLNC tufatufaina umi
O le umi o le faitau atoa e le o chimeric (FLNC) e faʻaalia ai le umi o le cDNA ile fausiaina o faletusi.Ole tufatufaina ole umi ole FLNC ose fa'ailoga taua ile iloiloga ole lelei ole fausiaina ole faletusi.
FLNC faitau tufatufa umi
2.Fa'ato'a fa'asoa fa'aitulagi o le ORF
Matou te fa'aogaina le TransDecoder e va'ai ai le polotini coding eria ma fa'asologa o amino acid e fa'atupu ai seti unigene, lea o lo'o i ai fa'amatalaga transcript atoatoa e le toe fa'aluaina i fa'ata'ita'iga uma.
Fa'ato'a fa'asoa fa'aitulagi o le ORF
3.KEGG ala fa'atamaoaigaina auiliiliga
E mafai ona iloa fa'amatalaga fa'aalia eseese (DETs) e ala i le fa'aogaina o fa'amaumauga fa'asologa o le RNA fa'avae NGS i seti fa'asologa atoa o lo'o gaosia e fa'amaumauga fa'asologa o PacBio.O nei DET e mafai ona fa'agasolo atili mo su'esu'ega fa'atino eseese, fa'ata'ita'iga, su'esu'ega fa'atamaoaigaina ala KEGG.
Fa'atamaoaigaina ala DET KEGG -Foto togi
BMK mataupu
Le fa'atupuina o le malosi o le Populus stem transcriptome
Lolomiina: La'au Biotechnology Journal, 2019
Fuafuaga fa'asologa:
Fa'ata'ita'iga aoina:vaega pito i luga: pito, pito muamua (IN1), lona lua (IN2), lona tolu (IN3), pito (IN4) ma le internode (IN5) mai le Nanlin895.
NGS-faasologa:RNA o tagata e 15 na fa'aputuina e fai ma fa'ata'ita'iga fa'aola.E tolu fa'ata'ita'iga fa'aola o mata ta'itasi na fa'agasolo mo le fa'asologa o le NGS
TGS-faasologa:Na vaevaeina vaega o a'a i vaega e tolu, e pei o le tumutumu, IN1-IN3 ma le IN4-IN5.O itulagi ta'itasi sa fa'agasolo mo le fa'asologa o PacBio fa'atasi ma ituaiga faletusi e fa: 0-1 kb, 1-2 kb, 2-3 kb ma le 3-10 kb.
I'uga autu
1. E 87150 le aofa'i o fa'amaumauga umi-umi na fa'ailoaina, lea, e 2081 tala fa'aisoforms ma 62058 tala fou fa'asoso'o so'o na fa'ailoa mai.
2.1187 lncRNA ma 356 fusion genes na iloa.
3. Mai le tuputupu aʻe muamua i le tuputupu aʻe lona lua, 15838 faʻamatalaga faʻaalia faʻapitoa mai le 995 faʻaaliga faʻapitoa na faʻaalia.I DEGs uma, 1216 o faʻamatalaga faʻamaumauga, o le tele o ia mea e leʻi lipotia.
4.GO su'esu'ega fa'atamaoaigaina na fa'aalia ai le taua o le vaeluaga o le cell ma le fa'ama'iina-fa'aitiitia fa'agasologa i le tuputupu a'e muamua ma lona lua.
O isi mea fa'apipi'i fa'atasi ma fa'aisoform ese'ese
WGCNA su'esu'ega i luga o fa'amaumauga
Fa'asinomaga
Chao Q, Gao ZF, Zhang D, ma isi.Le fa'atupuina o le malosi o le Populus stem transcriptome.La'au Biotechnol J. 2019;17(1):206-219.doi:10.1111/pbi.12958
