Le fa'asinomaga fa'avae mRNA Sequencing-Illumina

O le fa'asologa o le mRNA e fa'aaogaina le isi augatupulaga fa'asologa o metotia (NGS) e pu'e ai le avefe'au RNA(mRNA) fomu Eukaryote i se vaitaimi patino o lo'o fa'agaoioia ai nisi o galuega fa'apitoa.O le tusitusiga sili ona umi na fa'apipi'iina sa ta'ua o le 'Unigene' ma fa'aaogaina e fai ma fa'asologa fa'asologa mo su'esu'ega mulimuli ane, o se auala aoga lea e su'esu'e ai le masini mole mole ma feso'ota'iga fa'atulafonoina o le ituaiga e aunoa ma se fa'amatalaga.

Ina ua mae'a fa'aputuga fa'amaumauga fa'amaumauga ma fa'amatalaga galue unigene

(1) Su'esu'ega a le SSR, va'aiga CDS ma le fa'atulagaina o kenera o le a fa'atulaga muamua.

(2) Fa'atatauina o fa'amatalaga unigene i fa'ata'ita'iga ta'itasi o le a faia.

(3) Unigenes fa'aalia eseese i le va o fa'ata'ita'iga (po'o vaega) o le a maua e fa'atatau i fa'amatalaga unigene.

(4) Fa'avasegaina, fa'amatalaga fa'atino ma le fa'atamaoaigaina o su'esu'ega o unigenes fa'aalia eseese o le a faia.

Faafesootai matou

Au'aunaga Fa'amatalaga

Bioinformatics

I'uga Fa'aaliga

Tulaga Suesuega

Vaega

● Tuto'atasi mai so'o se genome fa'asinomaga,

● E mafai ona fa'aogaina fa'amaumauga e iloilo ai le fausaga ma fa'amatalaga o tusitusiga

● Su'esu'e nofoaga e kilipa fesuia'i

Tulaga Auaunaga

● Tu'uina atu i'uga fa'avae i le BMKCloud: O fa'ai'uga e tu'uina atu e fai ma faila fa'amaumauga ma lipoti feso'ota'i e ala ile BMKCloud platform, lea e fa'ataga ai le faitau fa'auiga o fa'amatalaga lavelave ma su'esu'ega fa'atatau ile la'uina o fa'amaumauga ile fa'avae o su'esu'ega bioinformatics masani.

● Au'aunaga ina ua mae'a fa'atau: Au'aunaga ina ua mae'a fa'atauga e aoga mo le 3 masina pe a mae'a galuega, e aofia ai le tulitatao o poloketi, fa'alavelave fa'alavelave, fa'ai'uga Q&A, ma isi.

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

≥ 20

≥ 0.5

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≥6.5;

Mo manu: RIN≥7.0;

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 fa'agata: Faitauga sela = 3×10⁷
* 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.

Su'ega toto:
PA×geneBloodRNATube;
6mLTRIzol ma le 2mL toto(TRIzol:Blood=3:1)

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. Dry-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 faʻamago i le RNA stabilization tube(eg RNAstable®) ma lafo i le vevela potu.

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

Muamua: Fa'asologa umi e le fa'ailoga-Illumina

Sosoo ai: Eukaryotic mRNA Sequencing-Illumina

Bioinformatics

1.mRNA(denovo) Mataupu Faavae o le Fonotaga

E ala i le Tolutasi, o faitauga ua vaevaeina i ni vaega laiti, ua taʻua o K-mer.O nei K-mers e faʻaaogaina e fai ma fatu e faʻalautele i contigs ona faʻavaeina ai lea o vaega i luga ole contig overlappings.Mulimuli ane, na faʻaaogaina De Bruijn iinei e iloa ai tusitusiga i vaega.

mRNA-(De-novo)-Otootoga-o le Tolutasi

mRNA (De novo) Vaaiga Aoao o le Tolutasi

2.mRNA (De novo) Tufatufaina o le Tulaga Fa'aaliga Gene

RNA-Seq e mafai ona ausia se faʻatusatusaga sili ona maʻaleʻale o faʻamatalaga o kenera.E masani lava, o le va'aiga iloagofie o fa'amatalaga fa'amatalaga FPKM e amata mai i le 10^-2 i le 10^6.

mRNA-(De-novo)-Tufatufa-o-FPKM-tetele-i-faata'ita'i-ta'itasi

mRNA (De novo) Tufatufaina o FPKM density i faʻataʻitaʻiga taʻitasi

3.mRNA (De novo) GO Fa'atamaoaigaina Iloiloga o DEGs

GO (Gene Ontology) database ose fa'atonuga fa'amatalaga olaola o lo'o i ai se gagana masani o ga'o ma ga'o oloa.O loʻo i ai le tele o laʻasaga, pe a maualalo le maualuga, o le sili atu ona patino o galuega.

mRNA-(De-novo)-GO-faavasegaina-o-DEGs-i-lua-tulaga

mRNA (De novo) GO faʻavasegaina o DEG i le tulaga lua

BMK mataupu

Transcriptome Analysis o Sucrose Metabolism i le taimi o le Fufula ma le Atinaʻe o le Pupula i le aniani (Allium cepa L.)

Lolomiina: tuaoi i le faasaienisi laau，2016

Fuafuaga fa'asologa

Illumina HiSeq2500

Fa'ata'ita'iga aoina

O le Utah Yellow Sweet Spain cultivar "Y1351" na faʻaaogaina i lenei suʻesuʻega.Ole numera o fa'ata'ita'iga na aoina
15 aso talu ona fulafula (DAS) o matauila (2-cm le lautele ma le 3-4 g mamafa), 30th DAS (5-cm le lautele ma le 100-110 g mamafa), ma ~3 i le 40th DAS (7-cm le lautele ma 260–300 kalama).

I'uga autu

1. i le ata Venn, e 146 DEG le aofa'i na maua i paga uma e tolu o la'asaga tau atina'e.
2. "O felauaiga o le carbohydrate ma le metabolism" na faʻatusalia e naʻo le 585 unigenes (ie, 7% o le COG faʻamatalaina).
3. Unigenes fa'amaonia manuia i le GO database na fa'avasegaina i ni vaega autu se tolu mo vaega eseese e tolu o le atina'eina o matauila.O le tele o fa'atusaina i le "biological process" vaega autu o le "metabolic process", sosoo ai ma le "cellular process".I le vaega autu o le "molecular function" o vaega e lua o loʻo sili ona faʻatusalia o le "saisai" ma le "gaioiga catalytic".

PB-uma-umi-RNA-Sequencing-mataupu-suʻesuʻega

Fa'asologa o fuifui o vaega orthologous (COG) fa'avasegaga

Histogram of gene ontology (GO) fa'avasegaina mo unigenes e maua mai pulu i ni vaega se tolu o atina'e.

Fa'atusa Venn o lo'o fa'aalia ai kenera o lo'o fa'aalia i so'o se vaega e lua o le atina'eina o le pulu aniani

Fa'asinomaga

Zhang C, Zhang H , Zhan Z , ma isi.Transcriptome Analysis o Sucrose Metabolism i le taimi o le Fufula ma le Atinaʻe o le Pupula i le aniani (Allium cepa L.)[J].Frontiers in Plant Science, 2016, 7:1425-.DOI: 10.3389/fpls.2016.01425