Lihlahisoa

Non-Reference based mRNA Sequencing-Illumina

tatellano ea mRNA e sebelisa mokhoa oa tatellano oa moloko o latelang (NGS) ho hapa lenģosa la RNA(mRNA) sebopeho sa Eukaryote ka nako e itseng eo mesebetsi e meng e khethehileng e ntseng e sebetsa.Sengoliloeng se selelele ka ho fetesisa se ne se bitsoa 'Unigene' 'me se sebelisitsoe e le tatelano ea litšupiso bakeng sa tlhahlobo e latelang, e leng mokhoa o sebetsang oa ho ithuta mokhoa oa limolek'hule le marang-rang a taolo ea mefuta ntle le ho buuoa.

Ka mor'a kopano ea data ea transcriptome le tlhaloso e sebetsang ea unigene

(1) Tlhahlobo ea SSR, ho bolela esale pele ka CDS le sebopeho sa liphatsa tsa lefutso li tla etsoa esale pele.

(2) Quantification ea unigene expression ho sampole ka 'ngoe e tla etsoa.

(3) Li-unigenes tse hlahisitsoeng ka mokhoa o fapaneng lipakeng tsa lisampole (kapa lihlopha) li tla fumanoa ho ipapisitsoe le polelo e fapaneng.

(4) Ho tla etsoa tlhahlobo ea lihlopha, tlhaloso e sebetsang le tlhahlobo ea ntlafatso ea li-unigenes tse hlalositsoeng ka mokhoa o fapaneng.

Iteanye le rona

Lintlha tsa Tšebeletso

Bioinformatics

Demo Results

Thuto-pale

Likaroloana

● E sa ipapise le genome efe kapa efe,

● Lintlha li ka sebelisoa ho hlahloba sebopeho le tlhaloso ea lingoliloeng

● Hlalosa libaka tse fapa-fapaneng tsa ho khaola

Melemo ea Tšebeletso

● Phatlalatso ea sephetho e thehiloeng ho BMKCloud: Liphetho li fanoa e le faele ea data le tlaleho e sebetsanang le sethala sa BMKCloud, se lumellang ho bala ha bonolo ha liphetho tsa tlhahlobo e rarahaneng le ho rafa ha data ho ipapisitseng le tlhahlobo e tloaelehileng ea bioinformatics.

● Litšebeletso tsa ka mor'a thekiso: Litšebeletso tsa ka mor'a thekiso li sebetsa likhoeli tse 3 ha morero o phethoa, ho kenyelletsa le ho latela merero, ho rarolla mathata, liphetho tsa Q&A, joalo-joalo.

Litlhoko tsa Mohlala le Phano

Litlhoko tsa Mohlala:

Li-nucleotides:

Conc.(ng/μl)

Chelete (μg)

Bohloeki

Botšepehi

≥20

≥ 0.5

OD260/280=1.7-2.5

OD260/230=0.5-2.5

Tšoaetso e fokolang kapa ha e na protheine kapa DNA e bontšitsoeng ho gel.

Bakeng sa limela: RIN≥6.5;

Bakeng sa liphoofolo: RIN≥7.0;

5.0≥28S/18S≥1.0;

bophahamo bo fokolang kapa bo se nang motheo

Sesepa: Boima ba 'mele(o omileng): ≥1 g
*Bakeng sa linama tse nyane ho feta 5 mg, re khothaletsa ho romela sampole ea lithane tse hatselitsoeng (ka naetrojene e metsi).

Ho emisoa ha lisele: Palo ea lisele = 3×10⁷
* Re khothaletsa ho romella li-lysate tse leqhoa.Haeba sele eo e le nyane ho feta 5×10⁵, ho kgothaletswa hore ho be le flash e hoamisitsweng ka hara metsi a naetrojene.

Mehlala ea mali:
PA×geneBloodRNATube;
6mLTRIzol le 2mL mali(TRIzol:Blood=3:1)

Tlhahiso ea Mehlala e khothalelitsoeng

Setshelo:
2 ml ea centrifuge tube (ha e khothalletsoe ho etsa foil)
Mohlala oa ho ngola: Sehlopha+katiso mohlala A1, A2, A3;B1, B2, B3....

Thomello:
1.Dry-ice: lisampole li hloka ho kenngoa ka mekotleng ebe li patoa ka leqhoa le omileng.
2.RNAstable tubes: RNA sampuli e ka omisoa ka RNA stabilization tube (mohlala, RNAstable®) le ho romelloa ka mocheso oa kamore.

Phallo ea Mosebetsi oa Ts'ebeletso

Moqapi oa liteko

Mohlala oa thomello

Tlhahiso ea RNA

Kaho ea laebrari

Tatelano

Tlhahlobo ea lintlha

Litšebeletso tsa ka mor'a thekiso

E fetileng: Tatelano ya nako e telele e sa khoutong-Illumina

E 'ngoe: Eukaryotic mRNA Sequencing-Illumina

Bioinformatics

1.mRNA(denovo) Molao-motheo oa Kopano

Ka Boraro-bo-bong, tse baloang li arotsoe ka likotoana tse nyane, tse tsejoang e le K-mer.Li-K-mers tsena li sebelisoa e le lipeo tse lokelang ho atolosoa ho li-contigs ebe karolo e ipapisitseng le li-contig overlappings.Qetellong, De Bruijn o ile a sebelisoa mona ho lemoha sengoloa sa likarolo.

mRNA-(De-novo)-Kakaretso-ka-Boraro-bo-bong

mRNA (De novo) Kakaretso ea Boraro-bo-bong

2.mRNA (De novo) Kabo ea Boemo ba Tlhaloso ea Gene

RNA-Seq e khona ho fihlela khakanyo e hlokolosi haholo ea polelo ea lefutso.Ka tloaelo, mefuta e mengata e fumanehang ea litlhaloso tsa FPKM e tloha ho 10 ^ -2 ho isa ho 10 ^ 6

mRNA-(De-novo) -Kabo-ea-FPKM-tšubuhlellano-ka mohlala o mong le o mong

mRNA (De novo) Kabo ea sekhahla sa FPKM ho sampole ka 'ngoe

3.mRNA (De novo) GO Enrichment Analysis of DEGs

Database ea GO (Gene Ontology) ke mokhoa o hlophisitsoeng oa litlhaloso tsa baeloji o nang le mantsoe a tloaelehileng a mesebetsi ea liphatsa tsa lefutso le lihlahisoa tsa lefutso.E na le mekhahlelo e mengata, moo boemo bo tlase bo leng teng, mesebetsi e tobileng haholoanyane.

mRNA-(De-novo)-GO-classification-of-DEGs-boemo-bobeli

mRNA (De novo) GO classification of DEGs boemong ba bobeli

Taba ea BMK

Tlhahlobo ea Transcriptome ea Sucrose Metabolism nakong ea Ho ruruha ha Bulb le Nts'etsopele ea Onion (Allium cepa L.)

E hatisitsoe: meeli ea saense ea limela,2016

Leano la tatellano

Illumina HiSeq2500

Pokello ea mohlala

Ho sebelisitsoe mofuta oa Utah Yellow Sweet Spain "Y1351" thutong ena.Palo ea lisampole tse bokeletsoeng e ne e
Letsatsi la bo15 ka mor'a ho ruruha (DAS) ea tleloubu (2-cm bophara le 3-4 g boima), 30th DAS (5-cm bophara le 100-110 g boima), le ~3 ka 40th DAS (7-cm bophara le 260-300 dikgerama).

Liphetho tsa bohlokoa

1. setšoantšong sa Venn, kakaretso ea li-DEGs tse 146 li ile tsa bonoa ho lipara tsohle tse tharo tsa mehato ea nts'etsopele.
2. "Lipalangoang tsa lik'habohaedreite le metabolism" li ne li emeloa ke li-unigene tsa 585 feela (ke hore, 7% ea COG e hlalositsoeng).
3. Li-Unigenes tse hlalositsoeng ka katleho polokelong ea polokelo ea li-GO li arotsoe ka lihlopha tse tharo tse ka sehloohong bakeng sa mekhahlelo e meraro e fapaneng ea ntlafatso ea tleloubu.Bongata bo emetsoeng sehlopheng se ka sehloohong sa "biological process" e ne e le "metabolism", e lateloang ke "mokhoa oa cellular".Mokhahlelong o ka sehloohong oa "mosebetsi oa limolek'hule" mekhahlelo e 'meli e neng e emetsoe haholo e ne e le "tlama" le "ts'ebetso ea catalytic".

Histogram ea lihlopha tsa lihlopha tsa orthologous (COG) lihlopha

Sehlopha sa histogram of gene ontology (GO) bakeng sa li-unigenes tse nkiloeng ho bulbs ka mekhahlelo e meraro ea nts'etsopele.

Sets'oants'o sa Venn se bonts'ang liphatsa tsa lefutso ka mokhoa o fapaneng methating efe kapa efe e 'meli ea nts'etsopele ea tleloubu ea onion

Referense

Zhang C, Zhang H , Zhan Z , et al.Tlhahlobo ea Transcriptome ea Sucrose Metabolism nakong ea Ho ruruha ha Bulb le Nts'etsopele ea Onion (Allium cepa L.)[J].Frontiers in Plant Science, 2016, 7:1425-.DOI: 10.3389/fpls.2016.01425