Ubushinwa Ntabwo bushingiye kuri mRNA ikurikirana-Uruganda rwa Illumina n'abakora |BMK

Ibidashingiye kuri mRNA ikurikirana-Illumina

Non-Reference based mRNA sequencing-Illumina Featured Image

mRNA ikurikirana ikoresha uburyo bukurikiraho bwo gukurikiranya (NGS) kugirango ifate intumwa RNA (mRNA) ifata Eukaryote mugihe runaka ibikorwa bimwe na bimwe bikora.Inyandiko ndende ndende yatanzwe yiswe 'Unigene' kandi ikoreshwa nk'uruhererekane rw'ibisobanuro byakurikiyeho, ubwo ni uburyo bwiza bwo kwiga uburyo bwa molekile hamwe n'urusobe rw'ibinyabuzima bitavuzwe.

Nyuma yimyandikire yamakuru hamwe na unigene ikora

(1) Isesengura rya SNP, isesengura rya SSR, guhanura CDS n'imiterere ya gene bizashyirwa ahagaragara.

(2) Kwuzuza imvugo ya unigene muri buri sample bizakorwa.

(3) Byerekanwe muburyo butandukanye hagati yintangarugero (cyangwa amatsinda) bizavumburwa hashingiwe kumvugo imwe

Twandikire

Ibisobanuro bya serivisi

Bioinformatics

Ibisubizo bya Demo

Inyigo

Ibiranga

ØYigenga kuri genome iyo ari yo yose,

ØAmakuru ashobora gukoreshwa mu gusesengura imiterere n'imvugo y'inyandiko-mvugo

ØMenya imbuga zo gukuramo impinduka

Inyungu za serivisi

ØGutanga ibisubizo bishingiye kuri BMKCloud: Ibisubizo bitangwa nka dosiye yamakuru hamwe na raporo yimikorere binyuze kuri platform ya BMKCloud, ituma abayikoresha basoma inshuti zisesenguye ibisubizo byimbitse hamwe nubucukuzi bwamakuru yihariye hashingiwe kubisesengura bisanzwe bya bioinformatics.

ØSerivise nyuma yo kugurisha: Serivisi nyuma yo kugurisha ifite agaciro mumezi 3 iyo umushinga urangiye, harimo imishinga ikurikirana, kurasa ibibazo, ibisubizo Q&A, nibindi.

Icyitegererezo gisabwa no gutanga

Icyitegererezo gisabwa:

Nucleotide:

Isuku	Ubunyangamugayo	Kwanduza	Umubare
OD260 / 280≥1.7-2.5 ； OD260 / 230≥0.5-2.5 ；	Kubimera: RIN≥6.5;Ku nyamaswa: RIN≥7;28S / 18S≥1.0;bigarukira cyangwa bidafite ishingiro	Ntarengwa cyangwa ntayo poroteyine cyangwa ADN yanduye kuri gel.	Umwanzuro.≥30 ng / μl;Inkingi ≥ 10 μl;Igiteranyo ≥ 1.5 μg

Tissue: Uburemere (bwumye): ≥1 g
* Kuri tissue ntoya ya mg 5, turasaba kohereza flash ya flash ikonje (muri azote yuzuye).

Guhagarika ingirabuzimafatizo: Kubara Akagari = 3 × 10⁷
* Turasaba kohereza lysate ya selile ikonje.Mugihe iyo selile ibara ntoya 5 × 10⁵, flash yahagaritswe muri azote yuzuye.

Ingero zamaraso:
PA × geneBloRNRNube;
6mLTRIzol n'amaraso 2mL (TRIzol: Amaraso = 3: 1)

Basabwe Gutanga Icyitegererezo

Ibirimwo:
2 ml centrifuge tube (Tin foil ntabwo isabwa)
Icyitegererezo cyerekana: Itsinda + kwigana urugero A1, A2, A3;B1, B2, B3 ... ...

Kohereza:
1.Ibara ryumye: Ingero zigomba gupakirwa mumifuka hanyuma zigashyingurwa mu rubura.
2.Imiyoboro ihamye: Ingero za RNA zirashobora gukama mumashanyarazi ya RNA (urugero RNAstable®) hanyuma ikoherezwa mubushyuhe bwicyumba.

Akazi ka serivisi

Igishushanyo mbonera

Icyitegererezo

Gukuramo RNA

Kubaka isomero

Urukurikirane

Isesengura ryamakuru

Serivisi nyuma yo kugurisha

Mbere: Umwanya muremure utari code-Illumina

Ibikurikira: Eukaryotic mRNA ikurikirana-Illumina

Bioinformatics

1.mRNA (denovo) Ihame ryInteko

Kubutatu, gusoma biracitsemo ibice bito, bizwi nka K-mer.Izi K-mers noneho zikoreshwa nkimbuto kugirango zongerwe muri contigs hanyuma ibice bishingiye kuri contig overlappings.Hanyuma, De Bruijn yakoreshejwe hano kugirango amenye inyandiko-mvugo mu bice.

mRNA-(De-novo)-Overview-of-Trinity

mRNA (De novo) Incamake y'Ubutatu

2.mRNA (De novo) Ikwirakwizwa rya Gene Urwego Urwego

RNA-Seq ishoboye kugera kubigereranyo byimbitse byerekana imiterere ya gene.Mubisanzwe, urutonde rwerekana inyandiko-mvugo FPKM iri hagati ya 10 ^ -2 kugeza 10 ^ 6

mRNA-(De-novo)-Distribution-of-FPKM-density-in-each-sample

mRNA (De novo) Ikwirakwizwa rya FPKM muri buri sample

3.mRNA (De novo) GO Gutezimbere Isesengura rya DEGs

GO (Gene Ontology) base base ni sisitemu yo gutondekanya ibinyabuzima ikubiyemo amagambo asanzwe ya gene nibikorwa bya gene.Irimo urwego rwinshi, aho urwego ruri hasi, nuburyo bwihariye imikorere iba.

mRNA-(De-novo)-GO-classification-of-DEGs-at-second-level

mRNA (De novo) GO gutondekanya DEG kurwego rwa kabiri

Urubanza rwa BMK

Isesengura rya Transcriptome ya Sucrose Metabolism mugihe cyo kubyimba no gutera imbere mu gitunguru (Allium cepa L.)

Byatangajwe: imipaka mubumenyi bwibimera， 2016

Ingamba zikurikirana

Illumina HiSeq2500

Icyegeranyo cy'icyitegererezo

Utah Yellow Sweet Espagne ubwoko bwa “Y1351” bwakoreshejwe muri ubu bushakashatsi.Umubare w'icyitegererezo wakusanyijwe ni
Umunsi wa 15 nyuma yo kubyimba (DAS) yigitereko (diametero 2 cm na 3-4 g), DAS ya 30 (cm 5 cm na 100-110 g), na ∼3 kuri 40 DAS (diameter 7 cm na Garama 260–300).

Ibisubizo by'ingenzi

1. ku gishushanyo cya Venn, hagaragaye 146 DEGs zose uko ari eshatu ziterambere
2. "Carbohydrate transport na metabolism" yari ihagarariwe na 585 gusa (ni ukuvuga 7% ya COG yatangajwe).
3.Abasangwabutaka basobanuriwe neza kububiko bwa GO bashyizwe mubyiciro bitatu byingenzi kubice bitatu bitandukanye byiterambere.Benshi bahagarariwe mubyiciro "biologiya inzira" byari "inzira ya metabolike", hagakurikiraho "inzira ya selile".Mu cyiciro cyibanze cy "imikorere ya molekuline" ibyiciro bibiri byerekanwe cyane "guhuza" n "ibikorwa bya catalitiki".

PB-full-length-RNA-Sequencing-case-study

Histogramu yama matsinda matsinda mato (COG)

Histogramu ya gene ontologiya (GO) itondekanya kuri unigenes ikomoka kumatara mubyiciro bitatu byiterambere

Igishushanyo cya Venn cyerekana genes zitandukanye muburyo bubiri bwo gukura kw igitunguru

Reba

Zhang C, Zhang H, Zhan Z, n'abandi.Isesengura rya Transcriptome ya Sucrose Metabolism mugihe cyo kubyimba no gutera imbere mu gitunguru (Allium cepa L.) [J].Imipaka mubumenyi bwibimera, 2016, 7: 1425-.DOI: 10.3389 / fpls.2016.01425