Imikhiqizo

I-Non-Reference based mRNA Sequencing-Illumina

Ukulandelana kwe-mRNA kusebenzisa inqubo yesizukulwane esilandelayo sokulandelanisa (NGS) ukuze ithwebule i-RNA(mRNA) yesithunywa ifomu i-Eukaryote ngesikhathi esithile imisebenzi ethile ekhethekile eyenza isebenze.Umbhalo omude kakhulu ohlukanisiwe wawubizwa ngokuthi 'i-Unigene' futhi wasetshenziswa njengochungechunge lwereferensi lokuhlaziya okwalandela, okuyindlela ephumelelayo yokufunda indlela yamangqamuzana nenethiwekhi yokulawula yohlobo ngaphandle kwereferensi.

Ngemuva kokuhlanganiswa kwedatha ye-transcriptome kanye nesichasiselo esisebenzayo se-unigene

(1) Ukuhlaziywa kwe-SSR, ukubikezela kwe-CDS kanye nesakhiwo sofuzo kuzokwenziwa kusengaphambili.

(2) Ukulinganisa kwenkulumo ye-unigene kusampula ngayinye kuzokwenziwa.

(3) Ama-unigene avezwe ngokuhlukile phakathi kwamasampula (noma amaqembu) azotholwa ngokusekelwe ekukhulumeni okungafani

(4) Ukuhlanganisa, isichasiselo esisebenzayo kanye nokuhlaziywa kokunothisa kwama-unigene avezwe ngokwehlukana kuzokwenziwa.

Xhumana nathi

Imininingwane Yesevisi

I-Bioinformatics

Imiphumela yedemo

Ucwaningo lwesigameko

Izici

● Ingazimele kunoma iyiphi i-genome yereferensi,

● Idatha ingase isetshenziselwe ukuhlaziya ukwakheka nokuvezwa kwemibhalo elotshiwe

● Khomba amasayithi okunamathisela aguquguqukayo

Izinzuzo Zesevisi

● Ukulethwa kwemiphumela okususelwe ku-BMKCloud: Imiphumela ilethwa njengefayela ledatha kanye nombiko osebenzisanayo ngenkundla ye-BMKCloud, evumela ukufundwa okusebenziseka kalula kwemiphumela yokuhlaziya eyinkimbinkimbi kanye nokumbiwa kwedatha okwenziwe ngendlela oyifisayo ngokusekelwe ekuhlaziyeni okuvamile kwe-bioinformatics.

● Amasevisi angemuva kokuthengisa: Amasevisi angemuva kokuthengisa asebenza izinyanga ezi-3 ngemva kokuqedwa kwephrojekthi, okuhlanganisa ukulandelela amaphrojekthi, ukuxazulula izinkinga, imiphumela ye-Q&A, njll.

Izidingo Zesampula Nokulethwa

Izimfuneko zesampula:

Ama-Nucleotides:

Conc.(ng/μl)

Inani (μg)

Ubumsulwa

Ubuqotho

≥ 20

≥ 0.5

OD260/280=1.7-2.5

OD260/230=0.5-2.5

Amaprotheni anomkhawulo noma awekho nhlobo noma ukungcola kwe-DNA okuboniswa kujeli.

Okwezitshalo: RIN≥6.5;

Okwezilwane: RIN≥7.0;

5.0≥28S/18S≥1.0;

ukuphakama okulinganiselwe noma okungenakho

Izicubu: Isisindo(somile): ≥1 g
*Ngezicubu ezincane kuno-5 mg, sincoma ukuthi uthumele isampula yezicubu eziqandisiwe (ku-nitrogen ewuketshezi).

Ukumiswa kweseli: Isibalo samaseli = 3×10⁷
*Sincoma ukuthi uthumele i-frozen cell lysate.Esimeni lapho lelo seli liba lincane kuno-5×10⁵, i-flash efriziwe ku-nitrogen ewuketshezi kuyanconywa.

Amasampula egazi:
PA×geneBloodRNATube;
I-6mLTRIzol kanye negazi elingu-2mL(TRIzol:Igazi=3:1)

Ukulethwa Kwesampula Okunconyiwe

Isitsha:
2 ml centrifuge tube (Tin foil ayinconywa)
Ukulebula okuyisampula: Ukuphindaphinda kweqembu+isb A1, A2, A3;B1, B2, B3....

Ukuthunyelwa:
1.Iqhwa elomile: Amasampula adinga ukupakishwa ezikhwameni futhi agqitshwe eqhweni elomile.
2.Amashubhu e-RNAstable: Amasampula e-RNA angomiswa ngeshubhu lokuzinzisa le-RNA(isb. i-RNAstable®) futhi athunyelwe ekamelweni lokushisa.

Ukugeleza Komsebenzi Wesevisi

Idizayini yokuhlola

Ukulethwa kwesampula

Ukukhishwa kwe-RNA

Ukwakhiwa komtapo wolwazi

Ukulandelanisa

Ukuhlaziywa kwedatha

Amasevisi angemuva kokuthengisa

Okwedlule: Ukulandelana okungafaki amakhodi okude-Illumina

Olandelayo: I-Eukaryotic mRNA Sequencing-Illumina

I-Bioinformatics

1.mRNA(denovo) Umgomo Womhlangano

NgoZiqu-zintathu, ukufunda kuhlukaniswa kube izingcezu ezincane, ezaziwa ngokuthi i-K-mer.Lawa ma-K-mers abe esesetshenziswa njengembewu ezonwetshwa ku-contigs bese kuba ingxenye esekelwe ekunqwabelaneni kwe-contig.Ekugcineni, u-De Bruijn wasetshenziswa lapha ukuze abone imibhalo ezingxenyeni.

mRNA-(De-novo)-Uhlolojikelele-kaZiqu-zintathu

mRNA (De novo) Uhlolojikelele lukaZiqu-zintathu

2.mRNA (De novo) Ukusatshalaliswa Kwezinga Lokubonakaliswa KweGene

I-RNA-Seq iyakwazi ukuzuza isilinganiso esibucayi kakhulu sokubonakaliswa kofuzo.Imvamisa, uhla olubonakalayo lwenkulumo elotshiweyo ye-FPKM isukela ku-10^-2 kuye ku-10^6

I-mRNA-(De-novo)-Ukusabalalisa-kwe-FPKM-ukuminyana-kusampula-nganye

mRNA (De novo) Ukusatshalaliswa kokuminyana kwe-FPKM kusampula ngayinye

3.mRNA (De novo) I-GO Enrichment Analysis yama-DEGs

Isizindalwazi se-GO (Gene Ontology) siwuhlelo oluhlelekile lwesichasiselo sebhayoloji oluqukethe ulwazimagama olujwayelekile lwemisebenzi yofuzo kanye nemikhiqizo yofuzo.Iqukethe amaleveli amaningi, lapho izinga liphansi, imisebenzi icaciswa kakhulu.

mRNA-(De-novo)-GO-classification-of-DEGs-ezingeni-lesibili

I-mRNA (De novo) GO ukuhlukaniswa kwama-DEGs ezingeni lesibili

Icala le-BMK

Ukuhlaziywa kwe-Transcriptome ye-Sucrose Metabolism ngesikhathi sokuvuvukala kwe-Bulb kanye nokuthuthukiswa ku-anyanisi (Allium cepa L.)

Kushicilelwe: imingcele kwisayensi yezitshalo,2016

Isu lokulandelanisa

Illumina HiSeq2500

Iqoqo lesampula

I-Utah Yellow Sweet Spain cultivar "Y1351" isetshenziswe kulolu cwaningo.Inani lamasampula aqoqiwe lali
Usuku lwe-15 ngemva kokuvuvukala (i-DAS) ye-bulb (ububanzi obungu-2-cm nesisindo esingu-3–4 g), i-DAS engu-30 (ububanzi obungu-5-cm nesisindo esingu-100–110 g), kanye no-3 ku-DAS yama-40 (ububanzi obungu-7-cm kanye 260-300 amagremu).

Imiphumela esemqoka

1. kumdwebo we-Venn, isamba sama-DEG angu-146 atholwe kuzo zonke izipheya ezintathu zezigaba zokuthuthuka.
I-2. "Ukuthuthwa kwe-carbohydrate kanye ne-metabolism" yayimelwe ama-unigenes angu-585 kuphela (okungukuthi, i-7% ye-COG echazwe).
3.Ama-Unigene achazwe ngempumelelo kusizindalwazi se-GO ahlukaniswa abe izigaba ezintathu eziyinhloko ezigabeni ezintathu ezihlukene zokuthuthukiswa kwamagilabhu.Iningi elimelelwe esigabeni esiyinhloko "senqubo yebhayoloji" kwakuyinqubo ye-metabolic ", elandelwa "inqubo yeselula".Esigabeni esiyinhloko "somsebenzi wamangqamuzana" izigaba ezimbili ezimelwe kakhulu "ukubopha" kanye "nomsebenzi we-catalytic".

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

I-Histogram yamaqoqo ama-orthologous groups (COG) ukuhlukaniswa

Ukuhlukaniswa kwe-histogram ye-gene ontology (GO) yama-unigene atholakala kuma-bulb ngezigaba ezintathu zokuthuthuka

Umdwebo we-Venn obonisa izakhi zofuzo ezivezwe ngokuhlukile kunoma yiziphi izigaba ezimbili zokuthuthukiswa kwebulb ka-anyanisi

Ireferensi

U-Zhang C, u-Zhang H, u-Zhan Z, nabanye.Ukuhlaziywa kwe-Transcriptome ye-Sucrose Metabolism ngesikhathi sokuvuvukala kwe-Bulb kanye nokuthuthukiswa ku-anyanisi (Allium cepa L.)[J].I-Frontiers ku-Plant Science, 2016, 7:1425-.I-DOI: 10.3389/fpls.2016.01425