Non-Reference esekelwe mRNA Sequencing-Illumina
Iimbonakalo
● Ukuzimela kuyo nayiphi na igenome yesalathiso,
● Idatha inokusetyenziselwa ukuhlalutya ubume kunye nokubonakaliswa kwemibhalo ebhaliweyo
● Chonga iisayithi zokucheba eziguquguqukayo
Iinkonzo eziluncedo
● Ukunikezelwa kweziphumo ezisekelwe kwi-BMKCloud: Iziphumo zihanjiswa njengefayile yedatha kunye nengxelo edibeneyo ngeqonga le-BMKCloud, elivumela ukufundwa komsebenzisi kweziphumo zohlalutyo olunzima kunye nokumbiwa kwedatha ngokwezifiso kwisiseko sohlalutyo oluqhelekileyo lwe-bioinformatics.
● Iinkonzo zokuthengisa emva kokuthengisa: Iinkonzo emva kokuthengiswa zivumelekile kwiinyanga ezi-3 ekugqityweni kweprojekthi, kubandakanywa ukulandelwa kweeprojekthi, ukuxazulula iingxaki, iziphumo ze-Q & A, njl.
IiMfuno zeSampulu kunye nokuhanjiswa
IiMfuno zeSampulu:
IiNucleotides:
| Conc.(ng/μl) | Imali (μg) | Ubunyulu | Ingqibelelo |
| ≥ 20 | ≥ 0.5 | OD260/280=1.7-2.5 OD260/230=0.5-2.5 Unyino okanye akukho protein okanye ungcoliseko lweDNA olubonakaliswe kwijeli. | Kwizityalo: RIN≥6.5; Kwizilwanyana: RIN≥7.0; 5.0≥28S/18S≥1.0; umphakamo osisiseko okanye ongekhoyo |
Izicubu: Ubunzima (obomileyo): ≥1 g
*Kwithishu ezincinci kuno-5 mg, sicebisa ukuba uthumele ifleshi ekhenkcezisiweyo (kwinitrogen engamanzi) isampulu yethishu.
Ukumiswa kweeseli: Ubalo lweeseli = 3×107
*Sicebisa ukuthumela i-lysate yeseli enomkhenkce.Kwimeko apho iseli ibala encinci kune-5 × 105, umbane okhenkcezisiweyo kulwelo lwenitrogen uyacetyiswa.
Iisampulu zegazi:
PA×geneBloodRNATube;
I-6mLTRIzol kunye ne-2mL yegazi(TRIzol:Igazi=3:1)
Unikezelo lweSampuli eCetyisiweyo
Isikhongozeli:
I-2 ml yetyhubhu ye-centrifuge (i-tin foil ayikhuthazwa)
Isampuli yokuleyibhela: Iqela+liphindaphinda umz. A1, A2, A3;B1, B2, B3... ...
Ukuthunyelwa ngenqanawa:
1.Umkhenkce owomileyo: Iisampuli kufuneka zipakishwe kwiingxowa kwaye zingcwatywe kwindawo eyomileyo.
I-2.RNAstable tubes: Iisampulu ze-RNA zingomiswa kwi-tube yokuzinzisa i-RNA (umzekelo, i-RNAstable®) kwaye ithunyelwe kwiqondo lokushisa.
Ukuhamba komsebenzi weNkonzo
Uyilo lovavanyo
Ukuhanjiswa kwesampuli
Ukukhutshwa kwe-RNA
Ukwakhiwa kwethala leencwadi
Ukulandelelanisa
Uhlalutyo lwedatha
Iinkonzo emva kokuthengisa
Bioinformatics
1.mRNA(denovo) UMgaqo weNdibano
NgoBathathu Emnye, ufundo luyahlulahlulwa lube ziziqwenga ezincinci, ezaziwa ngokuba yi-K-mer.Ezi K-mers emva koko zisetyenziswa njengembewu ukuze zondiselwe kwi-contigs kwaye ke icandelo elisekelwe kwi-contig overlappings.Ekugqibeleni, uDe Bruijn wasetyenziswa apha ukuqonda imibhalo kumacandelo.
mRNA (De novo) Isishwankathelo sikaBathathu Emnye
I-2.mRNA (De novo) Ukuhanjiswa kweNqanaba lokuZalisa iGene
I-RNA-Seq iyakwazi ukufezekisa uqikelelo olunovakalelo kakhulu lwentetho yemfuza.Ngokuqhelekileyo, uluhlu olubonakalayo lokubhaliweyo okubhaliweyo kwe-FPKM ukusuka kwi-10 ^ -2 ukuya kwi-10 ^ 6
mRNA (De novo) Ukuhanjiswa kwe-FPKM yoxinaniso kwisampulu nganye
3.mRNA (De novo) GO Enrichment Analysis of DEGs
Idatabase yeGO (Gene Ontology) yinkqubo ecwangcisiweyo yebhayoloji yenkcazo equlethe isigama esisemgangathweni semisebenzi yemfuza kunye nemveliso yemfuza.Iqulethe amanqanaba amaninzi, apho inqanaba elisezantsi likhona, ngakumbi imisebenzi ethile.
mRNA (De novo) GO ukuhlelwa kweeDEG kwinqanaba lesibini
Ityala le-BMK
Uhlalutyo lwe-Transcriptome ye-Sucrose Metabolism ngexesha lokuDumba kweBhulbhu kunye noPhuhliso kwi-anyanisi (Allium cepa L.)
Ipapashiwe: imida kwinzululwazi yezityalo, 2016
Isicwangciso sokulandelelana
Illumina HiSeq2500
Ingqokelela yesampuli
I-Utah Yellow Sweet Spain cultivar "Y1351" isetyenziswe kolu phononongo.Inani leesampuli eziqokelelweyo
Usuku lwe-15 emva kokudumba (DAS) kwebhalbhu (i-2-cm ububanzi kunye ne-3-4 g ubunzima), i-30 ye-DAS (i-5-cm ububanzi kunye ne-100-110 g ubunzima), kunye ne-3 kwi-DAS ye-40 (i-7-cm ububanzi kunye 260-300 grams).
Iziphumo eziphambili
1. kumzobo weVenn, iyonke ye-146 DEGs ichongiwe kuzo zontathu izibini zezigaba zophuhliso.
I-2. "Ukuthuthwa kwe-Carbohydrate kunye ne-metabolism" yayimelwe kuphela yi-585 unigenes (oko kukuthi, i-7% ye-COG echazwe).
3. Ii-unigenes ezichazwe ngempumelelo kwiziko ledatha ye-GO zahlelwa zaba ziindidi ezintathu eziphambili kumanqanaba amathathu awohlukeneyo ophuhliso lwebhalbhu.Uninzi olumelwe "kwinkqubo yezinto eziphilayo" udidi oluphambili "yinkqubo ye-metabolic", ilandelwa "yinkqubo yeselula".Kudidi oluphambili “lomsebenzi wemolekyuli” awona macandelo mabini ayemelwe kakhulu “yayibophelelayo” kunye “nenkqubo yokukhuthaza”.
 I-Histogram yamaqela e-orthologous groups (COG) ukuhlelwa |  I-Histogram ye-ontology yemfuza (GO) yokuhlelwa kwee-unigenes ezivela kwiibhalbhu kwizigaba ezithathu zophuhliso |
 Idayagram yeVenn ebonisa imizila yemfuza echazwe ngokwahlukileyo kuwo nawaphi na amanqanaba amabini ophuhliso lwebhalbhu yetswele |
Isalathiso
Zhang C, Zhang H, Zhan Z, et al.Uhlalutyo lwe-Transcriptome ye-Sucrose Metabolism ngexesha lokuDumba kweBhulbhu kunye noPhuhliso kwi-anyanisi (Allium cepa L.)[J].Imida kwiSayensi yeZityalo, i-2016, 7: 1425-.I-DOI: 10.3389/fpls.2016.01425
