Matsakaicin tushen mRNA Sequencing-Illumina
Siffofin
● Ba tare da kowane nau'in kwayar halitta ba,
Za'a iya amfani da bayanan don nazarin tsari da kuma bayanin rubutun
● Gano madaidaitan wuraren yanka
Amfanin Sabis
● Bayar da sakamako na tushen BMKCloud: Ana ba da sakamakon azaman fayil ɗin bayanai da rahoton hulɗa ta hanyar dandamali na BMKCloud, wanda ke ba da damar karantawa mai amfani mai amfani na abubuwan bincike mai rikitarwa da ma'adinan bayanan da aka keɓance akan tushen daidaitaccen bincike na bioinformatics.
● Bayan-sayar da sabis: Bayan-sayar da sabis na aiki na tsawon watanni 3 bayan kammala aikin, gami da bin diddigin ayyukan, harbin matsala, Q&A sakamakon, da sauransu.
Samfuran Bukatun da Bayarwa
Samfuran Bukatun:
Nucleotides:
| Conc.(ng/μl) | Adadin (μg) | Tsafta | Mutunci |
| ≥ 20 | ≥ 0.5 | OD260/280=1.7-2.5 OD260/230=0.5-2.5 Iyakance ko babu furotin ko gurɓataccen DNA da aka nuna akan gel. | Don tsire-tsire: RIN≥6.5; Na dabbobi: RIN≥7.0; 5.0≥28S/18S≥1.0; iyakance ko babu hawan tushe |
Nama: Nauyi (bushe): ≥1 g
* Don nama ƙasa da 5 MG, muna ba da shawarar aika samfurin nama daskararre (a cikin ruwa nitrogen).
Dakatar da salula: Ƙididdiga ta salula = 3×107
*Muna ba da shawarar jigilar daskararrun cell lysate.Idan wannan tantanin halitta ya ƙidaya ƙasa da 5 × 105, walƙiya daskararre a cikin ruwa nitrogen ana shawarar.
Samfuran jini:
PA×geneBloodRNATube;
6mLTRIzol da 2ml jini (TRIzol:Blood=3:1)
Isar da Samfurin Nasiha
Kwantena:
2 ml bututu centrifuge (Ba a ba da shawarar foil tin ba)
Alamar samfur: Ƙungiya+ kwafi misali A1, A2, A3;B1, B2, B3......
Jirgin ruwa:
1.Dry-ice: Ana buƙatar samfurori a cikin jaka kuma a binne su a bushe-kankara.
2.RNAstable tubes: Ana iya bushe samfuran RNA a cikin bututun daidaitawar RNA (misali RNAstable®) kuma a tura su cikin zafin jiki.
Gudun Aikin Sabis
Gwajin ƙira
Samfurin bayarwa
RNA cirewa
Gina ɗakin karatu
Jeri
Binciken bayanai
Bayan-sayar da sabis
Bioinformatics
1.mRNA(denovo) Ka'idar Taro
Ta Triniti, an raba karatun zuwa ƙananan ƙananan, wanda aka sani da K-mer.Ana amfani da waɗannan K-mers azaman tsaba da za'a faɗaɗa su zuwa ƙugiya sannan kuma ana dogara da abubuwan da ke kan juzu'i.A ƙarshe, an yi amfani da De Bruijn a nan don gane kwafi a cikin abubuwan.
mRNA (De novo) Bayanin Triniti
2.mRNA (De novo) Rarraba Matsayin Maganar Halitta
RNA-Seq yana da ikon cimma ƙima mai mahimmanci na maganganun kwayoyin halitta.A al'ada, iya gano kewayon bayanan kwafi FPKM yana jere daga 10^-2 zuwa 10^6.
mRNA (De novo) Rarraba yawan FPKM a cikin kowane samfurin
3.mRNA (De novo) GO Analysis Enrichment Analysis of DEGs
GO (Gene Ontology) ma'ajin bayanai shine ingantaccen tsarin bayanin halitta wanda ke ƙunshe da daidaitattun ƙamus na ayyukan samfuran kwayoyin halitta.Ya ƙunshi matakan da yawa, inda ƙananan matakin yake, mafi ƙayyadaddun ayyuka.
mRNA (De novo) GO rarrabuwa na DEGs a matakin na biyu
BMK Case
Nazari na Rubuce-rubuce na Sucrose Metabolism a Lokacin Bunƙasa Bulb da Ci gaban Albasa (Allium cepa L.)
Buga: iyakoki a kimiyyar shuka,2016
Dabarun jeri
Illumina HiSeq2500
Tarin samfurin
Utah Yellow Sweet Spain cultivar "Y1351" an yi amfani da ita a cikin wannan binciken.Yawan samfuran da aka tattara shine
15th Day bayan kumburi (DAS) na kwan fitila (2-cm diamita da 3-4 g nauyi), 30th DAS (5-cm diamita da 100-110 g nauyi), da ~ 3 a kan 40th DAS (7-cm diamita da 260-300 g).
Sakamako mai mahimmanci
1. a cikin zane na Venn, an gano jimlar 146 DEGs a duk nau'i-nau'i uku na matakan ci gaba.
2. "Carbohydrate Transport and metabolism" an wakilta ta kawai 585 unigenes (watau 7% na COG annotated).
3.Unigenes da aka samu nasarar bayyanawa ga GO database an kasaftasu zuwa manyan sassa uku don matakai daban-daban guda uku na ci gaban kwan fitila.Yawancin waɗanda aka wakilta a cikin “tsarin nazarin halittu” babban nau'in sune "tsarin ƙwayar cuta", sannan "tsarin salula".A cikin babban nau'in "ayyukan kwayoyin halitta" nau'ikan biyu da aka fi wakilta sune "dauri" da "aiki mai kara kuzari".
 Histogram na gungu na ƙungiyoyin orthologous (COG). |  Histogram na Gene Ontology (GO) rarrabuwa don unigenes da aka samo daga kwararan fitila a cikin matakai uku na haɓakawa. |
 Zane na Venn yana nuna kwayoyin halitta daban-daban da aka bayyana a kowane matakai biyu na haɓaka kwan fitilar albasa |
Magana
Zhang C, Zhang H, Zhan Z, da dai sauransu.Fassarar Fassarar Tattalin Arziki na Sucrose Metabolism a Lokacin Bunbulb da Ci Gaba a Albasa (Allium cepa L.) [J].Iyaka a Kimiyyar Shuka, 2016, 7:1425-.DOI: 10.3389/fpls.2016.01425
