Nā huahana

Non-Reference based mRNA Sequencing-Illumina

Hoʻohana ʻo mRNA sequencing i ke ʻano hana sequencing hanauna hou (NGS) e hopu i ka messenger RNA(mRNA) form Eukaryote i ka manawa kikoʻī e hoʻāla ana kekahi mau hana kūikawā.ʻO ka transcript lōʻihi loa i hoʻopili ʻia ua kapa ʻia ʻo 'Unigene' a hoʻohana ʻia e like me ke kaʻina kuhikuhi no ka nānā ʻana ma hope, ʻo ia ke ala kūpono e aʻo ai i ka mīkini molekala a me ka ʻoihana hoʻoponopono o ka ʻano me ka ʻole o ka kuhikuhi.

Ma hope o ka hōʻuluʻulu ʻana o ka ʻikepili transcriptome a me ka annotation hana unigene

(1) E hoʻokumu mua ʻia ka nānā ʻana o ka SSR, ka wānana CDS a me ke ʻano gene.

(2) E hana ʻia ka helu o ka ʻōlelo unigene i kēlā me kēia laʻana.

(3) E ʻike ʻia nā unigenes i hōʻike ʻia ma waena o nā laʻana (a i ʻole nā hui) ma muli o ka ʻōlelo unigene.

(4) E hoʻokō ʻia ka hui ʻana, ka wehewehe hana a me ka hoʻonui ʻana i nā unigenes i hōʻike ʻia.

Kāhea iā mā˚ou

Nā kikoʻī lawelawe

Bioinformatics

Nā hopena hōʻike

Nānā Hiʻohiʻona

Nā hiʻohiʻona

● Kūʻokoʻa i kekahi genome kuhikuhi,

● Hiki ke hoʻohana ʻia ka ʻikepili no ke kālailai ʻana i ke ʻano a me ka hōʻike ʻana o nā transcripts

● E ʻike i nā wahi ʻokiʻoki ʻano like ʻole

Pono lawelawe

● Hāʻawi i nā hualoaʻa ma muli o BMKCloud: Hāʻawi ʻia nā hopena ma ke ʻano he waihona ʻikepili a me ka hōʻike pili ma o ka paepae BMKCloud, e hiki ai i ka mea hoʻohana ke heluhelu pono i nā hopena hoʻopukapuka paʻakikī a me ka hoʻoili ʻana i ka ʻikepili pilikino ma ke kumu o ka loiloi bioinformatics maʻamau.

● Nā lawelawe ma hope o ke kūʻai aku: Manaʻo nā lawelawe ma hope o ke kūʻai aku no 3 mau mahina ma hope o ka pau ʻana o ka papahana, me ka hahai ʻana i nā papahana, ka hoʻopiʻi pilikia, nā hopena Q&A, etc.

Nā Koina Laʻana a me ka Hoʻouna ʻana

Nā Koina Laʻana:

Nucleotides:

Conc.(ng/μl)

Ka nui (μg)

Maemae

Pono pono

≥ 20

≥ 0.5

OD260/280=1.7-2.5

OD260/230=0.5-2.5

Hoʻopaʻa ʻia a ʻaʻole hoʻi i ka protein a i ʻole DNA contamination i hōʻike ʻia ma ka gel.

No nā mea kanu: RIN≥6.5;

No nā holoholona: RIN≥7.0;

5.0≥28S/18S≥1.0;

palena a ʻaʻole kiʻekiʻe kumu

Tissue: Kaumaha (maloʻo): ≥1 g
*No ka ʻiʻo liʻiliʻi ma mua o 5 mg, manaʻo mākou e hoʻouna i kahi hāpana kikoo uila (i loko o ka nitrogen wai).

Hoʻokuʻu ʻia ke kelepona: helu kelepona = 3×10⁷
* Manaʻo mākou e hoʻouna i ka lysate cell frozen.Inā ʻoi aku ka liʻiliʻi o kēlā cell ma mua o 5 × 10⁵, ʻōlelo ʻia ka flash frozen i loko o ka wai nitrogen.

Nā laʻana koko:
PA×geneBloodRNATube;
6mLTRIzol a me 2mL koko(TRIzol:Blood=3:1)

Manaʻo ʻia ka hāʻawi laʻana

ipu:
2 ml centrifuge paipu (ʻaʻole ʻōlelo ʻia ʻo Tin foil)
Laʻana lepili: Pūʻulu+hoʻopiʻi e laʻa me A1, A2, A3;B1, B2, B3... ...

Hoʻouna:
1.Dry-ice: Pono e hoʻopiliʻia nā laʻana i loko o nāʻeke a kanuʻia i loko o ka hau maloʻo.
2.RNAstable tubes: Hiki ke hoʻomaloʻo ʻia nā laʻana RNA ma ka RNA stabilization tube (eg RNAstable®) a hoʻouna ʻia i loko o ka lumi wela.

Holo Hana Hana

Hoʻolālā hoʻokolohua

Hāʻawi laʻana

RNA unuhi

Kūkulu hale waihona puke

Ke kaʻina ʻana

ʻIkepili ʻikepili

Nā lawelawe ma hope o ke kūʻai aku

Mua: Ka lōʻihi non-coding sequencing-Illumina

Aʻe: Eukaryotic mRNA Sequencing-Illumina

Bioinformatics

1.mRNA(denovo) Kumu o ka Hui

Na Trinity, ua ʻāpana ʻia nā heluhelu i nā ʻāpana liʻiliʻi, i kapa ʻia ʻo K-mer.Hoʻohana ʻia kēia mau K-mers ma ke ʻano he mau hua e hoʻonui ʻia i nā contigs a laila e hoʻokumu ʻia ka ʻāpana ma luna o nā overlappings contig.ʻO ka hope, ua noi ʻia ʻo De Bruijn ma aneʻi e ʻike i nā transcripts i nā ʻāpana.

mRNA-(De-novo)-Nānā-o-Trinity

mRNA (De novo) Nānā o ka Trinity

2.mRNA (De novo) Ka mahele ana o Gene Expression Level

Hiki iā RNA-Seq ke hoʻokō i kahi manaʻo koʻikoʻi o ka hōʻike gene.ʻO ka maʻamau, ʻike ʻia ka laulā o nā transcripts expression FPKM mai 10^-2 a i 10^6

mRNA-(De-novo)-Ka māhele-o-FPKM-density-i loko o kēlā me kēia laʻana.

mRNA (De novo) Ka mahele ʻana o ka FPKM density i kēlā me kēia hāpana

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

ʻO ka ʻikepili GO (Gene Ontology) kahi ʻōnaehana hōʻike biological i kūkulu ʻia i loko o nā huaʻōlelo maʻamau o nā hana gene a me nā huahana gene.Loaʻa i nā pae he nui, kahi i lalo ka pae, ʻoi aku ka kikoʻī o nā hana.

mRNA-(De-novo)-GO-hoʻokaʻawale-o-DEGs-ma ka pae ʻelua

mRNA (De novo) GO ka hoʻohālikelike ʻana o nā DEG ma ka pae ʻelua

Hihia BMK

Transcriptome Analysis of Sucrose Metabolism i ka wā o ka ʻōpuʻu ʻana a me ka hoʻomohala ʻana i ka Onion (Allium cepa L.)

Paʻi ʻia: palena i ka ʻepekema mea kanu，2016

Hoʻolālā kaʻina

Illumina HiSeq2500

ʻOhi laʻana

Ua hoʻohana ʻia ka cultivar Utah Yellow Sweet Spain "Y1351" i kēia haʻawina.ʻO ka nui o nā laʻana i hōʻiliʻili ʻia
ʻO ka lā 15 ma hope o ka pehu ʻana (DAS) o ka puʻupuʻu (2-cm anawaena a me 3-4 g kaumaha), 30th DAS (5-cm anawaena a me 100-110 g paona), a me ~3 ma ka 40th DAS (7-cm anawaena a 260-300 grams).

Nā hopena koʻikoʻi

1. ma ke kiʻikuhi Venn, ua ʻike ʻia he 146 DEG ma nā pae hoʻomohala ʻekolu.
2. Ua hōʻike ʻia ka "Carbohydrate transport and metabolism" e 585 unigenes wale nō (ʻo ia hoʻi, 7% o ka COG i hōʻike ʻia).
3. Ua hoʻokaʻawale maikaʻi ʻia ʻo Unigenes i ka ʻikepili GO i ʻekolu mau ʻāpana nui no nā pae like ʻole ʻekolu o ka hoʻomohala ʻana i ka puʻupuʻu.ʻO ka hapa nui i hōʻike ʻia ma ka "kaʻina hana biological" ʻo ia ka "kaʻina metabolic", a ukali ʻia e ka "kaʻina kelepona".Ma ka māhele nui o ka "molecular function" ʻelua mau ʻāpana i hōʻike ʻia he "binding" a me "catalytic activity".

Histogram of clusters of orthologous groups (COG) classification

Histogram of gene ontology (GO) classification for unigenes i loaʻa mai nā puʻupuʻu i ʻekolu pae hoʻomohala.

ʻO ke kiʻikuhi Venn e hōʻike ana i nā genes i hōʻike ʻokoʻa ʻia ma nā ʻanuʻu ʻelua o ka hoʻomohala ʻana i ka ʻumeke aniani

Kuhikuhi

Zhang C, Zhang H , Zhan Z , et al.Transcriptome Analysis of Sucrose Metabolism i ka wā o ka ʻōpuʻu ʻana a me ka hoʻomohala ʻana i loko o ka Onion (Allium cepa L.)[J].Frontiers ma Plant Science, 2016, 7:1425-.DOI: 10.3389/fpls.2016.01425