PacBio 2+3 Cikakken Tsawon mRNA Magani
Siffofin
● Tsarin karatu:
Samfurin da aka jera tare da PacBio don gano isoforms na kwafi
Samfurori daban-daban (kwafi da sharuɗɗan da za a gwada) tare da suNGS don ƙididdige maganganun kwafi
● Bibiyar PacBio a cikin yanayin CCS, samar da karatun HiFi
● Taswirar cikakken tsawon rubutun
● Bincike ba ya buƙatar bayanin kwayoyin halitta; duk da haka, ana iya amfani da shi
● Binciken bioinformatic ya haɗa da ba kawai magana a jinsin halitta da matakin isoform ba amma har ma nazarin lncRNA, fusions gene, poly-adenylation, da tsarin kwayoyin halitta.
Amfani
● Babban Daidaito: HiFi yana karantawa tare da daidaito> 99.9% (Q30), kwatankwacin NGS
● Madadin Binciken Rarraba: jera duk rubuce-rubucen yana ba da damar gano isoform da sifa.
● Haɗin Ƙarfin PacBio da NGS: ba da damar ƙididdige magana a matakin isoform, buɗe canjin da za a iya rufewa yayin nazarin dukkan maganganun kwayoyin halitta.
● Ƙwararrun Ƙwararru: tare da rikodin waƙa na kammala fiye da 1100 PacBio cikakken tsawon ayyukan kwafi da sarrafa samfuran 2300, ƙungiyarmu tana kawo ƙwarewar ƙwarewa ga kowane aikin.
● Tallafin Bayan Talla: alkawarinmu ya wuce bayan kammala aikin tare da lokacin sabis na watanni 3 bayan-sayar. A wannan lokacin, muna ba da bibiyar aikin, taimako na warware matsala, da kuma zaman Q&A don magance duk wata tambaya da ta shafi sakamakon.
Samfuran Bukatun da Bayarwa
| Laburare | Dabarun jeri | An ba da shawarar bayanai | Kula da inganci |
| PolyA ingantaccen ɗakin karatu na mRNA CCS | PacBio Sequel II PacBio Revio | 20/40 GB 5/10 M CCS | Q30≥85% |
| Poly A ya wadata | Farashin PE150 | 6-10 GB | Q30≥85% |
Nucleotides
|
| Conc.(ng/μl) | Adadin (μg) | Tsafta | Mutunci |
| Illumina Library | ≥ 10 | 0.2 | 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≥4.0; Na dabbobi: RIN≥4.5; 5.0≥28S/18S≥1.0; iyakance ko babu hawan tushe |
| Laburaren PacBio | ≥ 100 | ≥ 1.0 | OD260/280=1.7-2.5 OD260/230=0.5-2.5 Iyakance ko babu furotin ko gurɓataccen DNA da aka nuna akan gel. | Shuka: RIN≥7.5 Dabbobi: RIN≥8.0 5.0≥28S/18S≥1.0; iyakance ko babu hawan tushe |
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.
Kawo:
1. Busasshen ƙanƙara:Samfurori suna buƙatar tattarawa 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.
Ya haɗa da bincike mai zuwa:
Raw data ingancin iko
Alternative Polyadenylation Analysis (APA)
Binciken kwafin Fusion
Madadin Ƙwararren Bincike
Binciken Benchmarking Universal Single-Copy Orthologs (BUSCO).
Binciken kwafin sabon labari: Hasashen jerin coding (CDS) da bayanin aiki
Binciken lncRNA: Hasashen lncRNA da hari
Shaidar MicroSatelite (SSR)
Binciken Rubuce-rubuce daban-daban (DETs).
Binciken Halittun Halitta Masu Bambance-bambance (DEGs).
Bayanin aiki na DEGs da DETs
Binciken BUSCO
Madadin Ƙwararren Bincike
Alternative Polyadenylation Analysis (APA)
Halittar Halittar Halittu Daban-daban (DEGs) da Rubuce-rubucen (DeTs9 anlaysis
Cibiyoyin hulɗar Protein-Protein na DETs da DEGs
Bincika ci gaban da BMKGene's PacBio 2+3 mai cikakken tsawon mRNA ya daidaita ta hanyar tarin wallafe-wallafe.
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