Tsarin mRNA mai cikakken tsayi -PacBio

Da novojeri mai cikakken tsayin rubutu, wanda kuma aka sani daDa novoIso-Seq yana ɗaukar fa'idodin PacBio sequencer a cikin tsayin karantawa, wanda ke ba da damar jeri na cikakken tsawon kwayoyin cDNA ba tare da wani hutu ba.Wannan gaba ɗaya yana guje wa duk wani kurakurai da aka haifar a cikin matakan tattara bayanan rubutu kuma yana gina saiti na unigene tare da ƙudurin matakin isoform.Wannan saitin unigene yana ba da bayanan kwayoyin halitta masu ƙarfi a matsayin "kwayoyin halitta" a matakin rubutu.Bugu da kari, haɗe tare da bayanan jerin tsararru na gaba, wannan sabis ɗin yana ba da ƙarfin ƙididdige ƙimar matakin isoform.

Platform: PacBio Sequel II

Library: SMRT bell library

Tuntube Mu

Cikakkun Sabis

Sakamakon Demo

Nazarin Harka

Amfanin Sabis

● Karatu kai tsaye daga cikakken tsawon kwayoyin cDNA daga 3'- ƙarshen zuwa 5'- ƙarshen

● Ƙimar matakin iso-form a cikin tsarin tsari

● Rubuce-rubuce tare da babban daidaito da mutunci

● Mai dacewa sosai da nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in kifi da nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in kifi) da nau'in nau'in halitta

● Ƙaƙwalwar ƙididdiga mai girma tare da 4 PacBio Sequel II tsarin dandamali sanye take

● Kware sosai tare da ayyuka sama da 700 na tushen RNA na Pacbio

● Isar da sakamako na tushen BMKCloud: Ma'adinin bayanai na musamman da ake samu akan dandamali.

● Sabis na tallace-tallace yana aiki na tsawon watanni 3 bayan kammala aikin

Ƙayyadaddun Sabis

Platform: PacBio Sequel II

Laburaren jeri: Laburaren mRNA mai wadatar Poly A

Abubuwan da aka ba da shawarar: 20 Gb/samfuri (Ya danganta da nau'in)

FLNC (%): ≥75%

*FLNC: Cikakkun bayanan da ba na chimeric ba

Binciken bioinformatics

● Aikin sarrafa danyen bayanai

● Ƙirar rubutu

● Tsarin tsari

● Ƙimar Magana

● Bayanin Aiki

Samfuran Bukatun da Bayarwa

Samfuran Bukatun:

Nucleotides:

Conc.(ng/μl)

Adadin (μg)

Tsafta

Mutunci

≥ 120

≥ 0.6

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≥7.5;

Na dabbobi: RIN≥8.0;

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:Ƙididdigar salula = 3×10⁶- 1 × 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 ba da shawarar, wanda ya fi dacewa don hakar micro.

Samfuran jini:Girman ≥1 ml

Microorganism:Masa ≥ 1 g

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. Busasshen ƙanƙara: 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

Na baya: Eukaryotic mRNA Sequencing-Illumina

Na gaba: Cikakkun Tsawon mRNA Sequencing-Nanopore

1.FLNC tsawon rarraba

Tsawon cikakken tsayin karatun da ba na chimeric(FLNC) yana nuna tsawon cDNA a ginin ɗakin karatu.Rarraba tsawon FLNC alama ce mai mahimmanci a kimanta ingancin ginin ɗakin karatu.

mRNA-FLNC-karanta-tsawon-rarrabuwa

FLNC karanta tsawon rarraba

2.Cikakken rarraba tsawon yanki na ORF

Muna amfani da TransDecoder don tsinkaya yankuna masu coding furotin da madaidaitan jerin amino acid don samar da saitin unigene, wanda ya ƙunshi cikakkun bayanan kwafi marasa ƙari a duk samfuran.

mRNA-Cikakken-ORF-tsawon-rarrabuwa

Cikakken rarraba tsawon yanki na ORF

3.KEGG hanyoyin haɓaka haɓakar hanyar

Ana iya gano mabambantan kwafi (DETs) ta hanyar daidaita bayanai na tushen RNA na tushen NGS akan saitin rubutun cikakken tsayi wanda bayanan jeri na PacBio ya samar.Waɗannan DETs na iya ƙara sarrafa su don bincike na ayyuka daban-daban, misali nazarin haɓaka hanyoyin KEGG.

mRNA-DEG-KEGG-hanyar-haɓaka

DET KEGG haɓakar hanyar -Dot mãkirci

BMK Case

Haɓaka haɓakar haɓakawa na Populus stem transcriptome

Buga: Jaridar Biotechnology Journal, 2019

Dabarun tsarawa:
Tarin samfurin:yankuna masu tushe: koli, na farko internode (IN1), na biyu internode (IN2), internode na uku (IN3), internode (IN4) da kuma internode (IN5) daga Nanlin895
NGS-jeri:An tattara RNA na mutane 15 azaman samfurin halitta ɗaya.Kwafin halittu guda uku na kowane maki an sarrafa su don jerin NGS
TGS-jeri:An raba yankuna masu tushe zuwa yankuna uku, watau koli, IN1-IN3 da IN4-IN5.An sarrafa kowane yanki don tsarin PacBio tare da nau'ikan ɗakunan karatu guda huɗu: 0-1 kb, 1-2 kb, 2-3 kb da 3-10 kb.

Sakamako mai mahimmanci

1. An gano jimlar 87150 cikakkun kwafi, a cikin su, 2081 novel isoforms da 62058 novel madadin spliced isoforms aka gano.
2.1187 lncRNA da 356 fusion genes an gano su.
3.Daga girma na farko zuwa girma na biyu, 15838 da aka kwatanta daban-daban na rubuce-rubuce daga 995 da aka bayyana daban-daban an gano su.A cikin dukkan DEGs, 1216 sune abubuwan rubutawa, yawancin waɗanda har yanzu ba a ba da rahotonsu ba.
Binciken haɓaka haɓakar 4.GO ya bayyana mahimmancin rabon tantanin halitta da tsarin rage oxidation a cikin girma na farko da na biyu.