TRANSCRIPTOMICS
xwm
Kev sib txuas lus
Tag nrho-ntev transcript characterization ntawm SF3B1 kev hloov pauv hauv cov kab mob lymphocytic leukemia ntev qhia txog kev txo qis ntawm cov khoom khaws cia
Cov ntawv teev lus puv npo |Nanopor sequencing |Alternative isoform tsom xam
Keeb kwm
Somatic mutations nyob rau hauv splicing yam SF3B1 tau dav tshaj tawm rau koom nrog ntau yam qog nqaij hlav, nrog rau cov kab mob lymphocytic leukemia (CLL), uveal melanoma, mob qog noj ntshav mis, thiab lwm yam. Tsis tas li ntawd, cov kev tshawb fawb luv luv nyeem tau qhia tawm cov qauv sib txawv ntawm qhov tshwm sim los ntawm SF3B1 kev hloov pauv.Txawm li cas los xij, cov kev tshawb fawb ntawm cov qauv sib txawv no tau ntev tau txwv rau qhov xwm txheej-theem thiab tsis muaj kev paub txog isoform-theem vim muaj kev txwv ntawm cov ntawv nyeem luv luv.Ntawm no, nanopore sequencing platform tau qhia los tsim cov ntawv teev lus ntev, uas txhawb kev tshawb nrhiav ntawm AS isoforms.
Kev sim tsim
Kev sim
Kev pab pawg:1. CLL-SF3B1(WT) 2. CLL-SF3B1(K700E mutation);3. Ib txwm B-hlwb
Sequence Strategy:MinION 2D tsev qiv ntawv sequencing, PromethION 1D tsev qiv ntawv sequencing;luv-nyeem cov ntaub ntawv los ntawm tib cov qauv
Sequence platform:TSIS TAU TXAIS;ONT PromethION;
Bioinformatic Analysis
Cov txiaj ntsig
Atag nrho ntawm 257 lab nyeem tau tsim los ntawm 6 CLL cov qauv thiab 3 B-cells.Qhov nruab nrab ntawm 30.5% ntawm cov ntawv nyeem no tau txheeb pom tias yog cov ntawv sau ua tiav.
Full-length lwm txoj isoform tsom xam ntawm RNA (FLAIR) tau tsim los tsim cov txheej txheem ntawm kev ntseeg siab isoforms.FLAIR tuaj yeem ua tiav raws li:
Nanopore nyeem kev sib dhos: txheeb xyuas cov qauv sau ntawv dav dav raws li kev siv genome;
Splice junction kho: kom raug raws li qhov yuam kev (liab) nrog splice site los ntawm ob qho tib si annotated introns, introns los ntawm cov ntaub ntawv luv luv los yog ob qho tib si;
Collapse: summarize tus neeg sawv cev isoforms raws li splice junction chains (thawj-pass set).Xaiv kev ntseeg siab isofrom raws li tus naj npawb ntawm kev txhawb nqa nyeem (Threshold: 3).
Daim duab 1. FLAIR tsom xam txhawm rau txheeb xyuas cov isoforms tag nrho uas cuam tshuam nrog SF3B1 kev hloov pauv hauv CLL
FLAIR txheeb xyuas 326,699 qhov kev ntseeg siab ntawm cov isoforms, 90% ntawm cov isoforms tshiab.Feem ntau ntawm cov isoforms uas tsis tau sau tseg no tau pom tias yog kev sib txuas tshiab ntawm kev paub splice juntions (142,971), thaum lwm cov isoforms tshiab muaj ob qho tib si khaws cia intron (21,700) lossis tshiab exon (3594).
Long-read sequences txhawb kev txheeb xyuas cov mutant SF3B1-K700E -altered splice sites ntawm isoform-theem.35 lwm 3'SSs thiab 10 lwm 5'SSs tau pom tias muaj qhov sib txawv ntawm qhov sib txawv ntawm SF3B1-K700E thiab SF3B1-WT.33 ntawm 35 qhov kev hloov pauv tau raug tshawb pom tshiab los ntawm cov ntawv nyeem ntev.Hauv cov ntaub ntawv Nanopore, qhov kev faib tawm ntawm kev ncua deb ntawm SF3B1-K700E-hloov 3'SSs rau cov chaw canonical peaks yog nyob ib ncig ntawm -20 bp, uas yog qhov sib txawv ntawm kev tswj kev faib tawm, zoo ib yam li qhov tau tshaj tawm hauv CLL luv-nyeem cov kab ke.Isoforms ntawm ERGIC3 noob tau txheeb xyuas, qhov twg cov isoform tshiab uas muaj qhov sib thooj ntawm qhov chaw sib txuas tau pom muaj ntau dua hauv SF3B1-K700E.Ob qho tib si proximal thiab distal 3'SS tau cuam tshuam nrog cov qauv sib txawv AS tsim ntau cov isoforms.
Daim duab 2. Alternative 3′ splicing qauv qhia nrog nanopore sequencing cov ntaub ntawv
IR qhov xwm txheej siv kev txheeb xyuas tau raug txwv ntev los ntawm kev nyeem luv luv raws li kev tshuaj xyuas vim muaj kev ntseeg siab hauv IR kev txheeb xyuas thiab qhov ntau.Kev nthuav qhia ntawm IR isoforms hauv SF3B1-K700E thiab SF3B1-WT tau ntsuas raws li cov txheej txheem nanopore, nthuav tawm thoob ntiaj teb kev tswj hwm ntawm IR isoforms hauv SF3B1-K700E.
Daim duab 4. Kev ua liaj ua teb siv zog thiab kev sib txuas hauv network thoob plaws peb txoj kev ua liaj ua teb (A thiab B);Kev txheeb xyuas hav zoov Random (C) thiab Kev sib raug zoo ntawm kev siv ua liaj ua teb thiab AMF colonization (D)
Daim duab 3. Intron renttention txheej xwm muaj zog ntau downregulated nyob rau hauv CLL SF3B1-K700E
Technology
Nanopore Long-read Sequencing
Nanopore sequencing yog ib qho molecule real-time hluav taws xob teeb liab sequencing tshuab.
Double-stranded DNA los yog RNA yuav khi rau nanoporous protein nyob rau hauv biofilm thiab unwinding nyob rau hauv cov hlau lead ntawm lub cev muaj zog protein.
DNA / RNA strands dhau los ntawm nanopore channel protein ntawm qee tus nqi raws li qhov kev txiav txim ntawm qhov sib txawv voltage.
Molecules tsim hluav taws xob sib txawv raws li cov qauv tshuaj.
Real-time nrhiav kom tau raws li ib feem yog tiav los ntawm lub hauv paus hu.
Kev ua tau zoo ntawm tag nrho-length transcriptome sequencing
√ Cov ntaub ntawv Saturation
7-fold tsawg nyeem yuav tsum ncav cuag cov ntaub ntawv sib piv.
√ Daim ntawv teev cov qauv kev txheeb xyuas
Kev txheeb xyuas qhov sib txawv ntawm cov qauv sib txawv nrog kev pom zoo tag nrho-ntev nyeem ntawv ntawm txhua daim ntawv sau tseg
√ Cov ntawv sau-qib sib txawv ntawm kev txheeb xyuas - Qhia tawm cov kev hloov pauv zais los ntawm kev nyeem luv luv
Kev siv
Tang AD, Soulette CM, Baren MJV, et al.Tag nrho-ntev transcript characterization ntawm SF3B1 kev hloov pauv hauv cov kab mob lymphocytic leukemia ntev qhia txog kev txo qis ntawm kev khaws cia introns [J].Nature Communications.
Tech thiab Highlights Lub hom phiaj ntawm kev sib koom ua tiav daim ntawv thov tam sim no ntawm kev sib txawv high-throughput sequencing technologies nyob rau hauv ntau yam reseach arena nrog rau cov tswv yim ci ntsa iab hauv kev sim tsim thiab cov ntaub ntawv mining.
Post lub sij hawm: Jan-08-2022