TRANSCRIPTOMICS
nature
COCOMUNICATIONS
Yakazara-yakareba-yakanyorwa mavara eSF3B1 mutation mune isingaperi lymphocytic leukemia inoratidza kuderedzwa kweakachengetwa introns.
Yakazara-yakareba zvinyorwa|Nanopore sequencing|Alternative isoform analysis
Background
Somatic mutations in splicing factor SF3B1 yakave yakashumwa zvakanyanya kuti inobatanidza nekenza dzakasiyana-siyana, kusanganisira chronic lymphocytic leukemia (CLL), uveal melanoma, kenza yemazamu, nezvimwewo. Uyezve, zvidzidzo zvenguva pfupi-kuverenga zvinyorwa zvakaratidza aberrant splicing mapatani anokonzerwa neSF3B1 mutations.Nekudaro, zvidzidzo zveaya mamwe maitiro ekutsvedza zvagara zvakaganhurirwa kune chiitiko-chikamu uye kushaikwa kweruzivo pane isoform-chikamu nekuda kwekuganhurirwa kwepfupi-kuverenga-yakaunganidzwa zvinyorwa.Pano, nanopore sequencing chikuva chakaunzwa kuti igadzire yakazara-yakareba zvinyorwa, izvo zvakagonesa inverstigation paAS isoforms.
Kuedza Dhizaini
Miedzo
Kuronga1. CLL-SF3B1(WT) 2. CLL-SF3B1(K700E mutation);3. Kazhinji B-masero
Sequencing strategy:MinION 2D raibhurari sequencing, PromethION 1D raibhurari sequencing;data pfupi-yekuverenga kubva kumasampuli akafanana
Sequencing platform:ONT MinION;ONT PromethION;
Bioinformamatic Analysis
Results
A257 mamiriyoni ekuverenga akagadzirwa kubva ku6 CLL samples uye 3 B-masero.Paavhareji ye30.5% yezviverengero izvi zvakaonekwa sezvinyorwa zvehurefu hwakazara.
FUll-length alternative isoform analysis yeRNA(FLAIR) yakagadziridzwa kuti ibudise seti ye-high-confidence isoforms.FLAIR inogona kupfupikiswa se:
Nanopore anoverenga alignment: tsvaga general transcript chimiro chinoenderana nereferenzi genome;
Splice junction kururamisa: kwakaringana kutevedzana zvikanganiso (tsvuku) ine splice saiti kubva kune annotated introns, introns kubva pfupi-yekuverenga data kana zvese zviri zviviri;
Ckudonha: pfupikisa anomiririra isoforms zvichibva pane splice junction cheni (yekutanga-pass set).Sarudza yakakwira-ruvimbo isofrom yakavakirwa pahuwandu hwekutsigira kuverenga (Threshold: 3).
Mufananidzo 1. FLAIR kuongorora kuona yakazara-lenth isoforms yakabatana neSF3B1 mutation muCLL.
FLAIR Yakazivikanwa 326,699 yakakwirira-kuvimbika spliced isoforms, 90% ayo ari novel isoforms.Mazhinji eaya asina kunyorwa isoforms akawanikwa ari manyoro masanganiswa einozivikanwa splice juntions (142,971), nepo mamwe ese maisoforms anga aine akachengetwa intron (21,700) kana novel exon (3594).
LOng-kuverenga kutevedzana kunopa simba kuzivikanwa kwemutant SF3B1-K700E -yakashandurwa splice nzvimbo paisoform-level.35 mamwe ma3'SSs uye gumi mamwe ma5'SS akawanikwa aine musiyano wakanyanya pakati peSF3B1-K700E neSF3B1-WT.33 ye 35 shanduko dzaive dzichangowanikwa nekuverenga kwenguva refu kutevedzana.Mune data yeNanopore, kugoverwa kwechinhambwe pakati peSF3B1-K700E-yakashandurwa 3'SS kune canonical nzvimbo peaks yakatenderedza -20 bp, iyo yakasiyana zvakanyanya kubva pakugoverwa kwekutonga, yakafanana neyakashumwa muCLL pfupi-yekuverenga sequences.Isoforms yeERGIC3 gene yakaongororwa, apo inoveli isoform ine proximal splice saiti yakawanikwa yakawanda muSF3B1-K700E.Zvese zviri zviviri proximal uye distal 3'SS zvakabatana neakasiyana AS mapatani achigadzira akawanda isoforms.
Mufananidzo 2. Alternative 3′ splicing mapatani anoonekwa nenanopore sequencing data
IR chiitiko chekushandiswa kwekuongorora kwave kwenguva yakareba kushoma mupfupi-kuverenga-based ongororo nekuda kwekuvimba mukuzivikanwa kweIR uye huwandu.Tsanangudzo yeIR isoforms muSF3B1-K700E uye SF3B1-WT yakaverengerwa zvichienderana nenanopore sequence, zvichiratidza pasi-pasi-mutemo weIR isoforms muSF3B1-K700E.
Mufananidzo 4. Kusimba kwekurima uye kubatana kwetiweki mumasisitimu matatu ekurima (A neB);Random sango kuongororwa (C) uye Hukama pakati pekurima zvakanyanya uye AMF colonization (D)
Mufananidzo 3. Intron retention zviitiko zvakanyanya kuderedzwa zvakanyanya muCLL SF3B1-K700E
Vadivelu Comedy Technology
Nanopore Yakareba-Kuverenga Sequencing
Nanopore sequencing ndeye imwe molecule chaiyo-nguva yemagetsi chiratidzo chekuenzanisa tekinoroji.
Double-stranded DNA kana RNA inosunga kune nanoporous protein yakaiswa mubiofilm uye kusunungura pasi pekutungamira kwemota protein.
DNA/RNA tambo dzinopfuura nenanopore chiteshi protein pane imwe chiyero pasi pechiito chekusiyana kwemagetsi.
Mmaolecules anogadzira masaini akasiyana emagetsi zvinoenderana nemakemikari chimiro.
Rnguva-yenguva yekuona kutevedzana kunowanikwa nekufona kwekutanga.
Kuita kwehurefu hwakazara transcriptome sequencing
√ Data Saturation
7-kupeta mashoma kuverenga kunodiwa kuti usvike kuenzaniswa kwedata saturation.
√ Transcript Structure Identification
Kucherechedzwa kwezvakasiyana-siyana zvimiro nekubvumirana kuzere-kureba kuverenga kwechinyorwa chega chega
√ Transcript-level differential analysis -Ratidza shanduko dzakavanzwa nepfupi-kuverenga
Reference
Tang AD , Soulette CM , Baren MJV , et al.Yakazara-yakareba-yakanyorwa maratidziro eSF3B1 mutation mune isingaperi lymphocytic leukemia inoratidza kuderedzwa kweakachengetwa introns [J].Nature Communications.
Tech uye Highlights inovavarira kugovera zvichangobva kuitika zvemashandisirwo akasiyana-siyana epamusoro-soro ekutevedzanisa matekinoroji munzvimbo dzakasiyana siyana dzekutsvagisa pamwe chete nemazano akajeka mukugadzira dhizaini nekuchera data.
Nguva yekutumira: Jan-08-2022