TALIAGA
natura
FESOOTAIGA
O faʻamatalaga faʻamatalaga atoa o le suiga o le SF3B1 i le leukemia lymphocytic masani e faʻaalia ai le faʻaitiitia o le faʻatonutonuina o mea faʻapipiʻi.
Fa'amaumauga atoa|Nanopore sequencing|Su'e su'esu'ega isoform
Talaaga
Ssuiga omatic i splicing factor SF3B1 ua lipotia lautele e fesootai ma kanesa eseese, e aofia ai le lymphocytic leukemia (CLL), uveal melanoma, kanesa o le susu, ma isi.Ae ui i lea, o suʻesuʻega i nei isi faʻasologa faʻapipiʻi ua leva ona faʻatapulaʻaina i mea tutupu-tulaga ma le leai o se malamalama ile isoform-level ona o le faʻatapulaʻaina o tusitusiga faʻapipiʻi puʻupuʻu faitau.O iinei, o le nanopore sequencing platform na faʻalauiloaina e faʻatupu ai tusitusiga umi-umi, lea na faʻamalosia ai suʻesuʻega ile AS isoforms.
Fuafuaga Fa'ata'ita'i
Fa'ata'ita'iga
Fa'avasegaga:1. CLL-SF3B1(WT) 2. CLL-SF3B1(K700E suiga);3. sela B masani
Fuafuaga fa'asologa:MiniION 2D sequencing faletusi, PromethION 1D sequencing faletusi;fa'amatalaga pu'upu'u faitau mai fa'ata'ita'iga tutusa
Fa'asologa fa'asologa:ONT Minion;ONT PromethION;
Bioinformatic Iloiloga
I'uga
Aatoa ole 257 miliona faitau na gaosia mai 6 CLL samples ma 3 B-cells.I le averesi o le 30.5% o nei faitauga na faʻamaonia o ni tusitusiga atoa.
Full-umi su'esu'ega isoform su'esu'ega o le RNA(FLAIR) na fa'atupuina e fa'atupuina ai se seti o fa'atonuga fa'amaoni maualuga.FLAIR e mafai ona aoteleina e faapea:
Nanopore faitau alignment: fa'ailoa fa'asologa o tusitusiga fa'avae e fa'atatau ile genome fa'asino;
Sfa'asa'oga feso'ota'iga: fa'asa'o fa'asologa o mea sese(mumu) fa'atasi ai ma nofoaga fa'apipi'i mai fa'amatalaga fa'amatalaga, fa'ailoga mai fa'amatalaga pu'upu'u faitau po'o mea uma e lua;
Collapse: aotele fa'atusa fa'atusa e fa'avae i luga o filifili so'o fa'atasi (seti pasi muamua).Filifili le fa'atuatuaga maualuga e fa'atatau i le numera o faitau lagolago(Tula'au: 3).
Ata 1. Su'esu'ega FLAIR e fa'ailoa ai fa'aputuga atoa lenth e feso'ota'i ma suiga ole SF3B1 ile CLL
FLAIR Fa'ailoaina 326,699 maualuga fa'atuatuaina fa'apipi'i isoforms, 90% o ia mea fou isoforms.O le tele o nei fa'ailoga e le'i fa'ailoaina na maua o ni tu'ufa'atasiga fou o feso'ota'iga fa'apipi'i (142,971), a'o isi fa'asologa fou o lo'o iai i totonu ole intron(21,700) po'o le exon(3594).
Lfa'asologa fa'aauau e fa'amalosia ai le fa'ailoaina o le mutant SF3B1-K700E-suia nofoaga fa'apipi'i ile tulaga fa'aisoform.35 isi 3'SSs ma isi 10 isi 5'SSs na maua e matua'i va'aiga eseese i le va o le SF3B1-K700E ma le SF3B1-WT.O le 33 o le 35 suiga na faatoa maua e ala ua leva ona faitau fa'asologa.I faʻamaumauga a Nanopore, o le tufatufaina atu o le mamao i le va o SF3B1-K700E-suia 3'SSs i nofoaga faʻa-canonical pito i luga o le -20 bp, lea e matua ese lava mai le tufatufaina atu, e tutusa ma le mea na lipotia i le CLL pupuu faitau faasologa.Isoforms o le ERGIC3 gene na suʻesuʻeina, lea na maua ai se tala faʻapitoa o loʻo i ai le nofoaga vavalalata vavalalata na maua i le SF3B1-K700E.O 3'SS vavalalata ma le mamao sa feso'ota'i ma fa'ata'ita'iga AS fa'atupuina le tele o isoforms.
Ata 2. Su'ega 3′ fa'asologa fa'apipi'i fa'ailoa ile fa'asologa o fa'amaumauga o nanopore
Ua leva ona fa'atapula'aina le su'esu'ega o le fa'aogaina o mea na tupu ile su'esu'ega pu'upu'u faitau ona ole mautinoa ile fa'ailoaina ma le fa'atusatusaina ole IR.Faʻaaliga o IR isoforms i SF3B1-K700E ma SF3B1-WT na faʻatatauina faʻavae i luga o faʻasologa o nanopore, faʻaalia ai le lalolagi-faʻatonutonu lalo ole IR isoforms i SF3B1-K700E.
Ata 4. Fa'ato'aga malosi ma feso'ota'iga feso'ota'iga i faiga fa'ato'aga se tolu (A ma le B);Su'esu'ega fa'afuase'i o vaomatua(C) ma So'otaga i le va o le malosi o fa'ato'aga ma le nofoia o le AMF (D)
Ata 3. O fa'alavelave fa'amautu i totonu e sili atu ona fa'atulafonoina ile CLL SF3B1-K700E.
Tekinolosi
Nanopore Long-faitau Fa'asologa
Nanopore sequencing o se tasi mole mole taimi moni eletise sequencing sequencing tekinolosi.
DO le DNA poʻo le RNA o loʻo faʻapipiʻiina o le a fusifusia i porotini nanoporous o loʻo faʻapipiʻi i totonu o le biofilm ma faʻamavae i lalo o le taʻitaʻiga o le porotini afi.
DNA/RNA strands e pasi atu i le nanopore channel protein i se fua faatatau i lalo ole gaioiga ole eseesega ole voltage.
Molecules e fa'atupuina fa'ailoga eletise eseese e tusa ai ma le fa'atulagaina o vaila'au.
Real-taimi su'esu'ega o fa'asologa e ausia e ala i le vala'au faavae.
Fa'atinoga o fa'asologa fa'asologa atoa
√ Tulaga Fa'amatalaga
Fa'a 7 fa'aitiiti le faitau e mana'omia e o'o atu ai i fa'atusatusaga o fa'amaumauga.
√ Fa'amatalaga Fa'asologa o Fa'amaumauga
Fa'ailoaina o suiga eseese o le fa'atulagaga fa'atasi ai ma fa'atasiga fa'atasi le faitau atoa o tusitusiga ta'itasi
√ Transcript-level differential analysis -Fa'aalia suiga natia e tusi-faitau
Fa'asinomaga
Tang AD, Soulette CM, Baren MJV, ma isi.O faʻamatalaga faʻamatalaga atoa o le suiga o le SF3B1 i le leukemia lymphocytic masani e faʻaalia ai le faʻaitiitia o le taofiofia o introns [J].Fesootaiga Natura.
Tekinolosi ma Fa'ailoga e fa'atatau i le fa'asoaina o fa'aoga manuia lata mai o fa'atekonolosi fa'asologa fa'asolosolo maualuga i malae su'esu'e 'ese'ese fa'apea fo'i ma manatu fa'ata'ita'i ile fa'ata'ita'iga fa'ata'ita'iga ma su'esu'ega fa'amaumauga.
Taimi meli: Ian-08-2022