Zithunzi za TRANSCRIPTOMICS
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KUKAMBIRANA
Kufotokozera kwautali kwanthawi yayitali kwa SF3B1 mutation mu chronic lymphocytic leukemia kumawonetsa kutsika kwa introns zosungidwa.
Zolemba zazitali zonse|Nanopore sequencing|Kusanthula kwina kwa isoform
Mbiri
Somatic mutations in splicing factor SF3B1 yadziwika kuti ikugwirizana ndi khansa zosiyanasiyana, kuphatikizapo matenda a lymphocytic leukemia (CLL), uveal melanoma, khansa ya m'mawere, ndi zina zotero.Komabe, kafukufuku wokhudzana ndi kuphatikizika kosiyanasiyana kumeneku kwakhala kwanthawi yayitali pamlingo wa zochitika komanso kusowa kwa chidziwitso pamlingo wa isoform chifukwa cha kuchepa kwa zolembedwa zowerengedwa mwachidule.Apa, nsanja yotsatizana ya nanopore idayambitsidwa kuti ipange zolembedwa zazitali, zomwe zidapangitsa kuti inverstigation pa AS isoforms.
Mapangidwe Oyesera
Zoyesera
Kuyika m'magulu:1. CLL-SF3B1 (WT) 2. CLL-SF3B1 (K700E kusintha);3. Ma B-maselo abwinobwino
Njira yotsatirira:Kutsata laibulale ya MinION 2D, kutsatizana kwa library ya PromethION 1D;zowerengera zazifupi kuchokera ku zitsanzo zomwezo
Sequencing nsanja:ONT MinION;ONT PromethION;
Bioinformatics Analysis
Zotsatira
A257 miliyoni zowerengedwa zidapangidwa kuchokera ku zitsanzo 6 za CLL ndi ma B-cell atatu.Pa avareji 30.5% ya zowerengedwazi zidadziwika ngati zolembedwa zazitali.
FUll-length alternative isoform analysis of RNA(FLAIR) idapangidwa kuti ipange ma isoform odzidalira kwambiri.FLAIR ikhoza kufotokozedwa mwachidule monga:
Nanopore amawerenga mayanidwe: zindikirani kapangidwe kazolemba zonse kutengera ma genome;
Splice junction correction: zolakwika zolondola (zofiira) ndi tsamba la splice kuchokera ku ma introns olembedwa, ma introns ochokera ku data yowerengeka kapena zonse ziwiri;
Collapse: fotokozani mwachidule ma isoform oyimira kutengera maunyolo olumikizirana (gawo loyamba).Sankhani isofrom yodalirika kwambiri kutengera kuchuluka kwa zowerengera zothandizira (Threshold: 3).
Chithunzi 1. Kusanthula kwa FLAIR kuti muzindikire ma isoforms alenth athunthu okhudzana ndi kusintha kwa SF3B1 mu CLL
FLAIR Imadziwika 326,699 ma isoform odzidalira kwambiri, 90% omwe ndi ma isoforms atsopano.Ambiri mwa ma isoform omwe sanatchulidwewa adapezeka kuti ndi maphatikizidwe atsopano odziwika bwino (142,971), pomwe ma isoform ena onse anali ndi intron (21,700) kapena novel exon (3594).
LKuwerenga mosalekeza kumathandizira kuzindikira SF3B1-K700E -osinthidwa ma splice masamba pamlingo wa isoform.35 njira zina za 3'SS ndi 10 zina 5'SS zinapezeka kuti ndizosiyana kwambiri pakati pa SF3B1-K700E ndi SF3B1-WT.Zosintha 33 mwa 35 zidapezedwa kumene ndikutsatiridwa kwanthawi yayitali.Mu data ya Nanopore, kugawidwa kwa mtunda pakati pa SF3B1-K700E-osinthidwa 3'SSs kumalo ovomerezeka a malo ovomerezeka ndi pafupi -20 bp, yomwe imasiyana kwambiri ndi kugawidwa kolamulira, mofanana ndi zomwe zanenedwa mu CLL zowerengeka zochepa.Ma Isoforms a jini ya ERGIC3 adawunikidwa, pomwe buku la isoform lomwe lili ndi malo olumikizana ndi proximal splice adapezeka ochulukirapo mu SF3B1-K700E.Ma proximal ndi distal 3'SS anali olumikizidwa ndi mitundu yosiyana ya AS yotulutsa ma isoform angapo.
Chithunzi 2. Njira zina zolumikizirana 3′ zodziwika ndi data ya nanopore.
Kusanthula kagwiritsidwe ntchito ka zochitika za IR kwakhala kochepera kwakanthawi pakuwunika kowerengera mwachidule chifukwa chodalira chizindikiritso cha IR ndi kuchuluka kwake.Mafotokozedwe a ma isoform a IR mu SF3B1-K700E ndi SF3B1-WT adawerengedwa motengera ma nanopore, kuwulula kutsika kwapadziko lonse kwa ma isoform a IR mu SF3B1-K700E.
Chithunzi 4. Kuchulukira kwaulimi ndi kulumikizana kwa maukonde pamitundu itatu yaulimi (A ndi B);Kusanthula nkhalango mwachisawawa(C) ndi Ubale pakati pa ulimi ndi AMF colonization (D)
Chithunzi 3. Zochitika zosungirako za intron zimatsitsidwa kwambiri mu CLL SF3B1-K700E
Zamakono
Nanopore Kuwerenga Kwanthawi yayitali
Nanopore sequencing ndi molekyulu imodzi yeniyeni yotsatirira ma siginolo amagetsi.
DDNA yozungulira kapena RNA imamanga ku mapuloteni a nanoporous omwe ali mu biofilm ndikumasula motsogozedwa ndi mapuloteni amoto.
DZingwe za NA/RNA zimadutsa mu protein ya nanopore pamlingo wina chifukwa cha kusiyana kwa magetsi.
Mma elecules amapanga ma sign amagetsi osiyanasiyana malinga ndi kapangidwe kake.
RKuzindikira kwakanthawi kwakanthawi kumatheka ndi kuyimbira koyambira.
Kuchita kwa kutsatizana kwautali wonse wa transcriptome
√ Kuchulukitsa kwa data
Zowerengera zochepera 7 zomwe zimafunikira kuti mufikire kuchuluka kwa data.
√ Chizindikiritso cha Kapangidwe ka Zolemba
Kuzindikiritsa mitundu yosiyanasiyana yamapangidwe ndi kuwerengedwa kwautali wathunthu kwa cholembedwa chilichonse
√ Kusanthula kwa kusiyana kwa mulingo wa transcript -Kuwonetsa zosintha zobisika ndi kuwerenga kwakanthawi
Buku
Tang AD , Soulette CM , Baren MJV , et al.Kufotokozera kwautali kwanthawi yayitali kwa SF3B1 mutation mu chronic lymphocytic leukemia kumawonetsa kutsika kwa introns zosungidwa [J].Nature Communications.
Tech ndi Zowunikira Cholinga chake ndi kugawana njira zamakono zotsatirira bwino kwambiri m'mabwalo osiyanasiyana ofufuza komanso malingaliro anzeru pamapangidwe oyesera ndi migodi ya data.
Nthawi yotumiza: Jan-08-2022