NA KAUKA
ʻano ʻano
HOOLAHA
ʻO ke ʻano o ka transcript piha piha o ka mutation SF3B1 i ka maʻi maʻi lymphocytic maʻi e hōʻike ana i ka hoʻohaʻahaʻa ʻana o nā introns paʻa.
Nā kope lōʻihi|Nanopore sequencing|ʻO ka nānā ʻana isoform ʻē aʻe
Kāpae
SUa hōʻike nui ʻia nā hoʻololi omatic i ka splicing factor SF3B1 e pili pū me nā maʻi maʻi like ʻole, me ka maʻi maʻi lymphocytic maʻi (CLL), melanoma uveal, ka maʻi maʻi umauma, a me nā mea ʻē aʻe.Eia nō naʻe, ua kaupalena ʻia nā haʻawina e pili ana i kēia mau hiʻohiʻona splicing ʻokoʻa i ka pae hanana a me ka ʻike ʻole i ka pae isoform ma muli o ka palena o nā transcript i hōʻuluʻulu ʻia.Ma ʻaneʻi, ua hoʻokomo ʻia ka papa kuhikuhi nanopore e hoʻopuka i nā transcript lōʻihi piha, i hāʻawi i ka mana i ka inverstigation ma nā isoforms AS.
Hoʻolālā hoʻokolohua
Nā hoʻokolohua
Hui pū:1. CLL-SF3B1(WT) 2. CLL-SF3B1(K700E hoʻololi);3. Nā pūnaewele B maʻamau
Hoʻolālā kaʻina hana:ʻO ke kaʻina o ka waihona Minion 2D, ke ʻano o ka waihona waihona PromethION 1D;ʻikepili heluhelu pōkole mai nā laʻana like
Papahana kaʻina:ONT Minion;ONT PromethION;
ʻIkepili Bioinformatic
Nā hualoaʻa
AʻO ka huina o 257 miliona mau heluhelu i hana ʻia mai 6 CLL samples a me 3 B-cells.Ma ka awelika o 30.5% o kēia mau heluhelu i ʻike ʻia ma ke ʻano he transcript lōʻihi.
FUa hoʻomohala ʻia ka loiloi isoform ʻokoʻa lōʻihi o RNA(FLAIR) e hana i kahi pūʻulu o nā isoform hilinaʻi kiʻekiʻe.Hiki ke hōʻuluʻulu ʻia ʻo FLAIR e like me:
NHeluhelu ʻo anopore i ka hoʻolikelike ʻana: e ʻike i ka hoʻolālā transcript maʻamau e pili ana i ka genome kuhikuhi;
Ska hoʻoponopono ʻana i ka pilina pili: hoʻoponopono i nā hewa kaʻina hana (ʻulaʻula) me ka pūnaewele splice mai nā intron i hōʻike ʻia, nā intron mai nā ʻikepili heluhelu pōkole a i ʻole nā mea ʻelua;
Collapse: hōʻuluʻulu i nā isoform luna e pili ana i nā kaulahao hoʻokuʻi splice (hoʻonohonoho mua-pass).E koho i ka hilinaʻi kiʻekiʻe e pili ana i ka helu o nā heluhelu kākoʻo (Threshold: 3).
Kiʻi 1. Nānā FLAIR no ka ʻike ʻana i nā isoform lenth piha i pili me ka mutation SF3B1 ma CLL
FUa ʻike ʻia ʻo LAIR he 326,699 isoforms spliced kiʻekiʻe, 90% o ia mau isoforms hou.ʻO ka hapa nui o kēia mau isoforms i ʻike ʻole ʻia he hui pū ʻia o nā hui splice i ʻike ʻia (142,971), aʻo nā isoform hou i koe i loko o ka intron (21,700) a i ʻole novel exon (3594).
LʻO nā kaʻina o ka heluhelu ʻana e hāʻawi i ka ʻike o ka mutant SF3B1-K700E -altered splice sites ma isoform-level.Ua ʻike ʻia he 35 mau 3'SS a me 10 mau 5'SS ʻokoʻa ʻokoʻa ma waena o SF3B1-K700E a me SF3B1-WT.ʻO 33 o nā hoʻololi 35 i ʻike hou ʻia e nā kaʻina heluhelu lōʻihi.Ma ka ʻikepili Nanopore, ʻo ka puʻunaue o ka mamao ma waena o SF3B1-K700E-hoʻololi ʻia 3'SSs i nā pae canonical peaks ma kahi o -20 bp, kahi mea ʻokoʻa loa mai kahi puʻupuʻu mana, e like me ka mea i hōʻike ʻia ma nā kaʻina heluhelu pōkole CLL.Ua kālailai ʻia nā Isoforms o ka gene ERGIC3, kahi i ʻike nui ʻia ai kahi isoform hou i loaʻa ka pūnaewele splice proximal ma SF3B1-K700E.Hoʻopili ʻia ka 3'SS proximal a me ka distal me nā hiʻohiʻona AS ʻokoʻa e hana ana i nā isoforms lehulehu.
Kiʻi 2. ʻO nā hiʻohiʻona ʻokoʻa 3′ splicing i ʻike ʻia me ka ʻikepili sequencing nanopore
Ua kaupalena ʻia ka hoʻohana ʻana o ka hanana hanana IR i ka nānā ʻana i ka heluhelu pōkole ma muli o ka hilinaʻi i ka ʻike IR a me ka helu.Ua helu ʻia ka hōʻike ʻana o nā isoforms IR ma SF3B1-K700E a me SF3B1-WT ma muli o nā kaʻina nanopore, e hōʻike ana i kahi hoʻoponopono hoʻohaʻahaʻa honua o nā isoforms IR ma SF3B1-K700E.
Kiʻi 4. ʻO ka ikaika o ka mahiʻai a me ka pilina pūnaewele ma waena o nā ʻōnaehana mahiʻai ʻekolu (A a me B);Ka nānā 'ana i ka nahelehele (C) a me ka pilina ma waena o ka ikaika o ka mahi'ai a me ka noho 'ana o AMF (D)
Kiʻi 3. Hoʻohaʻahaʻa ikaika ʻia nā hanana hoʻopaʻa intron ma CLL SF3B1-K700E
ʻenehana
Nanopore Heluhelu lōʻihi
NʻO ke kaʻina hana anopore he ʻenehana hoʻokaʻina hōʻailona uila i ka manawa maoli.
DE hoʻopaʻa ʻia ʻo DNA a i ʻole RNA i hoʻopaʻa ʻia me ka nanoporous protein i hoʻokomo ʻia i loko o ka biofilm a wehe ʻia ma lalo o ke alakaʻi o ka protein motor.
DKe hele nei nā kaula NA/RNA i loko o ke kinikini nanopore channel ma kekahi ʻano ma lalo o ka hana o ka ʻokoʻa uila.
MHoʻopuka nā olecules i nā hōʻailona uila like ʻole e like me ke ʻano kemika.
RLoaʻa ka ʻike ʻana i nā kaʻina eal-time ma ke kelepona kumu.
ʻO ka hana ʻana o ke kaʻina transcriptome holoʻokoʻa
√ Hoʻohui ʻikepili
Heluhelu liʻiliʻi ʻo 7-fold no ka hiki ʻana i ka saturation data like.
√ Hōʻike Hoʻonohonoho Transcript
Ka ʻike ʻana i nā ʻano like ʻole me ka ʻae ʻana i ka heluhelu piha piha o kēlā me kēia transcript
√ Hōʻike ʻokoʻa ʻokoʻa o ka transcript-level -Hōʻike i nā loli i hūnā ʻia e nā heluhelu pōkole
Kuhikuhi
ʻO Tang AD, Soulette CM, Baren MJV, et al.Hōʻike ka hōʻike ʻana i ka transcript piha piha o ka mutation SF3B1 i ka maʻi leukemia lymphocytic maʻi i ka hoʻohaʻahaʻa ʻana o nā introns paʻa [J].Nature Communications.
ʻenehana a me nā mea koʻikoʻi manaʻo e kaʻana like i nā noi holomua hou loa o nā ʻenehana hoʻokaʻina kiʻekiʻe-throughput like ʻole ma nā kahua ʻimi noiʻi like ʻole a me nā manaʻo maikaʻi loa i ka hoʻolālā hoʻokolohua a me ka ʻimi ʻikepili.
Ka manawa hoʻouna: Jan-08-2022