Bambancin zaruruwan tsokar kwarangwal na ɗan adam bayan sarkar nauyi ta myosin

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Tsokar ƙashi wata nama ce mai bambancin halitta wadda ta ƙunshi galibin myofibrils, wanda a cikin mutane galibi ana rarraba ta zuwa nau'i uku: ɗaya "a hankali" (nau'in 1) da biyu "sauri" (nau'i 2A da 2X). Duk da haka, bambancin da ke tsakanin da kuma cikin nau'ikan myofibril na gargajiya har yanzu ba a fahimce shi sosai ba. Mun yi amfani da hanyoyin transcriptomic da proteomic zuwa ga myofibrils 1050 da 1038 daga ɗan adam vastus lateralis, bi da bi. Binciken proteomic ya haɗa da maza, kuma binciken transcriptomic ya haɗa da maza 10 da mata 2. Baya ga isoforms na sarkar myosin mai nauyi, mun gano sunadaran metabolism, sunadaran ribosomal, da sunadaran haɗin tantanin halitta a matsayin tushen bambancin intermyofibril mai girma dabam dabam. Bugu da ƙari, duk da gano tarin zaruruwa masu jinkiri da sauri, bayananmu sun nuna cewa zaruruwan nau'in 2X ba za a iya bambanta su da sauran zaruruwa masu sauri ba. Bugu da ƙari, rarrabuwar sarkar nauyi ta myosin bai isa ya bayyana yanayin myofiber a cikin myopathies na nemaline ba. Gabaɗaya, bayananmu suna nuna bambancin myofiber mai girma dabam-dabam, tare da tushen bambance-bambancen da suka wuce isoforms na sarkar nauyi ta myosin.
Bambancin ƙwayoyin halitta wani abu ne da ke cikin dukkan tsarin halittu, wanda ke ba da damar ƙwayoyin halitta su ƙware don biyan buƙatun daban-daban na kyallen takarda da ƙwayoyin halitta.1 Ra'ayin gargajiya game da bambancin zare na tsokar ƙashi shine cewa ƙwayoyin jijiyoyi masu motsi suna bayyana nau'in zare a cikin sashin motsi, kuma nau'in zare (watau nau'in 1, nau'in 2A, da nau'in 2X a cikin mutane) ana tantance shi ta hanyar halayen isoforms na sarkar nauyi ta myosin (MYH).2 Wannan ya fara ne akan rashin daidaiton pH ATPase, 3,4 kuma daga baya akan bayyanar kwayoyin halittarsu na MYH.5 Duk da haka, tare da ganowa da kuma karɓar zare masu "gauraye" waɗanda ke bayyana MYHs da yawa a cikin rabo daban-daban, ana ƙara kallon zare masu tsokar ƙashi a matsayin ci gaba maimakon nau'ikan zare daban-daban.6 Duk da haka, har yanzu filin ya dogara sosai akan MYH a matsayin babban mai rarrabawa don rarrabuwar myofiber, ra'ayi mai yiwuwa ya rinjayi ƙuntatawa da manyan son zuciya na nazarin farkon beraye waɗanda bayanin bayyanar MYH da kewayon nau'ikan zare suka bambanta da na mutane.2 Yanayin ya ƙara rikitarwa ta hanyar gaskiyar cewa tsokoki daban-daban na ɗan adam suna nuna nau'ikan zare daban-daban.7 Vastus lateralis tsoka ce mai gauraya wadda ke da matsakaicin yanayin bayyanar MYH (sabili da haka wakilci).7 Bugu da ƙari, sauƙin ɗaukar samfurinsa ya sa ya zama tsokar da aka fi nazari a kanta a cikin mutane.
Saboda haka, bincike mara son kai game da bambancin zare na tsokar ƙashi ta amfani da kayan aikin "omics" masu ƙarfi yana da matuƙar muhimmanci amma kuma yana da ƙalubale, wani ɓangare saboda yanayin ƙwayoyin tsokar ƙashi da yawa. Duk da haka, fasahar transcriptomics8,9 da proteomics10 sun fuskanci juyin juya hali a cikin 'yan shekarun nan saboda ci gaban fasaha daban-daban, wanda ya ba da damar nazarin tsokar ƙashi a ƙudurin zare ɗaya. Sakamakon haka, an sami ci gaba mai mahimmanci wajen siffanta bambancin zare ɗaya da kuma martaninsu ga abubuwan da ke haifar da atrophic da tsufa11,12,13,14,15,16,17,18. Abu mafi mahimmanci, waɗannan ci gaban fasaha suna da aikace-aikacen asibiti, wanda ke ba da damar ƙarin cikakkun bayanai da daidaito na bayyanar dysregulation da ke da alaƙa da cuta. Misali, ilimin cututtukan nemaline myopathy, ɗaya daga cikin cututtukan tsoka da aka gada mafi yawa (MIM 605355 da MIM 161800), yana da rikitarwa kuma yana da rikitarwa.19,20 Saboda haka, ingantaccen siffanta rashin daidaituwa na zare na tsokar ƙashi na iya haifar da ci gaba mai mahimmanci a fahimtarmu game da wannan cuta.
Mun ƙirƙiro hanyoyin nazarin ƙwayoyin tsoka guda ɗaya na transcriptomic da proteomic na zaruruwan tsoka guda ɗaya da aka ware da hannu daga samfuran biopsy na ɗan adam kuma muka yi amfani da su ga dubban zaruruwa, wanda hakan ya ba mu damar bincika bambancin ƙwayoyin tsoka na ɗan adam. A cikin wannan aikin, mun nuna ƙarfin transcriptomic da proteomic phenotyping na zaruruwan tsoka kuma mun gano sunadaran metabolism, ribosomal, da cell junctional proteins a matsayin manyan hanyoyin bambancin interfiber. Bugu da ƙari, ta amfani da wannan aikin proteomic, mun bayyana mahimmancin nematode myopathy a cikin zaruruwan tsoka guda ɗaya, wanda ya bayyana canji mai daidaituwa zuwa zaruruwan da ba sa yin oxidative ba tare da la'akari da nau'in zaruruwan ba bisa ga MYH.
Domin bincika bambancin zaruruwan tsokar ɗan adam, mun ƙirƙiri ayyuka guda biyu don ba da damar nazarin zaruruwan tsokar ƙashi ɗaya da na proteome (Hoto na 1A da Ƙarin Hoto na 1A). Mun haɓaka kuma mun inganta matakai da dama na hanyoyin aiki, daga adana samfura da adana RNA da amincin furotin zuwa inganta fitarwa don kowane hanya. Don nazarin zaruruwan, an cimma wannan ta hanyar saka barcode na ƙwayoyin halitta na musamman a matakin farko na rubutun baya, wanda ya ba da damar haɗa zaruruwa 96 don ingantaccen aiki a ƙasa. Jerin abubuwa masu zurfi (± karanta miliyan 1 a kowace zare) idan aka kwatanta da hanyoyin ƙwayoyin halitta guda ɗaya na gargajiya sun ƙara wadatar da bayanan zaruruwan. 21 Don proteomics, mun yi amfani da ɗan gajeren gradient chromatographic (minti 21) tare da tattara bayanai na DIA-PASEF akan na'urar auna taro ta timsTOF don inganta zurfin proteome yayin da muke ci gaba da samar da babban fitarwa. 22,23 Domin bincika bambancin zare na tsokar ƙashi mai lafiya, mun siffanta zare na zare 1,050 daga masu ba da gudummawa ga manya 14 masu lafiya da kuma proteomes na zare 1,038 daga masu ba da gudummawa ga manya 5 masu lafiya (Jadawali na Ƙarin 1). A cikin wannan takarda, ana kiran waɗannan bayanan a matsayin transcriptomes na zare 1,000 da proteomes, bi da bi. Hanyarmu ta gano jimlar bayanan 27,237 da furotin 2,983 a cikin nazarin transcriptomic da proteomic na zare 1,000 (Hoto na 1A, Ƙarin Bayanan 1-2). Bayan tace bayanan transcriptomic da proteomic don > kwayoyin halitta da aka gano >1,000 da ƙimar inganci 50% ga kowace zare, an yi nazarin bioinformatics na gaba don zare 925 da 974 a cikin rubutun transcriptome da proteome, bi da bi. Bayan tacewa, an gano matsakaicin kwayoyin halitta 4257 ± 1557 da furotin 2015 ± 234 (matsakaicin ± SD) a kowace zare, tare da iyakancewar bambancin tsakanin mutane (Karin Figures 1B–C, Ƙarin Bayanan Bayanai 3–4). Duk da haka, bambancin cikin abin da ake magana a kai ya fi bayyana a tsakanin mahalarta, wataƙila saboda bambance-bambancen da ke cikin yawan RNA/protein tsakanin zaruruwa masu tsayi daban-daban da yankunan da ke haɗaka. Ga yawancin furotin (>2000), ma'aunin bambancin ya kasance ƙasa da 20% (Karin Hoto na 1D). Duk hanyoyin biyu sun ba da damar kama nau'ikan rubuce-rubuce da furotin masu ƙarfi tare da alamun da aka bayyana sosai waɗanda ke da mahimmanci ga matsewar tsoka (misali, ACTA1, MYH2, MYH7, TNNT1, TNNT3) (Karin Figures 1E–F). Yawancin siffofin da aka gano sun kasance iri ɗaya tsakanin bayanan bayanai na transcriptomic da proteomic (Karin Hoto na 1G), kuma matsakaicin ƙarfin UMI/LFQ na waɗannan fasalulluka suna da alaƙa mai kyau (r = 0.52) (Karin Hoto na 1H).
Tsarin aiki na Transcriptomics da Proteomics (wanda aka ƙirƙira tare da BioRender.com). Lanƙwasa na kewayon BD Dynamic range don MYH7, MYH2, da MYH1, da kuma ƙididdigar iyakokin aikin nau'in fiber. E, F Rarraba bayyanar MYH a tsakanin zaruruwa a cikin bayanan transcriptomics da proteomics. G, H Shirye-shiryen kimantawa da hasashen bambancin ra'ayi (UMAP) don zane-zanen transcriptomics da proteomics masu launi ta hanyar nau'in fiber mai tushen MYH. I, J Shirye-shiryen fasali waɗanda ke nuna bayyanar MYH7, MYH2, da MYH1 a cikin bayanan transcriptomics da proteomics.
Da farko mun fara sanya nau'in zare mai tushen MYH ga kowane zare ta amfani da hanyar da aka inganta wacce ke amfani da babban ƙarfin ji da kuma yanayin ƙarfin magana na MYH a cikin bayanan bayanai na omics. Nazarin da suka gabata sun yi amfani da iyakokin da ba a saba ba don yiwa zare alama a matsayin tsantsar nau'in 1, nau'in 2A, nau'in 2X, ko gauraye bisa ga ƙayyadadden kaso na bayyanar MYHs11,14,24 daban-daban. Mun yi amfani da wata hanya daban inda aka sanya bayanin kowace zare ta hanyar MYHs da muka yi amfani da su don rubuta zare: MYH7, MYH2, da MYH1, wanda ya dace da nau'in 1, nau'in 2A, da nau'in 2X zare, bi da bi. Daga nan muka ƙididdige ƙananan wurin juyawa na kowane lanƙwasa da ya haifar kuma muka yi amfani da shi a matsayin maƙalli don sanya zare a matsayin tabbatacce (sama da maƙalli) ko korau (ƙasa da maƙalli) ga kowane MYH (Hoto na 1B-D). Waɗannan bayanan sun nuna cewa MYH7 (Hoto na 1B) da MYH2 (Hoto na 1C) suna da ƙarin bayanin bayyanar kunnawa/kashewa a matakin RNA idan aka kwatanta da matakin furotin. Hakika, a matakin furotin, ƙananan zaruruwa ba su bayyana MYH7 ba, kuma babu wani zare da ke da bayyanar MYH2 100%. Na gaba mun yi amfani da ƙa'idodin bayyanawa da aka ƙayyade don sanya nau'ikan zaruruwa masu tushen MYH ga dukkan zaruruwa a cikin kowane bayanai. Misali, an sanya zaruruwa masu tushen MYH7+/MYH2-/MYH1- ga nau'in 1, yayin da aka sanya zaruruwa masu tushen MYH7-/MYH2+/MYH1+ ga nau'in 2A/2X gauraye (duba Tebur na Ƙarin 2 don cikakken bayani). Tare da tattara dukkan zaruruwa, mun lura da rarraba nau'ikan zaruruwa masu tushen MYH iri ɗaya a matakan RNA (Hoto na 1E) da furotin (Hoto na 1F), yayin da haɗin nau'ikan zaruruwa masu tushen MYH ya bambanta a tsakanin mutane, kamar yadda aka zata (Hoto na Ƙarin 2A). An rarraba yawancin zaruruwa a matsayin ko dai tsarkakakken nau'in 1 (34-35%) ko nau'in 2A (36-38%), kodayake an gano adadi mai yawa na zaruruwa masu hade iri 2A/2X (16-19%). Wani bambanci mai ban mamaki shine cewa zaruruwan nau'in 2X tsarkakakku za a iya gano su ne kawai a matakin RNA, amma ba a matakin furotin ba, wanda ke nuna cewa saurin bayyanar MYH an tsara shi aƙalla bayan an rubuta shi.
Mun tabbatar da hanyar buga firam ɗin MYH mai tushen proteomics ta amfani da ɗigon ɗigon da aka yi wa rigakafi, kuma duka hanyoyin sun cimma daidaito 100% wajen gano zaruruwan nau'in 1 da nau'in 2A tsarkakakku (duba Ƙarin Hoto na 2B). Duk da haka, hanyar da aka yi wa proteomics ta fi sauƙi, ta fi inganci wajen gano zaruruwan gauraye, da kuma ƙididdige rabon kowace kwayar halittar MYH a cikin kowace zaruruwa. Waɗannan bayanai suna nuna ingancin amfani da hanyar da ta dogara da omics mai ma'ana, mai matuƙar tasiri don kwatanta nau'ikan zaruruwan tsoka.
Sai muka yi amfani da haɗin bayanan da aka bayar ta hanyar transcriptomics da proteomics don rarraba myofibers bisa ga cikakken transcriptome ko proteome ɗinsu. Ta amfani da hanyar uniform manifold approximation and projection (UMAP) don rage girman girma zuwa manyan sassa shida (Ƙarin Figures 3A-B), mun sami damar hango bambancin myofiber a cikin transcriptome (Hoto na 1G) da proteome (Hoto na 1H). Abin lura, mahalarta ba su haɗa myofibers ta hanyar rukuni ba (Ƙarin Figures 3C-D) ko kwanakin gwaji (Ƙarin Figure 3E) a cikin ko dai bayanan transcriptomics ko proteomics, wanda ke nuna cewa bambancin cikin-batun a cikin zaruruwan tsokar kwarangwal ya fi bambancin tsakanin-batun. A cikin shirin UMAP, ƙungiyoyi biyu daban-daban da ke wakiltar myofibers masu sauri da "sannu" sun bayyana (Hoto na 1G-H). An haɗa ƙwayoyin myofiber na MYH7+ (a hankali) a kan sandar UMAP1 mai kyau, yayin da aka haɗa ƙwayoyin myofiber na MYH2+ da MYH1+ (a hankali) a kan sandar UMAP1 mara kyau (Figures 1I-J). Duk da haka, ba a sami wani bambanci tsakanin nau'ikan zare masu sauri (watau nau'in 2A, nau'in 2X, ko gauraye 2A/2X) bisa ga bayyanar MYH ba, yana nuna cewa bayyanar MYH1 (Hoto na 1I-J) ko wasu alamomin myofiber na gargajiya 2X kamar ACTN3 ko MYLK2 (Hoto na Ƙarin 4A-B) ba ta bambanta tsakanin nau'ikan myofiber daban-daban lokacin da ake la'akari da dukkan ƙwayoyin transcriptome ko proteome. Bugu da ƙari, idan aka kwatanta da MYH2 da MYH7, ƙananan rubuce-rubuce ko furotin sun kasance suna da alaƙa mai kyau da MYH1 (Hoto na Ƙarin 4C-H), yana nuna cewa yawan MYH1 bai nuna cikakken bayanin myofiber transcriptome/proteome ba. An cimma irin wannan ƙarshe lokacin da aka tantance gaurayen bayyanar nau'ikan isoforms guda uku na MYH a matakin UMAP (Ƙarin Hotuna 4I–J). Saboda haka, yayin da za a iya gano zaruruwan 2X a matakin rubutun bisa ga ƙididdige MYH kawai, ba a iya bambanta zaruruwan MYH1+ da sauran zaruruwan sauri ba idan aka yi la'akari da dukkan zaruruwan rubutun ko proteome.
A matsayin binciken farko na bambancin zare a hankali fiye da MYH, mun tantance sunadaran guda huɗu da aka kafa a hankali waɗanda ke da takamaiman nau'in zare: TPM3, TNNT1, MYL3, da ATP2A22. Nau'ikan zare a hankali sun nuna alaƙar Pearson mai girma, kodayake ba cikakke ba, da MYH7 a cikin duka transcriptomics (Siffa ta Ƙarin 5A) da proteomics (Siffa ta Ƙarin 5B). Kimanin kashi 25% da 33% na zare a hankali ba a rarraba su a matsayin zare a hankali tsantsa ta dukkan nau'ikan kwayoyin halitta/furotin a cikin transcriptomics ba (Siffa ta Ƙarin 5C) da proteomics (Siffa ta Ƙarin 5D), bi da bi. Saboda haka, rarrabuwar zare a hankali bisa ga nau'ikan kwayoyin halitta/furotin da yawa yana gabatar da ƙarin rikitarwa, har ma ga sunadaran da aka sani da takamaiman nau'in zare. Wannan yana nuna cewa rarrabuwar zare bisa ga isoforms na dangin kwayar halitta/furotin guda ɗaya bazai iya nuna ainihin bambancin zaren tsokar kwarangwal ba.
Domin ƙarin bincike kan bambancin ƙwayoyin tsokar kwarangwal na ɗan adam a sikelin dukkan samfurin omics, mun yi rage girman bayanai ba tare da son kai ba ta amfani da babban nazarin sassan (PCA) (Hoto na 2A). Kamar yadda aka yi a cikin zane-zanen UMAP, babu wanda ya yi nasara ko kuma ranar gwaji da ya yi tasiri ga tarin zare a matakin PCA (Hoto na Ƙarin 6A–C). A cikin bayanan guda biyu, an bayyana nau'in zare mai tushen MYH ta hanyar PC2, wanda ya nuna tarin zare mai saurin juyawa nau'in 1 da rukuni na biyu wanda ya ƙunshi nau'in 2A mai sauri, nau'in 2X, da gaurayen zare mai 2A/2X (Hoto na 2A). A cikin bayanan guda biyu, waɗannan rukunoni guda biyu an haɗa su ta hanyar ƙaramin adadin zare mai gauraye nau'in 1/2A. Kamar yadda aka zata, nazarin wakilcin da ya wuce kima na manyan direbobin PC ya tabbatar da cewa PC2 yana motsawa ta hanyar sa hannun kwantiragi da na rayuwa (Hoto na 2B da Hotuna na Ƙarin 6D–E, Set ɗin Bayanai na Ƙarin 5–6). Gabaɗaya, an gano cewa nau'in fiber mai tushen MYH ya isa ya bayyana ci gaba da bambancin tare da PC2, ban da abin da ake kira zaruruwan 2X waɗanda aka rarraba a cikin transcriptome a cikin rukunin sauri.
A. Babban nazarin sassa (PCA) na bayanan transcriptome da proteome da aka yi wa launi bisa ga nau'in zare bisa ga MYH. B. Binciken haɓaka na direbobin kwafi da furotin a cikin PC2 da PC1. An gudanar da nazarin ƙididdiga ta amfani da fakitin clusterProfiler da ƙimar p-daidaitaccen Benjamini-Hochberg. C, D. Shirye-shiryen PCA masu launi bisa ga sharuɗɗan haɗin gwiwar kwayoyin halitta (GO) a cikin sharuɗɗan transcriptome da costamere GO a cikin proteome. Kibiyoyi suna wakiltar direbobin kwafi da furotin da alkiblarsu. E, F. Tsarin kwatancen da tsinkayar iri ɗaya (UMAP) na siffofi masu dacewa a asibiti waɗanda ke nuna gradients na bayyanawa ba tare da la'akari da nau'in zare mai jinkiri/sauri ba. G, H. Alaƙa tsakanin direbobin PC2 da PC1 a cikin takaddun kwafi da furotin.
Ba zato ba tsammani, nau'in myofiber mai tushen MYH ya bayyana mataki na biyu mafi girma na bambancin (PC2), yana nuna cewa wasu abubuwan halitta da ba su da alaƙa da nau'in myofiber mai tushen MYH (PC1) suna taka muhimmiyar rawa wajen daidaita bambancin fiber na tsokar ƙashi. Binciken da aka yi kan manyan direbobi a cikin PC1 ya nuna cewa bambancin a cikin PC1 an ƙaddara shi ne ta hanyar mannewar ƙwayoyin halitta da abubuwan ribosome a cikin transcriptome, da kuma sunadaran costameres da ribosomal a cikin proteome (Hoto na 2B da Hotunan Ƙarin 6D–E, Saitin Bayanan Ƙarin 7). A cikin tsokar ƙashi, costameres suna haɗa faifan Z zuwa sarcolemma kuma suna da hannu a cikin watsawa da siginar ƙarfi. 25 Shirye-shiryen PCA da aka yi bayani ta amfani da mannewar ƙwayoyin halitta (transcriptome, Hoto na 2C) da costamere (proteome, Hoto na 2D) sun bayyana ƙarfin canjin hagu a cikin PC1, yana nuna cewa waɗannan fasalulluka suna da wadata a cikin wasu zaruruwa.
Binciken da aka yi dalla-dalla game da tarin myofiber a matakin UMAP ya nuna cewa yawancin fasaloli sun nuna bambancin bayyanar MYH mai zaman kansa maimakon takamaiman myofiber subcluster. An lura da wannan ci gaba ga wasu kwayoyin halitta da ke da alaƙa da yanayin cututtuka (Hoto na 2E), kamar CHCHD10 (cutar neuromuscular), SLIT3 (ƙarancin tsoka), CTDNEP1 (cutar tsoka). An kuma lura da wannan ci gaba a cikin furotin, gami da sunadaran da ke da alaƙa da cututtukan jijiyoyi (UGDH), siginar insulin (PHIP), da kuma rubutun bayanai (HIST1H2AB) (Hoto na 2F). Gabaɗaya, waɗannan bayanai suna nuna ci gaba a cikin bambancin saurin juyawa mai zaman kansa na nau'in fiber a cikin myofibers daban-daban.
Abin sha'awa, kwayoyin halittar da ke cikin PC2 sun nuna kyakkyawar alaƙar transcriptome-proteome (r = 0.663) (Hoto na 2G), wanda ke nuna cewa nau'ikan zare masu juyawa a hankali da sauri, musamman ma waɗanda ke da alaƙa da ƙashi da kuma abubuwan da ke cikin ƙwayoyin tsoka, ana sarrafa su ta hanyar rubutu. Duk da haka, kwayoyin halittar da ke cikin PC1 ba su nuna alaƙar transcriptome-proteome ba (r = -0.027) (Hoto na 2H), yana nuna cewa bambance-bambancen da ba su da alaƙa da nau'ikan zare masu juyawa a hankali/sauri ana sarrafa su ta hanyar rubutu bayan rubutu. Saboda bambance-bambancen da ke cikin PC1 an yi bayanin su ne ta hanyar kalmomin ribosomal gene ontology, kuma idan aka yi la'akari da cewa ribosomes suna taka muhimmiyar rawa a cikin tantanin halitta ta hanyar shiga cikin da kuma rinjayar fassarar furotin, 31 za mu fara bincike kan wannan bambancin ribosomal da ba a zata ba.
Da farko mun yi wa tsarin nazarin manyan sinadaran proteomics fenti bisa ga yawan sunadaran da ke cikin kalmar GOCC "cytoplasmic ribosome" (Hoto na 3A). Duk da cewa wannan kalma ta wadatar a gefen PC1 mai kyau, wanda ya haifar da ƙaramin gradient, sunadaran ribosomal suna haifar da rabuwa a bangarorin biyu na PC1 (Hoto na 3A). Sunadaran ribosomal da aka wadatar a gefen mara kyau na PC1 sun haɗa da RPL18, RPS18, da RPS13 (Hoto na 3B), yayin da RPL31, RPL35, da RPL38 (Hoto na 3C) su ne manyan abubuwan da ke haifar da PC1 mai kyau. Abin sha'awa, RPL38 da RPS13 sun bayyana sosai a cikin tsokar kwarangwal idan aka kwatanta da sauran kyallen takarda (Hoto na Ƙarin 7A). Waɗannan sa hannun ribosomal masu ban mamaki a cikin PC1 ba a lura da su a cikin rubutun ba (Hoto na Ƙarin 7B), yana nuna ƙa'idar bayan rubutun.
A. Babban binciken sassan jiki (PCA) wanda aka yi wa launi bisa ga kalmomin cytoplasmic ribosomal gene ontology (GO) a fadin proteome. Kibiyoyi suna nuna alkiblar bambancin da furotin ke shiga tsakani a cikin taswirar PCA. Tsawon layi ya yi daidai da babban maki na sinadaran da aka bayar ga wani furotin da aka bayar. B, C. Tsarin fasalin PCA don RPS13 da RPL38. D. Binciken tarin sunadarai na cytoplasmic ribosomal marasa kulawa. E. Tsarin tsarin ribosome na 80S (PDB: 4V6X) yana nuna sunadaran ribosomal tare da yalwar yawa a cikin zaruruwan tsoka. F. Sunadaran ribosomal tare da stoichiometry daban-daban da aka gano kusa da tashar fita ta mRNA.
An gabatar da ra'ayoyin bambancin ribosomal da ƙwarewa a baya, inda kasancewar ƙananan ribosome (nau'in ribosomal) na iya yin tasiri kai tsaye ga fassarar furotin a cikin kyallen takarda daban-daban32 da ƙwayoyin halitta33 ta hanyar zaɓin fassarar takamaiman mRNA transcript pools34 (ƙwararriyar ribosome). Don gano ƙananan sunadaran ribosomal waɗanda aka bayyana tare a cikin zaruruwan tsoka, mun yi nazarin tarin sunadaran ribosomal marasa kulawa a cikin proteome (Hoto na 3D, Saitin Bayanan Ƙarin 8). Kamar yadda aka zata, sunadaran ribosomal ba su taru ta hanyar nau'in fiber ba bisa ga MYH. Duk da haka, mun gano ƙungiyoyi uku daban-daban na sunadaran ribosomal; rukuni na farko (ribosomal_cluster_1) an daidaita shi tare da RPL38 kuma saboda haka yana da ƙaruwar magana a cikin zaruruwa tare da bayanin PC1 mai kyau. Rukunin na biyu (ribosomal_cluster_2) an daidaita shi tare da RPS13 kuma an ɗaukaka shi a cikin zaruruwa tare da bayanin PC1 mara kyau. Rukunin ribosomal na uku (ribosomal_cluster_3) ba ya nuna bambancin ra'ayi a cikin zaruruwan tsokar kwarangwal kuma ana iya ɗaukarsa a matsayin furotin ribosomal na tsokar kwarangwal "ainihin". Duk rukunin ribosomal guda 1 da 2 suna ɗauke da sunadaran ribosomal waɗanda aka nuna a baya suna daidaita fassarar madadin (misali, RPL10A, RPL38, RPS19, da RPS25) kuma suna tasiri ga ci gaba (misali, RPL10A, RPL38).34,35,36,37,38 Daidai da sakamakon PCA, wakilcin da aka lura na waɗannan sunadaran ribosomal a cikin zaruruwa suma sun nuna ci gaba (Hoto na Ƙarin 7C).
Domin ganin inda furotin ribosomal iri-iri ke cikin ribosome, mun yi amfani da tsarin tsarin ribosome na ɗan adam 80S (Bankin Bayanan Protein: 4V6X) (Hoto na 3E). Bayan ware furotin ribosomal na rukunin ribosomal daban-daban, wuraren da suke ba su daidaita sosai ba, wanda ke nuna cewa hanyarmu ta gaza samar da wadata ga wasu yankuna/gudaje na ribosome. Abin sha'awa, duk da haka, rabon manyan furotin na subunit a cikin rukuni na 2 ya yi ƙasa da na rukuni na 1 da 3 (Hoto na Ƙarin 7D). Mun lura cewa sunadarai masu canza stoichiometry a cikin zaruruwan tsoka an fi mayar da su zuwa saman ribosome (Hoto na 3E), daidai da ikonsu na hulɗa da abubuwan shiga ribosome na ciki (IRES) a cikin al'ummomin mRNA daban-daban, don haka suna daidaita fassarar zaɓi. 40, 41 Bugu da ƙari, yawancin furotin tare da canza stoichiometry a cikin zaruruwan tsoka suna kusa da yankunan aiki kamar ramin fita na mRNA (Hoto na 3F), wanda ke tsara tsawaita fassara da kama takamaiman peptides. 42 A taƙaice, bayananmu sun nuna cewa stoichiometry na furotin ribosomal na tsokar ƙashi yana nuna bambancin ra'ayi, wanda ke haifar da bambance-bambance tsakanin zaruruwan tsokar ƙashi.
Na gaba, mun fara gano sa hannun zare mai sauri da jinkiri da kuma bincika hanyoyin da ake bi wajen daidaita rubutunsu. Idan muka kwatanta tarin zare mai sauri da jinkiri da UMAP ta ayyana a cikin bayanan guda biyu (Figures 1G-H da 4A-B), nazarin zare mai sauri da kuma proteomic ya gano siffofi daban-daban na 1366 da 804, bi da bi (Figures 4A-B, Ƙarin Bayanan 9-12). Mun lura da bambance-bambancen da ake tsammani a cikin sa hannu da suka shafi sarcomeres (misali, tropomyosin da troponin), haɗin haɗakar motsawa da damuwa (SERCA isoforms), da metabolism na makamashi (misali, ALDOA da CKB). Bugu da ƙari, zare mai sauri da kuma furotin da ke daidaita ubiquitination na furotin an bayyana su daban-daban a cikin zare mai sauri da jinkiri (misali, USP54, SH3RF2, USP28, da USP48) (Figures 4A-B). Bugu da ƙari, kwayar halittar furotin mai ƙwayoyin cuta RP11-451G4.2 (DWORF), wacce a baya aka nuna tana bayyana ta daban-daban a cikin nau'ikan zare na tsoka na rago43 kuma tana haɓaka aikin SERCA a cikin tsokar zuciya44, an inganta ta sosai a cikin zare na tsokar kwarangwal mai jinkirin aiki (Hoto na 4A). Hakazalika, a matakin zare na mutum ɗaya, an lura da bambance-bambance masu mahimmanci a cikin sa hannu da aka sani kamar isoforms na lactate dehydrogenase masu alaƙa da metabolism (LDHA da LDHB, Hoto na 4C da Hoto na Ƙarin 8A)45,46 da kuma sa hannu na musamman na nau'in zare da ba a san su ba a baya (kamar IRX3, USP54, USP28, da DPYSL3) (Hoto na 4C). Akwai babban haɗuwa na fasalulluka da aka bayyana daban-daban tsakanin bayanan transcriptomic da proteomic (Hoto na Ƙarin 8B), da kuma alaƙar canjin ninkawa wanda galibi ke haifar da bayyanar bambancin fasalin sarcomere (Hoto na Ƙarin 8C). Abin lura shi ne, wasu sa hannu (misali USP28, USP48, GOLGA4, AKAP13) sun nuna ƙaƙƙarfan ƙa'idar bayan an rubuta su ne kawai a matakin proteomic kuma suna da bayanin martaba na musamman na nau'in zare mai saurin juyawa (Karin Hoto na 8C).
Tsarin dutsen mai aman wuta na A da B yana kwatanta gungu masu jinkiri da sauri waɗanda aka gano ta hanyar makircin kwatancen da tsinkayar (UMAP) iri ɗaya a cikin Hotuna 1G-H. Digogi masu launi suna wakiltar rubuce-rubuce ko sunadarai waɗanda suka bambanta sosai a FDR < 0.05, kuma digogi masu duhu suna wakiltar rubuce-rubuce ko sunadarai waɗanda suka bambanta sosai a canjin log > 1. An gudanar da nazarin ƙididdiga ta hanyoyi biyu ta amfani da gwajin DESeq2 Wald tare da ƙimar p da aka daidaita ta Benjamini-Hochberg (transscriptomics) ko hanyar samfurin layi na Limma tare da nazarin Bayesian na empirical sannan aka biyo baya da daidaitawar Benjamini-Hochberg don kwatantawa da yawa (proteomics). C Tsarin sa hannu na kwayoyin halitta ko sunadarai da aka zaɓa waɗanda aka bayyana daban-daban tsakanin zaruruwa masu jinkiri da sauri. D Binciken haɓaka bayanai da sunadarai da aka bayyana daban-daban. Ana wadatar da ƙimar da ke haɗuwa a cikin bayanan guda biyu, ƙimar transcriptome ana wadatar da ita ne kawai a cikin rubutun, kuma ƙimar proteome ana wadatar da ita ne kawai a cikin proteome. An gudanar da nazarin ƙididdiga ta amfani da fakitin clusterProfiler tare da ƙimar p-da aka daidaita ta Benjamini-Hochberg. E. Abubuwan da aka gano na musamman na nau'in zare ta hanyar SCENIC bisa ga ma'aunin takamaiman mai kula da SCENIC da kuma bambancin bayyanar mRNA tsakanin nau'ikan zare. F. Bayyana abubuwan da aka zaɓa na zare da aka bayyana daban-daban tsakanin zare mai jinkiri da sauri.
Daga nan muka yi nazarin yawan wakilcin kwayoyin halitta da sunadaran da aka wakilta daban-daban (Hoto na 4D, Ƙarin Bayanan Saiti na 13). Inganta hanyoyin da suka bambanta tsakanin bayanan guda biyu ya bayyana bambance-bambancen da ake tsammani, kamar fatty acid β-oxidation da ketone metabolism processes (slow zaruruwa), myofilament/muscle contract (sauri da jinkirin zaruruwa, bi da bi), da kuma carbohydrate catabolic processes (fari zaruruwa). An kuma ƙara yawan aikin furotin Serine/threonine phosphatase a cikin zaruruwa masu sauri, waɗanda siffofi kamar subunits na regulatory da catalytic phosphatase (PPP3CB, PPP1R3D, da PPP1R3A) suka haifar, waɗanda aka san suna daidaita metabolism na glycogen (47) (Ƙarin Figures 8D–E). Sauran hanyoyin da aka wadatar da zare masu sauri sun haɗa da tsarin sarrafawa (P-) jikuna (YTHDF3, TRIM21, LSM2) a cikin proteome (Ƙarin Hoto na 8F), mai yuwuwar shiga cikin tsarin bayan-transcription (48), da kuma aikin factor transcription (SREBF1, RXRG, RORA) a cikin transcriptome (Ƙarin Hoto na 8G). An wadatar da zare masu jinkiri a cikin aikin oxidoreductase (BDH1, DCXR, TXN2) (Ƙarin Hoto na 8H), ɗaure amide (CPTP, PFDN2, CRYAB) (Ƙarin Hoto na 8I), matrix na extracellular (CTSD, ADAMTSL4, LAMC1) (Ƙarin Hoto na 8J), da kuma aikin receptor-ligand (FNDC5, SPX, NENF) (Ƙarin Hoto na 8K).
Domin samun ƙarin fahimta game da ƙa'idar rubutun da ke ƙarƙashin halayen nau'in zare mai sauri/sauri, mun yi nazarin haɓaka abubuwan rubutu ta amfani da SCENIC49 (Karin Bayanan Saiti 14). Yawancin abubuwan rubutu sun kasance masu ƙarfi sosai tsakanin zare mai sauri da jinkirin tsoka (Hoto na 4E). Wannan ya haɗa da abubuwan rubutu kamar MAFA, wanda a baya aka danganta shi da haɓakar zare mai sauri na tsoka,50 da kuma wasu abubuwan rubutu da ba a taɓa alaƙa da su ba a baya da shirye-shiryen kwayoyin halitta na musamman na zare mai tsoka. Daga cikin waɗannan, PITX1, EGR1, da MYF6 sune abubuwan rubutu mafi wadata a cikin zare mai sauri (Hoto na 4E). Sabanin haka, ZSCAN30 da EPAS1 (wanda aka fi sani da HIF2A) sune abubuwan rubutu mafi wadata a cikin zare mai sauri na tsoka (Hoto na 4E). Daidai da wannan, an bayyana MAFA a matakai mafi girma a yankin UMAP wanda ya dace da zare mai sauri na tsoka, yayin da EPAS1 yana da tsarin magana akasin haka (Hoto na 4F).
Baya ga sanannun kwayoyin halittar da ke samar da sunadaran gina jiki, akwai nau'ikan halittun RNA marasa tsari da yawa waɗanda za su iya shiga cikin daidaita ci gaban ɗan adam da cututtuka. 51, 52 A cikin bayanan da aka tattara, RNA da yawa marasa tsari da ke nuna takamaiman nau'in fiber (Hoto na 5A da Ƙarin Bayanan 15), gami da LINC01405, wanda yake da takamaiman musamman ga zaruruwa masu jinkiri kuma an ruwaito cewa yana raguwa a cikin tsoka daga marasa lafiya da ke da cutar mitochondrial myopathy. 53 Sabanin haka, RP11-255P5.3, wanda ya dace da kwayar halittar lnc-ERCC5-5 (https://lncipedia.org/db/transcript/lnc-ERCC5-5:2) 54, yana nuna takamaiman nau'in fiber mai sauri. Dukansu LINC01405 (https://tinyurl.com/x5k9wj3h) da RP11-255P5.3 (https://tinyurl.com/29jmzder) suna nuna takamaiman tsokar kwarangwal (Ƙarin Figures 9A–B) kuma ba su da wani sanannen kwayoyin halitta masu ƙunƙu ...
A. An tsara takardun RNA marasa lambar sirri sosai a cikin zaruruwan tsoka masu juyawa a hankali da sauri. B. Hotunan RNAscope masu wakiltar suna nuna takamaiman nau'in zare mai juyawa a hankali da sauri na LINC01405 da RP11-255P5.3, bi da bi. Ma'aunin sikelin = 50 μm. C. Ƙididdige bayyanar RNA mara lambar sirri ta nau'in myofiber kamar yadda RNAscope ta ƙaddara (n = biopsies 3 daga mutane masu zaman kansu, suna kwatanta zaruruwan tsoka masu sauri da jinkirin a cikin kowane mutum). An gudanar da nazarin ƙididdiga ta amfani da gwajin t-test na ɗalibi mai wutsiya biyu. Zane-zanen akwati suna nuna matsakaicin da kwata na farko da na uku, tare da gashin baki yana nuna mafi ƙarancin da matsakaicin ƙima. D. Tsarin aikin gano furotin na ƙwayoyin cuta na De novo (wanda aka ƙirƙira tare da BioRender.com). E. An bayyana furotin mai ƙwayoyin cuta LINC01405_ORF408:17441:17358 musamman a cikin zaruruwan tsoka masu kwarangwal a hankali (n=biopsi 5 daga mahalarta masu zaman kansu, suna kwatanta zaruruwan tsoka masu sauri da jinkirin a cikin kowane mahalarta). An gudanar da nazarin ƙididdiga ta amfani da hanyar samfurin layi na Limm tare da hanyar Bayesian ta gwaji, sannan aka bi hanyar Benjamini-Hochberg don kwatantawa da yawa tare da daidaita ƙimar p. Zane-zanen akwati suna nuna matsakaicin, na farko da na uku, tare da gashin baki yana nuna matsakaicin/mafi ƙarancin ƙima.
Kwanan nan, bincike ya nuna cewa rubuce-rubuce da yawa na rashin lambar sirri suna ɓoye sunadaran ƙwayoyin cuta da aka rubuta, waɗanda wasu daga cikinsu ke daidaita aikin tsoka. 44, 55 Domin gano sunadaran ƙwayoyin cuta masu yuwuwar takamaiman nau'in zare, mun bincika bayanan mu na fiber proteome 1000 ta amfani da fayil ɗin FASTA na musamman wanda ke ɗauke da jerin bayanan da ba na lambar sirri ba (n = 305) da aka samo a cikin bayanan fiber transcriptome 1000 (Hoto na 5D). Mun gano sunadaran ƙwayoyin cuta 197 daga cikin bayanan 22 daban-daban, 71 daga cikinsu an daidaita su daban-daban tsakanin zaren tsoka mai jinkirin da sauri (Hoto na Ƙarin 9C da Saitin Bayanan Ƙarin 16). Ga LINC01405, an gano samfuran sunadaran ƙwayoyin cuta guda uku, ɗaya daga cikinsu ya nuna irin wannan takamaiman zaren a hankali ga rubutunsa (Hoto na 5E da Hoto na Ƙarin 9D). Don haka, mun gano LINC01405 a matsayin kwayar halitta da ke ɓoye sunadaran ƙwayoyin cuta waɗanda ke takamaiman ƙwayoyin cuta don zaren tsoka mai jinkirin.
Mun ƙirƙiri cikakken tsarin aiki don babban fasalin furotin na zaruruwan tsoka daban-daban da kuma gano masu kula da bambancin zaruruwan fiber a cikin yanayi masu lafiya. Mun yi amfani da wannan tsarin aiki don fahimtar yadda nemaline myopathies ke shafar bambancin zaruruwan tsoka. Nemaline myopathies cututtukan tsoka ne da aka gada waɗanda ke haifar da raunin tsoka kuma, a cikin yara da abin ya shafa, suna da matsaloli iri-iri ciki har da wahalar numfashi, scoliosis, da ƙarancin motsi na gaɓoɓi. 19,20 Yawanci, a cikin nemaline myopathies, bambance-bambancen cututtuka a cikin kwayoyin halitta kamar actin alpha 1 (ACTA1) suna haifar da rinjayen abun da ke cikin myofiber na fiber a hankali, kodayake wannan tasirin yana da bambanci. Wani abin da aka fi sani shine troponin T1 nemaline myopathy (TNNT1), wanda ke da rinjayen zaruruwan sauri. Don haka, fahimtar bambancin da ke ƙarƙashin rashin daidaituwar zaruruwan tsoka da aka lura a cikin nemaline myopathies na iya taimakawa wajen warware alaƙar da ke tsakanin waɗannan cututtuka da nau'in myofiber.
Idan aka kwatanta da ingantattun hanyoyin sarrafawa (n=3 ga kowace ƙungiya), ƙwayoyin myofiber da aka ware daga marasa lafiya na nemaline myopathy waɗanda ke da maye gurbi a cikin kwayoyin halittar ACTA1 da TNNT1 sun nuna ƙarancin myofiber ko dystrophy mai yawa (Hoto na 6A, Tebur na Ƙarin 3). Wannan ya gabatar da ƙalubalen fasaha masu mahimmanci don nazarin proteomic saboda ƙarancin adadin kayan da ake da su. Duk da haka, mun sami damar gano sunadarai 2485 a cikin ƙwayoyin myofiber 272 na kwarangwal. Bayan tace aƙalla sunadarai 1000 da aka ƙididdige a kowace zare, an yi wa zaruruwa 250 nazarin bioinformatics na gaba. Bayan tacewa, an ƙididdige matsakaicin furotin 1573 ± 359 ga kowace zare (Hoto na Ƙarin 10A, Ƙarin Bayanan Saiti 17–18). Abin lura, duk da raguwar girman zare, zurfin proteome na samfuran marasa lafiya na nemaline myopathy ya ragu kaɗan. Bugu da ƙari, sarrafa waɗannan bayanai ta amfani da fayilolin FASTA ɗinmu (gami da bayanan da ba na coding ba) ya ba mu damar gano furotin guda biyar na ƙwayoyin cuta a cikin ƙwayoyin cuta na myofibers daga marasa lafiya na nemaline myopathy (Karin Bayanan Saiti 19). Tsarin aiki na proteome ya fi faɗi sosai, kuma jimillar furotin a cikin rukunin kulawa ya yi daidai da sakamakon binciken proteome na fiber 1000 da ya gabata (Karin Hoto na 10B–C).
A. Hotunan microscopic da ke nuna atrophy ko dystrophy na fiber da kuma rinjayen nau'ikan zare daban-daban bisa ga MYH a cikin ACTA1 da TNNT1 nemaline myopathies (NM). Ma'aunin sikelin = 100 μm. Don tabbatar da sake haifar da tabo a cikin marasa lafiya na ACTA1 da TNNT1, an yi wa marasa lafiya uku fenti sau biyu zuwa uku (sassan huɗu a kowane hali) kafin a zaɓi hotuna masu wakilci. B. Rarraba nau'in zare a cikin mahalarta bisa ga MYH. C. Babban nazarin sassan (PCA) na zare na tsokar kwarangwal a cikin marasa lafiya da ciwon nemaline myopathies da sarrafawa. D. Zare na tsokar kwarangwal daga marasa lafiya da ciwon nemaline myopathies da sarrafawa da aka zana akan taswirar PCA da aka ƙaddara daga zare 1000 da aka bincika a Hoto na 2. Misali, zare na dutsen mai aman wuta yana kwatanta bambance-bambance tsakanin mahalarta da ciwon nemaline myopathies da sarrafawa na ACTA1 da TNNT1, da kuma tsakanin mahalarta da ciwon nemaline myopathies na ACTA1 da TNNT1. Da'irori masu launi suna nuna sunadaran da suka bambanta sosai a π < 0.05, kuma dige-dige masu duhu suna nuna sunadaran da suka bambanta sosai a FDR < 0.05. An gudanar da nazarin ƙididdiga ta amfani da hanyar samfurin layi na Limma da hanyoyin Bayesian na gwaji, sannan aka gyara darajar p don kwatantawa da yawa ta amfani da hanyar Benjamini-Hochberg. H. Binciken haɓaka sunadaran da aka bayyana sosai a cikin dukkan furotin da kuma a cikin zaruruwan nau'in 1 da 2A. An gudanar da nazarin ƙididdiga ta amfani da fakitin clusterProfiler da ƙimar p da aka daidaita na Benjamini-Hochberg. I, J. Babban nazarin sassan (PCA) zane-zane masu launi ta hanyar matrix na extracellular da kalmomin mitochondrial gene ontology (GO).
Domin ƙwayoyin cuta na nemaline na iya yin tasiri ga yawan nau'ikan ƙwayoyin cuta na MYH da ke bayyana a cikin tsokar ƙashi, 19,20 mun fara bincika nau'ikan ƙwayoyin cuta na MYH da ke bayyana a cikin marasa lafiya da ke da ƙwayoyin cuta na nemaline da kuma sarrafawa. Mun tantance nau'in ƙwayoyin cuta na myofiber ta amfani da wata hanya mara son kai da aka bayyana a baya don gwajin myofiber 1000 (Ƙarin Hotuna 10D–E) kuma mun sake kasa gano ƙwayoyin cuta na 2X masu tsabta (Hoto na 6B). Mun lura da tasirin ƙwayoyin cuta na nemaline akan nau'in ƙwayoyin cuta na myofiber, kamar yadda marasa lafiya biyu da ke da maye gurbin ACTA1 suka sami ƙaruwar yawan ƙwayoyin cuta na nau'in 1, yayin da marasa lafiya biyu da ke da cutar nemaline ta TNNT1 suka sami raguwar yawan ƙwayoyin cuta na nau'in 1 (Hoto na 6B). Hakika, bayyanar MYH2 da isoforms masu sauri na troponin (TNNC2, TNNI2, da TNNT3) ya ragu a cikin myopathies na ACTA1-nemaline, yayin da aka rage bayyanar MYH7 a cikin myopathies na TNNT1-nemaline (Hoto na Ƙarin 11A). Wannan ya yi daidai da rahotannin da suka gabata na canjin nau'in myofiber daban-daban a cikin myopathies na nemaline.19,20 Mun tabbatar da waɗannan sakamakon ta hanyar immunohistochemistry kuma mun gano cewa marasa lafiya da ke da cutar ACTA1-nemaline myopathy suna da rinjaye na myofibers na nau'in 1, yayin da marasa lafiya da ke da cutar TNNT1-nemaline myopathy suna da akasin haka (Hoto na 6A).
A matakin furotin guda ɗaya, ƙwayoyin tsoka na ƙashi daga marasa lafiya na ACTA1 da TNNT1 nemaline myopathy sun haɗu da yawancin zaruruwan sarrafawa, inda zaruruwan nemaline myopathy na TNNT1 galibi sune suka fi shafa (Hoto na 6C). Wannan ya bayyana musamman lokacin da aka tsara manyan nazarin sassan (PCA) na zaruruwan da aka yi wa fenti ga kowane majiyyaci, tare da marasa lafiya na TNNT1 nemaline myopathy 2 da 3 da suka bayyana mafi nisa daga samfuran sarrafawa (Hoto na Ƙarin 11B, Saitin Bayanan Ƙarin 20). Don fahimtar yadda zaruruwan daga marasa lafiya na myopathy suka kwatanta da zaruruwan lafiya, mun yi amfani da cikakkun bayanai da aka samu daga nazarin proteomic na zaruruwa 1,000 daga mahalarta manya masu lafiya. Mun yi hasashen zaruruwa daga bayanan myopathy (ACTA1 da TNNT1 nemaline myopathy marasa lafiya da sarrafawa) akan taswirar PCA da aka samu daga nazarin proteomic na zaruruwa 1000 (Hoto na 6D). Rarraba nau'ikan fiber na MYH tare da PC2 a cikin zaruruwan sarrafawa yayi kama da rarrabawar fiber da aka samu daga nazarin furotin na fiber 1000. Duk da haka, yawancin zaruruwa a cikin marasa lafiya na nemaline myopathy sun koma ƙasa da PC2, suna haɗuwa da zaruruwa masu lafiya masu saurin juyawa, ba tare da la'akari da nau'in fiber na MYH na asali ba. Don haka, kodayake marasa lafiya da ke fama da cutar myopathy ta ACTA1 nemaline sun nuna canji zuwa zaruruwa na nau'in 1 lokacin da aka auna su ta amfani da hanyoyin da aka dogara da MYH, duka ACTA1 nemaline myopathy da TNNT1 nemaline myopathy sun canza furotine na tsokar kwarangwal zuwa zaruruwa masu saurin juyawa.
Daga nan muka kwatanta kowace ƙungiyar marasa lafiya kai tsaye da ingantattun hanyoyin kula da lafiya kuma muka gano sunadaran da aka bayyana daban-daban guda 256 da 552 a cikin ƙwayoyin cuta na nemaline na ACTA1 da TNNT1, bi da bi (Hoto na 6E-G da Hoto na Ƙarin 11C, Saitin Bayanan Ƙarin 21). Binciken wadatar kwayoyin halitta ya nuna raguwar haɗin kai a cikin sunadaran mitochondrial (Hoto na 6H-I, Saitin Bayanan Ƙarin 22). Abin mamaki, duk da bambancin rinjayen nau'ikan fiber a cikin ƙwayoyin cuta na nemaline na ACTA1 da TNNT1, wannan raguwar ta kasance ba ta da alaƙa da nau'in fiber da aka samo daga MYH (Hoto na 6H da Hotuna na Ƙarin 11D-I, Saitin Bayanan Ƙarin 23). An kuma tsara sunadaran ƙwayoyin cuta guda uku a cikin ƙwayoyin cuta na nemaline na ACTA1 ko TNNT1. Biyu daga cikin waɗannan ƙananan furotin, ENSG00000215483_TR14_ORF67 (wanda kuma aka sani da LINC00598 ko Lnc-FOXO1) da ENSG00000229425_TR25_ORF40 (lnc-NRIP1-2), sun nuna bambancin yawa ne kawai a cikin nau'in myofibers na nau'in 1. An ruwaito cewa ENSG00000215483_TR14_ORF67 suna taka rawa a cikin daidaita zagayowar ƙwayoyin halitta. 56 A gefe guda kuma, an ƙara ENSG00000232046_TR1_ORF437 (wanda ya yi daidai da LINC01798) a cikin nau'in myofibers na nau'in 1 da na nau'in 2A a cikin ACTA1-nemaline myopathy idan aka kwatanta da sarrafawa masu lafiya (Hoto na Ƙarin 12A, Saitin Bayanan Ƙarin 24). Sabanin haka, sunadaran ribosomal ba su da wani tasiri a kan myopathy na nemaline, kodayake an rage RPS17 a cikin ACTA1 nemaline myopathy (Hoto na 6E).
Binciken ƙarin ƙarfi ya kuma nuna ƙaruwar tsarin garkuwar jiki a cikin ƙwayoyin cuta na ACTA1 da TNNT1 nemaline, yayin da aka ƙara mannewar ƙwayoyin halitta a cikin ƙwayoyin cuta na TNNT1 nemaline (Hoto na 6H). An nuna wadatar waɗannan abubuwan da ke cikin ƙwayoyin halitta ta hanyar sunadaran matrix na extracellular da ke canza PCA a cikin PC1 da PC2 a cikin mummunan alkibla (watau, zuwa ga mafi yawan zaruruwan da abin ya shafa) (Hoto na 6J). Duk ƙungiyoyin marasa lafiya sun nuna ƙaruwar bayyanar sunadaran extracellular da ke da hannu a cikin martanin garkuwar jiki da hanyoyin gyara sarcolemmal, kamar annexins (ANXA1, ANXA2, ANXA5)57,58 da furotin ɗinsu mai hulɗa da juna S100A1159 (Karin Figures 12B–C). An riga an ruwaito cewa wannan tsari yana inganta a cikin ƙwayoyin cuta na tsoka60 amma, a saninmu, ba a taɓa danganta shi da ƙwayoyin cuta na nemaline ba a da. Ana buƙatar aikin yau da kullun na wannan injina na kwayoyin halitta don gyaran sarcolemmal bayan rauni da kuma haɗa sabbin ƙwayoyin cuta na myocytes da myofibers58,61. Saboda haka, ƙaruwar ayyukan wannan tsari a cikin ƙungiyoyin marasa lafiya guda biyu yana nuna amsawar ramawa ga raunin da rashin kwanciyar hankali na myofiber ya haifar.
Tasirin kowace cutar nemaline myopathy yana da alaƙa mai kyau (r = 0.736) kuma ya nuna haɗuwa mai ma'ana (Ƙarin Figures 11A–B), yana nuna cewa cutar nemaline ta ACTA1 da TNNT1 suna da irin wannan tasiri akan proteome. Duk da haka, an tsara wasu sunadarai ne kawai a cikin ACTA1 ko TNNT1 nemaline myopathy (Ƙarin Figures 11A da C). Protein MFAP4 na profibrotic yana ɗaya daga cikin furotin da aka fi daidaita a cikin cutar nemaline ta TNNT1 amma bai canza ba a cikin cutar nemaline ta ACTA1. SKIC8, wani ɓangare na hadaddun PAF1C wanda ke da alhakin daidaita kwafi na kwayar halittar HOX, an rage shi a cikin cutar nemaline ta TNNT1 amma ba a shafi cutar nemaline ta ACTA1 ba (Ƙarin Figure 11A). Kwatanta kai tsaye na ACTA1 da TNNT1 nemaline myopathy ya nuna raguwar sunadarai masu yawa a cikin mitochondrial da ƙaruwa a cikin furotin masu garkuwar jiki a cikin TNNT1 nemaline myopathy (Hoto na 6G-H da Hotunan Ƙarin 11C da 11H-I). Waɗannan bayanan sun yi daidai da babban atrophy/dystrophy da aka lura a cikin TNNT1 nemaline myopathy idan aka kwatanta da TNNT1 nemaline myopathy (Hoto na 6A), yana nuna cewa TNNT1 nemaline myopathy yana wakiltar nau'in cutar mafi tsanani.
Domin tantance ko tasirin da aka lura da shi na nemaline myopathy ya ci gaba da kasancewa a matakin tsoka gaba ɗaya, mun yi babban bincike na proteomic na biopsies na tsoka daga rukuni ɗaya na marasa lafiya na TNNT1 nemaline myopathy kuma muka kwatanta su da sarrafawa (n=3 kowace ƙungiya) (Ƙarin Hoto na 13A, Ƙarin Bayanan Saiti na 25). Kamar yadda aka zata, sarrafawa suna da alaƙa sosai a cikin babban binciken sassan, yayin da marasa lafiya na TNNT1 nemaline myopathy sun nuna babban bambancin samfuri iri ɗaya kamar wanda aka gani a cikin nazarin zare ɗaya (Ƙarin Hoto na 13B). Binciken girma ya sake haifar da furotin da aka bayyana daban-daban (Ƙarin Hoto na 13C, Ƙarin Bayanan Saiti na 26) da hanyoyin halittu (Ƙarin Hoto na 13D, Ƙarin Bayanan Saiti na 27) wanda aka nuna ta hanyar kwatanta zare ɗaya, amma sun rasa ikon bambance tsakanin nau'ikan zare daban-daban kuma sun kasa yin lissafin tasirin cututtuka daban-daban a cikin zare.
Idan aka haɗa waɗannan bayanai, waɗannan bayanai sun nuna cewa proteomics na myofiber guda ɗaya na iya bayyana fasalulluka na asibiti waɗanda ba za a iya gano su ta hanyoyin da aka yi niyya kamar immunoblotting ba. Bugu da ƙari, waɗannan bayanan suna nuna iyakokin amfani da nau'in fiber na actin (MYH) kaɗai don bayyana daidaitawar phenotypic. Hakika, kodayake sauyawar nau'in fiber ya bambanta tsakanin actin da troponin nemaline myopathies, duka nemaline myopathies suna raba nau'in fiber na MYH daga metabolism na ƙwayar tsoka zuwa furotin tsoka mai sauri da ƙarancin oxidative.
Bambancin ƙwayoyin halitta yana da matuƙar muhimmanci ga kyallen takarda don biyan buƙatunsu daban-daban. A cikin tsokar ƙashi, ana bayyana wannan a matsayin nau'ikan zare waɗanda ke da matakai daban-daban na samar da ƙarfi da gajiya. Duk da haka, a bayyane yake cewa wannan yana bayyana ƙaramin ɓangare ne kawai na bambancin zaren tsokar ƙashi, wanda ya fi canzawa, rikitarwa da fuskoki da yawa fiye da yadda aka zata a baya. Ci gaban fasaha yanzu ya haskaka abubuwan da ke daidaita zaren tsokar ƙashi. Hakika, bayananmu sun nuna cewa zaren nau'in 2X bazai zama wani nau'in zaren tsokar ƙashi daban ba. Bugu da ƙari, mun gano sunadaran metabolism, sunadaran ribosomal da sunadarai masu alaƙa da tantanin halitta a matsayin manyan abubuwan da ke haifar da bambancin zaren tsokar ƙashi. Ta hanyar amfani da aikinmu na proteomic ga samfuran marasa lafiya tare da myopathy na nematode, mun ƙara nuna cewa nau'in zaren da ke tushen MYH ba ya nuna cikakken bambancin tsokar ƙashi, musamman lokacin da tsarin ya rikice. Hakika, ba tare da la'akari da nau'in zaren da ke tushen MYH ba, myopathy na nematode yana haifar da sauyawa zuwa zaren da sauri da ƙarancin oxidative.
An rarraba zaruruwan tsokar ƙashi tun ƙarni na 19. Binciken omics na baya-bayan nan ya ba mu damar fara fahimtar bayanin bayyanar nau'ikan zaruruwan MYH daban-daban da kuma martaninsu ga abubuwan da ke haifar da su daban-daban. Kamar yadda aka bayyana a nan, hanyoyin omics kuma suna da fa'idar ƙarin ji na auna alamun nau'in zare fiye da hanyoyin gargajiya na rigakafi, ba tare da dogaro da ƙididdige alamomi guda ɗaya (ko kaɗan) don ayyana nau'in zaren tsokar ƙashi ba. Mun yi amfani da ayyukan aiki na transcriptomic da proteomic tare da haɗa sakamakon don bincika ƙa'idar rubutu da bayan-rubutu na bambancin zaren fiber a cikin zaruruwan tsokar ƙashi na ɗan adam. Wannan aikin ya haifar da gazawar gano zaruruwan nau'in 2X tsarkakakku a matakin furotin a cikin vastus lateralis na ƙungiyar samarinmu masu lafiya. Wannan ya yi daidai da binciken zaruruwa guda ɗaya da suka gabata wanda ya gano kusan kashi 1% zaruruwan 2X tsarkakakku a cikin vastus lateralis masu lafiya, kodayake ya kamata a tabbatar da wannan a cikin wasu tsokoki a nan gaba. Bambancin da ke tsakanin gano zaruruwan 2X tsarkakakku kusan a matakin mRNA da zaruruwan 2A/2X haɗe kawai a matakin furotin yana da rikitarwa. Bayyanar mRNA isoform ta MYH ba circadian ba ce,67 tana nuna cewa da wuya mu "rasa" siginar farawa ta MYH2 a cikin zaruruwa masu tsabta 2X a matakin RNA. Wata hujja mai yiwuwa, kodayake zato ne kawai, na iya zama bambance-bambance a cikin daidaiton furotin da/ko mRNA tsakanin isoforms na MYH. Hakika, babu wani zare mai sauri da ke da tsarki 100% ga kowane isoform na MYH, kuma ba a san ko matakan bayyanar mRNA na MYH1 a cikin kewayon 70-90% zai haifar da daidaiton yawan MYH1 da MYH2 a matakin furotin ba. Duk da haka, lokacin da ake la'akari da dukkan transcriptome ko proteome, nazarin rukuni zai iya gano ƙungiyoyi biyu daban-daban waɗanda ke wakiltar zaruruwan tsoka masu jinkiri da sauri, ba tare da la'akari da ainihin abun da ke cikin MYH ba. Wannan ya yi daidai da nazarin ta amfani da hanyoyin transcriptomic guda ɗaya, waɗanda galibi ke gano ƙungiyoyin myonuclear guda biyu daban-daban. 68, 69, 70 Bugu da ƙari, kodayake binciken da aka yi a baya na proteomic ya gano zaruruwan nau'in 2X, waɗannan zaruruwan ba sa taruwa daban da sauran zaruruwan sauri kuma suna nuna ƙaramin adadin sunadaran da suka bambanta idan aka kwatanta da sauran nau'ikan zaruruwan bisa ga MYH. 14 Waɗannan sakamakon sun nuna cewa ya kamata mu koma ga ra'ayin farkon ƙarni na 20 game da rarrabuwar zaruruwan tsoka, wanda ya raba zaruruwan tsokar ɗan adam ba zuwa azuzuwa uku daban-daban bisa ga MYH ba, amma zuwa ƙungiyoyi biyu bisa ga halayen metabolism da contractile. 63
Mafi mahimmanci, ya kamata a yi la'akari da bambancin myofiber a fannoni daban-daban. Nazarin "omics" da suka gabata sun nuna a wannan alkibla, suna nuna cewa zaruruwan tsoka ba sa samar da ƙungiyoyi daban-daban amma an shirya su ne a kan ci gaba. 11, 13, 14, 64, 71 A nan, mun nuna cewa, ban da bambance-bambance a cikin halayen ƙanƙancewa da na rayuwa na tsokar ƙashi, ana iya bambance myofibers ta hanyar fasaloli da suka shafi hulɗar tantanin halitta da hanyoyin fassara. Hakika, mun sami bambancin ribosome a cikin zaruruwan tsokar ƙashi wanda ke ba da gudummawa ga bambancin ba tare da la'akari da nau'ikan zaruruwa masu jinkiri da sauri ba. Babban dalilin wannan babban bambancin myofiber, ba tare da la'akari da nau'in zaruruwa mai jinkiri da sauri ba, har yanzu ba a fayyace shi ba, amma yana iya nuna tsarin sarari na musamman a cikin ƙwayoyin tsoka waɗanda ke amsawa ga takamaiman ƙarfi da kaya,72 sadarwa ta musamman ta tantanin halitta ko ta musamman ga gabobin jiki tare da wasu nau'ikan tantanin halitta a cikin yanayin tsoka 73,74,75 ko bambance-bambance a cikin ayyukan ribosome a cikin myofibers na mutum ɗaya. Hakika, ribosomal heteroplasmy, ko dai ta hanyar maye gurbin RPL3 da RPL3L mai kama da juna ko kuma a matakin 2′O-methylation na rRNA, an nuna yana da alaƙa da hawan tsokar ƙashi76,77. Aikace-aikacen Multi-omic da sarari tare da halayyar aiki na myofibers na mutum ɗaya zai ƙara haɓaka fahimtarmu game da ilimin halittar tsoka a matakin Multi-omic78.
Ta hanyar nazarin furotin na myofibers guda ɗaya daga marasa lafiya da ke fama da ciwon nemaline, mun kuma nuna amfani, inganci, da kuma amfani da myofiber proteomics guda ɗaya don bayyana yanayin cututtukan ƙwayoyin tsoka. Bugu da ƙari, ta hanyar kwatanta aikinmu da nazarin proteomics na duniya, mun sami damar nuna cewa myofiber proteomics guda ɗaya yana samar da zurfin bayanai iri ɗaya kamar proteomics na nama na duniya kuma yana faɗaɗa wannan zurfin ta hanyar lissafin bambancin interfiber da nau'in myofiber. Baya ga bambance-bambancen da ake tsammani (kodayake masu canzawa) a cikin rabon nau'in fiber da aka lura a cikin myopathies na nemaline ACTA1 da TNNT1 idan aka kwatanta da sarrafawa masu lafiya,19 mun kuma lura da sake fasalin oxidative da extracellular ba tare da la'akari da canjin nau'in fiber da MYH ke gudanarwa ba. An riga an ruwaito cutar Fibrosis a cikin TNNT1 nemaline myopathies.19 Duk da haka, bincikenmu ya gina akan wannan binciken ta hanyar bayyana ƙarin matakan furotin da ke da alaƙa da damuwa a cikin ƙwayoyin halitta, kamar annexins, waɗanda ke da hannu a cikin hanyoyin gyara sarcolemmal, a cikin myofibers daga marasa lafiya da ke da cutar ACTA1 da TNNT1 nemaline myopathies.57,58,59 A ƙarshe, ƙaruwar matakan annexin a cikin myofibers daga marasa lafiya da ke da cutar nemaline myopathy na iya wakiltar martanin ƙwayoyin halitta ga gyara myofibers masu matuƙar atrophic.
Ko da yake wannan binciken yana wakiltar mafi girman nazarin ƙwayoyin tsoka guda ɗaya na ɗan adam har zuwa yau, ba tare da iyakancewa ba. Mun ware ƙwayoyin tsoka na kwarangwal daga ƙaramin samfurin mahalarta da tsoka ɗaya (vastus lateralis). Saboda haka, ba zai yiwu a ware wanzuwar takamaiman ƙwayoyin zare a cikin nau'ikan tsoka da kuma a cikin matsanancin ilimin halittar tsoka ba. Misali, ba za mu iya ware yiwuwar wani ɓangare na ƙwayoyin zare masu sauri (misali, zare masu tsabta 2X) da ke fitowa a cikin masu tsere masu horo sosai da/ko 'yan wasa masu ƙarfi79 ko a lokacin rashin aiki na tsoka66,80. Bugu da ƙari, ƙarancin girman samfurin mahalarta ya hana mu bincika bambance-bambancen jinsi a cikin bambancin ƙwayoyin zare, kamar yadda aka san rabon nau'in ƙwayoyin zare ya bambanta tsakanin maza da mata. Bugu da ƙari, ba mu iya gudanar da nazarin ƙwayoyin tsoka da ƙwayoyin zare a kan zare ko samfuran tsoka ɗaya daga mahalarta ɗaya ba. Yayin da mu da wasu muke ci gaba da inganta nazarin ƙwayoyin halitta guda ɗaya da na myofiber guda ɗaya ta amfani da nazarin omics don cimma ƙarancin samfurin shigarwa (kamar yadda aka nuna a nan a cikin nazarin zaruruwa daga marasa lafiya da ke fama da cutar mitochondrial myopathy), damar haɗa hanyoyin multi-omics (da aiki) a cikin zaruruwan tsoka guda ɗaya ta bayyana.
Gabaɗaya, bayananmu suna gano kuma suna bayyana abubuwan da ke haifar da bambancin ƙwayoyin tsoka ta hanyar rubutu da kuma bayan rubutawa. Musamman, muna gabatar da bayanai waɗanda ke ƙalubalantar wata ƙa'ida ta daɗe a fannin ilimin halittar tsokar ƙashi da ke da alaƙa da ma'anar nau'ikan zare na gargajiya bisa tushen MYH. Muna fatan sabunta muhawarar kuma a ƙarshe mu sake tunani game da fahimtarmu game da rarrabuwar zare da bambancin ƙwayoyin tsoka.
Mahalarta taron Caucasian guda goma sha huɗu (maza 12 da mata 2) sun amince da kansu su shiga cikin wannan binciken. Kwamitin Ɗabi'a na Asibitin Jami'ar Ghent (BC-10237) ya amince da wannan binciken, ya bi ka'idar Helsinki ta 2013, kuma an yi masa rijista a ClinicalTrials.gov (NCT05131555). An gabatar da halayen mahalarta a cikin Tebur na Ƙarin Bayani na 1. Bayan samun izini na baki da na rubutu, mahalarta sun yi gwajin lafiya kafin a haɗa su a cikin binciken. Mahalarta taron matasa ne (shekaru 22-42), masu lafiya (ba su da wata matsala ta lafiya, babu tarihin shan taba), kuma suna aiki a jiki matsakaici. An ƙayyade yawan shan iskar oxygen ta amfani da na'urar auna ƙarfin jiki kamar yadda aka bayyana a baya. 81
An tattara samfuran biopsy na tsoka a lokacin hutu da kuma a cikin yanayin azumi sau uku, kwanaki 14 a tsakanin juna. Saboda an tattara waɗannan samfuran a matsayin wani ɓangare na babban bincike, mahalarta sun sha placebo (lactose), wani antagonist na H1-receptor antagonist (540 mg fexofenadine), ko kuma antagonist na H2-receptor (40 mg famotidine) mintuna 40 kafin biopsy. Mun riga mun nuna cewa waɗannan antagonist na histamine receptor antagonists ba sa shafar lafiyar tsokar ƙashi mai hutawa81, kuma ba a lura da wani rukuni da ya shafi jihar a cikin tsare-tsaren kula da inganci ba (Karin Figures 3 da 6). An ci abinci mai daidaitaccen tsari (41.4 kcal/kg nauyin jiki, 5.1 g/kg nauyin jiki carbohydrate, 1.4 g/kg furotin nauyin jiki, da 1.6 g/kg nauyin jiki mai nauyin jiki) na tsawon awanni 48 kafin kowace ranar gwaji, kuma an sha karin kumallo mai daidaitaccen tsari (1.5 g/kg nauyin jiki carbohydrate) da safe na ranar gwaji. A ƙarƙashin maganin sa barci na gida (0.5 ml 1% lidocaine ba tare da epinephrine ba), an samo ƙwayoyin tsoka daga tsokar vastus lateralis ta amfani da aspiration na percutaneous Bergström.82 An saka samfuran tsoka nan da nan a cikin RNA daga baya kuma an adana su a zafin 4°C har sai an raba zare da hannu (har zuwa kwana 3).
An canja wurin fakitin myofiber da aka ware zuwa sabon RNAlater medium a cikin wani abincin gargajiya. Daga nan aka yanke myofibers ɗin da hannu ta amfani da na'urar stereomicroscope da ƙananan tweezers. An yanke zare ashirin da biyar daga kowane biopsy, tare da mai da hankali na musamman wajen zaɓar zare daga wurare daban-daban na biopsy. Bayan an yanke, an nutsar da kowace zare a hankali a cikin μl 3 na lysis buffer (SingleShot Cell Lysis Kit, Bio-Rad) wanda ke ɗauke da proteinase K da enzymes na DNase don cire sunadaran da ba a so da DNA. Daga nan aka fara cire ƙwayoyin halitta da kuma cire furotin/DNA ta hanyar yin vortex na ɗan lokaci, juya ruwan a cikin microcentrifuge, sannan aka kunna shi a zafin ɗaki (minti 10). Daga nan aka sanya lysate ɗin a cikin na'urar zagayowar zafi (T100, Bio-Rad) a 37°C na minti 5, 75°C na minti 5, sannan aka adana nan take a -80°C har sai an ci gaba da sarrafa shi.
An shirya ɗakunan karatu na RNA masu jituwa da Illumina daga 2 µl na myofiber lysate ta amfani da Kit ɗin Shirya Laburare na QuantSeq-Pool 3′ mRNA-Seq (Lexogen). Ana iya samun cikakkun hanyoyin a cikin littafin jagorar masana'anta. Tsarin yana farawa da haɗakar cDNA ta farko ta hanyar kwafi na baya, wanda a lokacin ake gabatar da abubuwan gano kwayoyin halitta na musamman (UMIs) da barcodes na musamman na i1 don tabbatar da haɗa samfuran da rage bambancin fasaha yayin sarrafa su. Sannan ana haɗa cDNA daga myofibers 96 kuma ana tsarkake su da beads na maganadisu, bayan haka ana cire RNA kuma ana yin haɗakar segment ta biyu ta amfani da firam ɗin bazuwar. Ana tsarkake ɗakin karatu da beads na maganadisu, ana ƙara alamun i5/i7 na musamman a cikin tafkin, kuma ana ƙara PCR. Mataki na ƙarshe na tsarkakewa yana samar da ɗakunan karatu masu jituwa da Illumina. An tantance ingancin kowane ɗakin karatu ta amfani da Kit ɗin Nazarin DNA na Babban Sensitivity Small Fragment (Agilent Technologies, DNF-477-0500).
Dangane da ƙididdige Qubit, an ƙara haɗa wuraren waha a yawan daidaito (2 nM). Daga nan aka jera wurin da aka samo sakamakon a kan kayan aikin NovaSeq 6000 a yanayin da aka saba amfani da shi ta amfani da Kayan Reagent na NovaSeq S2 (nucleotides 1 × 100) tare da nauyin nM 2 (4% PhiX).
Tsarin bututunmu ya dogara ne akan bututun nazarin bayanai na QuantSeq Pool na Lexogen (https://github.com/Lexogen-Tools/quantseqpool_analysis). An fara cire bayanai da bcl2fastq2 (v2.20.0) bisa ga ma'aunin i7/i5. Sannan an cire Read 2 da idemux (v0.1.6) bisa ga samfurin barcode na i1 kuma an cire jerin UMI da umi_tools (v1.0.1). Sannan an rage karanta da cutadapt (v3.4) a zagaye da yawa don cire gajerun karatu (<20 a tsayi) ko karantawa da ta ƙunshi jerin adaftar kawai. Daga nan aka daidaita karatun zuwa ga kwayar halittar ɗan adam ta amfani da STAR (v2.6.0c) kuma an yi wa fayilolin BAM fihirisa da SAMtools (v1.11). An cire kwafi na karanta ta amfani da umi_tools (v1.0.1). A ƙarshe, an yi ƙidayar daidaitawa ta amfani da featureCounts a cikin Subread (v2.0.3). An gudanar da sarrafa inganci ta amfani da FastQC (v0.11.9) a matakai daban-daban na matsakaicin bututun.
An gudanar da duk wasu ayyukan sarrafa bayanai da kuma gani na bioinformatics a cikin R (v4.2.3), galibi ta amfani da tsarin aiki na Seurat (v4.4.0). 83 Saboda haka, an canza dabi'un UMI da matrices na metadata zuwa abubuwan Seurat. An cire kwayoyin halitta da aka bayyana a cikin ƙasa da kashi 30% na dukkan zaruruwa. An cire samfuran marasa inganci bisa ga mafi ƙarancin matakin ƙimar UMI 1000 da kwayoyin halitta 1000 da aka gano. A ƙarshe, zaruruwa 925 sun wuce duk matakan tace inganci. An daidaita dabi'un UMI ta amfani da hanyar Seurat SCTransform v2, 84 gami da duk fasalulluka 7418 da aka gano, kuma an rage bambance-bambancen tsakanin mahalarta. Ana iya samun duk bayanan bayanai masu dacewa a cikin Ƙarin Bayanan 28.