I-Olink Hlola Ukulandelanisa usebenzisa i-NextSeq 550
Isingeniso
Ukusetshenziswa okuhlosiwe
I-Olink® Explore iyinkundla ye-multiplex immunoassay yokutholwa kwe-biomarker yamaprotheni omuntu. Umkhiqizo uhloselwe Ukusetshenziswa Kocwaningo Kuphela, futhi hhayi ukusetshenziswa ezinqubweni zokuxilonga. Umsebenzi waselabhorethri uzoqhutshwa kuphela abasebenzi baselabhorethri abaqeqeshiwe. Ukucubungula idatha kuzokwenziwa kuphela abasebenzi abaqeqeshiwe. Imiphumela ihloselwe ukusetshenziswa abacwaningi ngokuhlanganyela nokunye okutholwe emitholampilo noma elabhorethri.
Mayelana nale manuwali
Le Manuwali Yomsebenzisi inikeza imiyalelo edingekayo ukuze i-Olink® Explore Libraries ilandelelane ku-Illumina® NextSeq™ 550. Imiyalo kufanele ilandelwe ngokuqinile nangokucacile. Noma yikuphi ukuchezuka kuzo zonke izinyathelo zaselabhorethri kungase kubangele idatha eyonakele. Ngaphambi kokuqala ukuhamba komsebenzi kulabhorethri, thintana ne-Olink® Explore Overview Incwadi Yomsebenzisi yesingeniso seplatifomu, okuhlanganisa ulwazi mayelana nama-reagents, okokusebenza kanye nemibhalo edingekayo, i-overview wokuhamba komsebenzi, kanye nemihlahlandlela yaselabhorethri. Ukuze uthole imiyalelo yokuthi uwasebenzise kanjani amakhithi e-Olink® Explore Reagent, bheka i-Olink® Explore User Manual esebenzayo. Ngokucubungula idatha nokuhlaziywa kwemiphumela yokulandelana kwe-Olink® Explore, bheka ku-Olink® MyData Cloud User Guide. Zonke izimpawu zokuthengisa namalungelo okushicilela aqukethwe kulolu hlelo lokusebenza kuyimpahla ye-Olink® Proteomics AB, ngaphandle uma kushiwo ngenye indlela.
Ukusekela kwezobuchwepheshe
Ukuze uthole ukwesekwa kobuchwepheshe, xhumana ne-Olink Proteomics ku- support@olink.com.
Iziyalezo zaselabhorethri
Lesi sahluko sinikeza imiyalelo yokuthi ungalandelanisa kanjani i-Olink Libraries ku-NextSeq™ 550 usebenzisa i-NextSeq™ 500/550 High Output Kit v2.5 (75 Cycles). Iphrothokholi esetshenziselwa ukulandelana iwukujwayela kokuhamba komsebenzi kwe-Illumina® NGS okujwayelekile kwe-Illumina® NextSeq™ 550. Ngaphambi kokuqhubeka nokulandelanisa, qiniseka ukuthi ikhwalithi Yelabhulali ecwengiwe iqinisekisiwe. Bheka i-Olink Explore User Manual esebenzayo ukuze uthole imiyalelo mayelana nokulawula ikhwalithi.
Hlela ukugijima kokulandelana
Ilabhulali eyodwa ye-Olink ingalandelana ngeseli ye-NextSeq™ 550 High Output flow kanye nokugijima ngakunye. Inani lamaseli okukhiphayo Okuphezulu nokugijima okudingekayo ukuze kulandelelane amakhithi e-Olink Explore Reagent ahlukene kuchazwe kuThebula 1. Uma kudingeka ukugijima okungaphezu kokukodwa, phinda imiyalelo echazwe kule manuwali.
Ithebula 1.Ukulandelanisa ukuhlela kokuqalisa:
| Ikhithi ye-Olink® Hlola iReagent | Inombolo Yemitapo yolwazi ye-Olink | Inani lamaseli agelezayo kanye nokuqalisa |
| I-Olink® Explore 384 Reagent Kit | 1 | 1 |
| I-Olink® Explore 4 x 384 Reagent Kit | 4 | 4 |
| I-Olink® Explore 1536 Reagent Kit | 4 | 4 |
| Ikhithi ye-Olink® Explore Expansion Reagent | 4 | 4 |
| I-Olink® Explore 3072 Reagent Kit | 8 | 8 |
Faka iresiphi yangokwezifiso ye-Olink®
Phakathi nalesi sinyathelo, iresiphi yangokwezifiso ye-Olink® ifakwa ku-NextSeq™ 550. Lesi sinyathelo sidinga ukwenziwa kanye kuphela, ngaphambi kokuthi kwenziwe ukugijima kokulandelana kwe-Olink okokuqala ngqa.
QAPHELA: Iresiphi yangokwezifiso ye-Olink izosebenza kuphela ne-NextSeq™ 500/550 High Output Kits kanye ne-NextSeq™ Control Software 4.0.
- Vula iziphu bese ubeke iresiphi yangokwezifiso ye-Olink Olink_NSQ550_HighOutput_V1 kufolda elandelayo yensimbi ye-NextSeq™ 550: C:\Program Files\Illumina\NextSeq Control Software\Recipe\Custom\High\.
- Ngaphansi Kokwenza Ngezifiso Isistimu > Phatha Ithuluzi, nika amandla Amaresiphi Okwezifiso. Uma ingakhethiwe, inketho yeresiphi yangokwezifiso ngeke ivele ngesikhathi sokusetha.
QAPHELA: Kunguqulo yesofthiwe ye-NCS 4.0, inketho yokukhetha iresiphi yangokwezifiso izokwenzeka kuphela ngemva kokuba ikhatriji ye-reagent ilayishiwe, hhayi ekhasini lokusetha langaphambilini.
QAPHELA: Ukugijima kufanele kusethwe kumodi eyenziwa ngesandla ukuze kuvunyelwe amaresiphi angokwezifiso.
Lungiselela ukulandelana kwama-reagents
Phakathi nalesi sinyathelo, i-cartridge ye-reagent equkethe ama-reagents e-clustering kanye nokulandelana iyancibilika futhi iseli eligelezayo liyalungiswa.
Lungisa i-cartridge ye-reagent
ISEXWAYISO: I-cartridge ye-reagent iqukethe amakhemikhali angaba yingozi. Gqoka izinto ezivikelayo ezanele futhi ulahle ama-reagents asetshenzisiwe ngokuhambisana namazinga asebenzayo. Ukuze uthole ulwazi olwengeziwe, bheka i-Illumina NextSeq 550 System Guide (idokhumenti #15069765).
Lungiselela ibhentshi
- 1x NextSeq™ 500/550 High Output Reagent Cartridge v2 (imijikelezo engu-75).
Iziyalezo
- Faka i-cartridge ye-reagent eqandisiwe icwiliswe emanzini apholile ekamelweni futhi uyivumele incibilike ihora elingu-1. Qinisekisa ukuthi wonke ama-reagent reservoirs ama-cartridges ancibilikisiwe ngokuphelele.
QAPHELA: Ukuze kube lula, ncibilikisa i-cartridge ngayizolo bese uyigcina ebusuku ku-4 °C. Kuleli zinga lokushisa, ama-reagents azinzile kuze kube yisonto elilodwa. - Yomisa isisekelo se-cartridge kahle ngethawula lephepha bese wesula ama-foil seal omile ngezicubu ezingena-lint uma kudingeka.
- Guqula i-cartridge izikhathi eziyishumi ukuze uhlanganise kahle ama-reagents ancibilikisiwe ngaphakathi.
- Thepha i-cartridge ngobumnene ebhentshini ukuze ususe amabhamuza omoya. Gcina i-cartridge ekamelweni lokushisa uma izosetshenziswa phakathi namahora angu-4.
Lungiselela iseli eligelezayo
Lungiselela ibhentshi
- 1x NextSeq™ 500/550 Iseli Eligelezayo Lokukhipha Okuphezulu v2.5.
Iziyalezo
- Letha i-flow cell efrijini ekamelweni lokushisa imizuzu engu-30.
- Gqoka amagilavu amasha angenayo impushana (ukugwema ukungcolisa ingilazi yeseli lokugeleza).
- Uma usulungele ukulayisha iseli eligelezayo ensimbini, susa iseli eligelezayo ephaketheni kanye ne-clamshell yepulasitiki.
- Hlola i-flow cell. Uma izinhlayiya noma uthuli lubonakala kunoma iyiphi indawo yengilazi, hlanza indawo esebenzayo nge-isopropyl alcohol engena-lint futhi uyomise ngethishu elebhu ene-lint ephansi.
Lungiselela i-Olink® Library ukuze ilandelelane
Phakathi nalesi sinyathelo, ukuhlanjululwa kwe-NaOH ne-Tris-HCl kuyalungiswa, futhi i-Olink Library ehlanzekile nelawulwa ngekhwalithi iyahlanjululwa futhi ishintshwe ngezinyathelo ezilandelanayo.
Lungiselela i-NaOH dilution
I-dilution ye-NaOH isetshenziselwa ukusho igama elithi Amalabhulali.
Lungiselela ibhentshi
- 1 N NaOH isitoko
- Amanzi e-MilliQ
- 1x Ishubhu le-Microcentrifuge (1.5 mL)
- I-pipette eyenziwe ngesandla (10-100 μL)
- Hlunga amathiphu we-pipette
Ngaphambi kokuthi uqale
Maka ithubhu ye-microcentrifuge "NaOH".
Iziyalezo
- Lungiselela ukuhlanjululwa kwe-0.2 N NaOH ku-NaOH Tube ngokweThebula 2.
- Vortex i-NaOH Tube kahle futhi ujikeleze phansi. Sebenzisa phakathi kwamahora angu-12.
Ithebula 2. 0.2 I-N NaOH dilution
| I-Reagent | Ivolumu (μL) |
| Amanzi e-MilliQ | 80 |
| 1 N NaOH isitoko | 20 |
2.4.2 Lungiselela i-Tris-HCl dilution
I-Tris-HCl dilution isetshenziselwa ukwenza ilabhulali engashintshiwe ibe lula.
Lungiselela ibhentshi
- 1 M Tris-HCl pH 7.0 isitoko (isixazululo se-Trizma® hydrochloride)
- Amanzi e-MilliQ
- 1x Ishubhu le-Microcentrifuge (1.5 mL)
- I-pipette eyenziwe ngesandla (10-100 μL)
- Hlunga amathiphu we-pipette
Ngaphambi kokuthi uqale
- Maka ishubhu le-microcentrifuge “Tris-HCl”
Iziyalezo
- Lungiselela ukuhlanjululwa kwe-Tris-HCl okungu-200 mM kuThubhu ye-Tris-HCl ngokweThebula 3.
- Vortex i-Tris-HCl Tube kahle bese uyiphotha phansi.
Ithebula 3. 200 mM Tris-HCl dilution:
| I-Reagent | Ivolumu (μL) |
| Amanzi e-MilliQ | 80 |
| I-1M Tris-HCl pH 7.0 isitoko (isixazululo se-Trizma® hydrochloride) | 20 |
Nciphisa Imitapo yolwazi ye-Olink®
Phakathi nalesi sinyathelo, i-Olink Library ehlanziwe nekhwalithi ihlanjululwa ngo-1:33.
Lungiselela ibhentshi
- I-Lib Tube, elungiselelwe ngokuya nge-Olink Explore User Manual esebenzayo
- Amanzi e-MilliQ
- 1x Ishubhu le-Microcentrifuge (1.5 mL)
- Amapayipi okwenziwa ngesandla (0.5-10 kanye no-100-1000 μL)
- Hlunga amathiphu we-pipette
Ngaphambi kokuthi uqale
- Ncibilikisa i-Lib Tube uma ifriziwe.
- Maka ithubhu entsha ye-microcentrifuge: "Dil".
Iziyalezo
- Engeza u-96 μL wamanzi we-MilliQ ku-Dil Tube.
- Vortex the Lib Tube bese uyiphotha phansi kafushane.
- Dlulisa u-3 μL kusuka ku-Lib Tube kuya ku-Dil Tube.
- Vortex the Dil Tube bese uyiphotha phansi kafushane
Dlulisa futhi unciphise Ilabhulali ye-Olink® ekugxiliseni kokugcina kokulayisha
Ngalesi sinyathelo, i-Olink Library ehlanjululwe iguqulwa futhi iphinde ihlanjululwe ekugxilweni kokugcina kokulayisha.
Lungiselela ibhentshi
- I-Dil Tube, elungiselelwe esinyathelweni sangaphambilini
- 0.2 I-N NaOH dilution, esanda kulungiswa esinyathelweni sangaphambilini
- 200 mM Tris-HCl (pH 7.0) dilution, elungiswe esinyathelweni sangaphambilini
- Ibhafa ye-Hybridization 1 (HT1) ifakwe ku-NextSeq™ Accessory Box v2
- 2x amashubhu e-Microcentrifuge (1.5 mL no-2 mL)
- Amapayipi okwenziwa ngesandla (0.5-10 kanye no-100-1000 μL)
- Hlunga amathiphu we-pipette
Ngaphambi kokuthi uqale
- Ncibilikisa isigcinalwazi esiqandisiwe se-HT1 ezingeni lokushisa legumbi. Gcina ku +4 °C uze usetshenziswe.
- Maka ishubhu entsha ye-microcentrifuge engu-1.5 mL: “I-Den” (yeMtapo wezincwadi oyi-denaturated).
- Maka ishubhu entsha ye-microcentrifuge engu-2 mL: “Seq” (ukuthi ilungele ukulayisha Umtapowolwazi).
Iziyalezo
- Dlulisa i-5 μL kusuka ku-Dil Tube kuya ku-Den Tube.
- Engeza u-5 μL ka-0.2 N NaOH ku-Den Tube.
- Vortex the Den Tube bese uyiphotha phansi kafushane.
- Fukamela i-Den Tube imizuzu emi-5 ekamelweni lokushisa ukuze uguqule Ilabhulali.
- Engeza u-5 μL ka-200 mM Tris-HCl (pH 7.0) ku-Den Tube ukuze unciphise ukusabela.
- Vortex the Den Tube bese uyiphotha phansi kafushane.
- Engeza u-985 μL we-HT1 eshisiwe ku-Den Tube.
- Vortex the Den Tube bese uyiphotha phansi kafushane. Ishubhu ingagcinwa ku +4 °C ize isetshenziswe (ngalolo suku).
- Dlulisa u-205 μL usuka ku-Den Tube uye ku-Seq Tube.
- Engeza u-1095 μL we-HT1 eshisiwe ku-Seq Tube.
- Guqula i-Seq Tube ukuze uhlanganise ama-reagents bese uyiphotha phansi kafushane. Ivolumu yokugcina yokulayisha ingu-1.3 mL.
- Ngokushesha qhubekela ku-2.5 Yenza ukugijima kokulandelana kwe-Olink®.
Yenza ukugijima kokulandelana kwe-Olink®
Phakathi nalesi sinyathelo, i-buffer cartridge, i-flow cell kanye ne-reagent cartridge elungisiwe equkethe i-Olink Library ilayishwa ku-NextSeq 550, futhi ukugijima kokulandelana kuqalwa kusetshenziswa iresiphi yangokwezifiso ye-Olink.
Lungiselela ibhentshi
- I-Seq Tube (esilungele ukulayisha Umtapowolwazi), ilungiswe esinyathelweni sangaphambilini
- I-1x NextSeq™ 500/550 High Output Reagent Cartridge v2, elungiswe esinyathelweni sangaphambilini
- 1x NextSeq™ 500/550 High Output Flow Cell v2.5, elungiswe esinyathelweni sangaphambilini
- I-1x NextSeq™ 500/550 Buffer Cartridge v2 (imijikelezo engu-75), ekamelweni lokushisa
Setha amapharamitha wokugijima wokulandelanisa
Phakathi nalesi sinyathelo, amapharamitha wokugijima wokulandelanisa akhethwa ku-NextSeq™ 550.
- Esikrinini sasekhaya se-NextSeq™ 550, khetha okuthi Hlola.
- Esikrinini esithi Khetha Ukuhlola, khetha Ukulandelana.
- Ekhasini elithi Run Setup, khetha Imodi yokuqalisa mathupha bese emva kwalokho Okulandelayo.
- Setha amapharamitha wokugijima ngendlela elandelayo:
- Esigabeni se-Run Name, faka i-ID yokuhlola eyingqayizivele.
- Kunkambu ye-ID yelabhulali, faka i-ID Yelabhulali oyisebenzisayo (kuyakhetheka).
- Kunkambu ethi Funda, khetha inketho ethi Funda Okukodwa.
- Faka inombolo yemijikelezo kanje:
- Funda ku-1:24
- Inkomba 1: 0
- Inkomba 2: 0
- Funda ku-2:0
- Gcina ibhokisi lokuhlola leziqalo zangokwezifiso lingakhethiwe.
- Setha indawo yefolda yokukhiphayo ukuze uthole idatha esetshenziswayo yamanje. Khetha Phequlula ukuze uguqule indawo yefolda yokuphumayo.
- Musa ukumisa i-Sample Sheet.
- Khetha okuthi Hlanza okusebenzisekayo kulokhu kuqalisa.
- Khetha Okulandelayo.
Layisha iseli eligelezayo ku-NextSeq™ 550
- Susa iseli eligelezayo elisetshenzisiwe kusukela ekuqaliseni kwangaphambilini.
- Beka i-flow cell entsha elungisiwe ku-stage.
- Khetha Layisha. Umnyango uvaleka ngokuzenzakalelayo.
- Uma i-ID yeseli eligelezayo ivela esikrinini futhi izinzwa zibhekwa ziluhlaza, khetha Okulandelayo
Thulula isiqukathi se-reagent
ISEXWAYISO: Leli sethi lama-reagents liqukethe amakhemikhali angaba yingozi. Gqoka izinto ezivikelayo ezanele futhi ulahle ama-reagents asetshenzisiwe ngokuhambisana namazinga asebenzayo. Ukuze uthole ulwazi olwengeziwe, bheka i-Illumina NextSeq 550 System Guide.
- Vula umnyango wekhompathimenti yebhafa, susa isiqukathi sezinto ezisetshenziswayo esichithiwe engxenyeni engezansi, futhi ulahle okuqukethwe ngokuvumelana nezindinganiso ezisebenzayo.
- Shelelezisa isiqukathi se-reagent esingenalutho usibuyisele endaweni yebhafa engezansi. Ukuchofoza okuzwakalayo kubonisa ukuthi isiqukathi sibekwe ngendlela efanele.
Layisha i-buffer cartridge
- Susa i-cartridge yebhafa esetshenzisiwe endaweni engaphezulu yebhafa futhi ulahle okuqukethwe ngokuvumelana nezindinganiso ezisebenzayo.
- Shelelezisa i-cartridge entsha ye-buffer endaweni engaphezulu yebhafa. Ukuchofoza okuzwakalayo kubonisa ukuthi i-cartridge ibekwe ngendlela efanele. Qinisekisa ukuthi i-ID ye-buffer cartridge iyavela esikrinini nokuthi izinzwa zihlolwe ngokuluhlaza okotshani.
- Vala umnyango wekhompathimenti yebhafa bese ukhetha Okulandelayo.
Layisha i-cartridge ye-reagent
- Vula umnyango wekhompathimenti ye-reagent, susa i-cartridge ye-reagent esetshenzisiwe, futhi ulahle okuqukethwe okungasetshenzisiwe ngokuvumelana nezindinganiso ezisebenzayo. Ichibi elisendaweni yesi-6 liyakhishwa ukuze kube lula ukulahlwa okuphephile.
- Gcoba uphawu lwechibi #10 olubhalwe ukuthi “Layisha Umtapo Wezincwadi Lapha” ngethiphu elihlanzekile elingu-1 mL.
- Layisha u-1.3 mL we-Olink Library osuka ku-Seq Tube uye endaweni yokugcina amanzi #10 ebhalwe ngokuthi “Layisha Umtapowolwazi Lapha”.
- Shelelisela i-cartridge ye-reagent entsha endaweni ye-reagent bese uvala umnyango wegumbi le-reagent.
- Khetha okuthi Layisha bese ulinda imizuzwana engu-~30 kuze kuvele i-ID ye-cartridge ye-reagent esikrinini nezinzwa zihlolwa zibe luhlaza.
- Kuhlu oluya phansi Lweresiphi, khetha inketho yeresiphi ethi [Ngokwezifiso] “Olink_NSQ550_HighOutput_V1”.
- Khetha Okulandelayo.
Qala ukugijima okulandelanayo
- Qinisekisa amapharamitha wokugijima aboniswe ku-Review isikrini. Ukuze uhlele noma yimaphi amapharamitha, cindezela Emuva ukuze ubuyele esikrinini se-Run Setup.
- Khetha Okulandelayo. Ukuqalisa kuqala ngemva kokuhlolwa kwangaphambilini okuzenzakalelayo. Isikhathi sokusebenza sokulandelana sicishe sibe yimizuzu eyi-7h30.
- Hlanza indawo yokusebenza.
QAPHELA: Lapho ukugijima kokulandelana sekuqediwe, isofthiwe iqala ukugeza okuzenzakalelayo ngemva kokugijima isebenzisa izixazululo zokugeza ezinikezwe kukhathriji yesilondolozi kanye ne-NaOCl ehlinzekwe kukhathriji ye-reagent. Lokhu kugeza kuthatha cishe imizuzu engama-90. Inkinobho ethi Ikhaya iqala ukusebenza uma ukugeza sekuqediwe. Ama-cartridges asetshenzisiwe kanye neseli eligelezayo lingashiywa endaweni kuze kube sekugijimeni okulandelayo.
Qaphela ukuqhubeka kokugijima
U-Olink usebenzisa i-NGS njengokufunda ukuze alinganise inani lokulandelana okwaziwayo ukuze alinganisele ukuhlangana kwephrotheni enikeziwe ku-s.amples (okuhlobene nezinye iziampkancane). Ikhwalithi yedatha evela ekugijimeni ngakunye kokulandelana kwe-Hlola inqunywa ikakhulukazi amapharamitha e-QC ahlukile kubuchwepheshe be-Olink. Ngakho-ke, amamethrikhi ajwayelekile okulawula ikhwalithi asetshenziswa ku-NGS evamile, njenge-Q-score, awabalulekile kangako.
Umlando wokubuyekeza
| Inguqulo | Usuku | Incazelo |
| 1.1 | 2021-12-13 | Izinguquko zokuhlela |
| 1.0 | 2021-12-01 | Okusha |
www.olink.com
Okokusetshenziswa Kocwaningo Kuphela. Ayisetshenziselwa Izinqubo Zokuxilonga.
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© Copyright 2021 Olink Proteomics AB. Zonke izimpawu zokuthengisa zezinkampani zangaphandle ziyimpahla yabanikazi bazo. Olink Proteomics, Dag Hammarskjölds väg 52B , SE-752 37 Uppsala, Sweden
1192, v1.1, 2021-12-13
Amadokhumenti / Izinsiza
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I-Olink Hlola Ukulandelanisa usebenzisa i-NextSeq 550 [pdf] Imaniwali yosebenzisayo Hlola Ukulandelanisa usebenzisa i-NextSeq 550, Hlola Ukulandelanisa, usebenzisa i-NextSeq 550, NextSeq 550 |
