I-Olink Phonononga ulandelelwano usebenzisa i-NextSeq 550
Intshayelelo
Ukusetyenziswa okucetywayo
I-Olink® Phonononga liqonga le-multiplex immunoassay lokufumanisa iprotein biomarker yomntu. Imveliso yenzelwe ukuSebenza koPhando kuphela, kwaye kungekhona ukusetyenziswa kwiinkqubo zokuxilonga. Umsebenzi waselabhoratri uya kuqhutywa kuphela ngabasebenzi baselabhoratri abaqeqeshiweyo. Ukusetyenzwa kwedatha kuya kwenziwa kuphela ngabasebenzi abaqeqeshiweyo. Iziphumo zenzelwe ukuba zisetyenziswe ngabaphandi ngokubambisana nezinye iziphumo zeklinikhi okanye elebhu.
Malunga nale ncwadana
Le Ncwadi yoMsebenzisi ibonelela ngemiyalelo efunekayo ukulandelelanisa i-Olink® Explore Libraries kwi-Illumina® NextSeq™ 550. Imiyalelo mayilandelwe ngokungqongqo nangokucacileyo. Nakuphi na ukutenxa kuwo onke amanyathelo elabhoratri kunokubangela ukonakaliswa kwedatha. Phambi kokuba uqalise ukuhamba kwelabhoratri, jongana ne-Olink® Explore Overview Incwadi yoMsebenzisi yentshayelelo eqongeni, kubandakanya ulwazi malunga neerejenti, izixhobo kunye namaxwebhu afunekayo, ngaphezuluview wokuhamba komsebenzi, kunye nezikhokelo zaselabhoratri. Ukufumana imiyalelo malunga nendlela yokusebenzisa i-Olink® Explore Reagent Kits, bhekisa kwi-Olink® Explore User Manual esebenzayo. Ukulungiswa kwedatha kunye nohlalutyo lwe-Olink® Hlola iziphumo zokulandelelana, bhekisa kwi-Olink® MyData Cloud User Guide. Zonke iimpawu zokuthengisa kunye namalungelo okushicilela aqulethwe kwesi sixhobo yipropati ye-Olink® Proteomics AB, ngaphandle kokuba kuchazwe ngenye indlela.
Uxhaso lobuchwepheshe
Ngenkxaso yobugcisa, qhagamshelana ne-Olink Proteomics ku support@olink.com.
Imiyalelo yeLebhu
Esi sahluko sibonelela ngemiyalelo engokulandelelanisa iiLayibrari ze-Olink kwi-NextSeq™ 550 usebenzisa i-NextSeq™ 500/550 iKit yeZiphumo eziPhezulu v2.5 (Imijikelo engama-75). Iprothokholi esetyenziselwa ukulandelelanisa i-adaptation ye-Illumina® standard NGS workflow for Illumina® NextSeq™ 550. Ngaphambi kokuba uqhubeke nokulandelelanisa, qiniseka ukuba umgangatho weThala leeNcwadi elisulungekileyo liqinisekisiwe. Jonga kwi-Olink esebenzayo Phonononga Incwadi Yomsebenzisi ngemiyalelo malunga nolawulo lomgangatho.
Cwangcisa ukubaleka kolandelelwano
Ithala leencwadi le-Olink elinye linolandelelwano ngokwe-NextSeq™ 550 yeSeli yokuPhuma okuPhakamileyo kunye nokubaleka ngakunye. Inani leeseli ze-High Output flow flow and runs ezifunekayo ukulandelelanisa i-Olink Explore Reagent Kits ichazwe kwiTheyibhile 1. Ukuba kufuneka ukubaleka okungaphezulu kwesinye, phinda imiyalelo echazwe kule ncwadana.
Itheyibhile 1. Ukulandelelanisa isicwangciso sokuqhuba:
| Olink® Phonononga iReagent Kit | Inani leelayibrari ze-Olink | Inani lokuqukuqela (ii)seli kunye ne(s) |
| I-Olink® Phonononga i-384 Reagent Kit | 1 | 1 |
| I-Olink® Phonononga i-4 x 384 yeReagent Kit | 4 | 4 |
| I-Olink® Phonononga i-1536 Reagent Kit | 4 | 4 |
| Olink® Phonononga Ukwandiswa kweReagent Kit | 4 | 4 |
| I-Olink® Phonononga i-3072 Reagent Kit | 8 | 8 |
Faka iresiphi yesiqhelo ye-Olink®
Ngeli nyathelo, i-Olink® iresiphi yesiko ifakwe kwi-NextSeq™ 550. Eli nyathelo lifuna ukwenziwa kanye kuphela, ngaphambi kokuba ulandelelwano lwe-Olink lwenziwe okokuqala.
PHAWULA: Iresiphi yesiko le-Olink iya kusebenza kuphela nge-NextSeq™ 500/550 yeeKits zeZiphumo eziPhezulu kunye ne-NextSeq™ Control Software 4.0
- Vula i-zip kwaye ubeke iresiphi yesiko ye-Olink Olink_NSQ550_HighOutput_V1 kwifolda elandelayo yesixhobo se-NextSeq™ 550: C:\Program Files\Illumina\NextSeq Control Software\Recipe\Custom\High\.
- Ngaphantsi kweNkqubo yokuLungisa> Lawula iSixhobo, vumela iiRecipes zesiNtu. Ukuba ayikhethwanga, ukhetho lweresiphi yesiko aluyi kuvela ngexesha lokuseta.
PHAWULA: Kwi-software ye-NCS 4.0, ukhetho lokukhetha iresiphi yesiko luya kwenzeka kuphela emva kokuba i-cartridge ye-reagent ilayishiwe, kungekhona kwiphepha lokucwangcisa kwangaphambili.
PHAWULA: Ukubaleka kufuneka kusekwe kwimowudi yesandla ukuvumela iiresiphi zesiko.
Lungiselela ulandelelwano reagents
Ngethuba le nyathelo, i-cartridge ye-reagent equkethe i-clustering kunye ne-reagents yokulandelelanisa iyancibilika kwaye iseli yokuhamba iyalungiswa.
Lungisa ikhatriji yereagent
ISILUMKISO: Ikhatriji yereagent iqulethe iikhemikhali ezinokuba yingozi. Nxiba izixhobo ezifanelekileyo zokukhusela kwaye ulahle ii-reagents ezisetyenzisiweyo ngokuhambelana nemigangatho esebenzayo. Ngolwazi oluthe kratya, bhekisa kwiSikhokelo seNkqubo ye-Illumina NextSeq 550 (uxwebhu #15069765).
Lungisa ibhentshi
- I-1x i-NextSeq™ 500/550 ye-Reagent ye-High Output Reagent Cartridge v2 (imijikelo engama-75).
Imiyalelo
- Beka i-cartridge ye-reagent enomkhenkce kwisiqingatha-ifakwe emanzini apholileyo kwaye uyivumele inyibilike iyure enye. Qinisekisa ukuba zonke iireservoirs zeekhatriji ziyanyibilika ngokupheleleyo.
PHAWULA: Ukwenzela lula, yinyibilikisa ikhatriji kusuku olungaphambili kwaye uyigcine ngobusuku kwi-4 °C. Kulo bushushu, ii-reagents zizinzile ukuya kwiveki enye. - Yomisa isiseko sekhatriji ngokucokisekileyo ngetawuli yephepha kwaye ucime i-foil etywiniweyo yomile ngethishu engena-lint xa kukho imfuneko.
- Guqula ikhatriji kalishumi ukuze udibanise ngokucokisekileyo ii-reagents ezinyibilikisiweyo ngaphakathi.
- Thepha i-cartridge ngobunono kwibhentshi ukususa amaqamza omoya. Gcina i-cartridge kwindawo yokushisa ukuba iya kusetyenziswa kwiiyure ze-4.
Lungisa i-flow cell
Lungisa ibhentshi
- 1x I-NextSeq™ 500/550 iSeli yokuPhuhlisa okuPhakamileyo okuPhumayo v2.5.
Imiyalelo
- Yizisa i-flow cell efrijini kwiqondo lokushisa kwegumbi imizuzu engama-30.
- Nxiba iiglavu ezintsha zasimahla (ukunqanda ukungcolisa umphezulu weglasi yeseli yokuhamba).
- Xa ulungele ukulayisha i-flow cell kwisixhobo, susa i-flow cell kwi-package kunye ne-clamshell yeplastiki.
- Jonga i-flow cell. Ukuba i-particle okanye uthuli lubonakala kuyo nayiphi na imigangatho yeglasi, coca indawo esebenzayo nge-isopropyl alcohol-free isopropyl yosula kwaye uyomise nge-low-lint lab tissue.
Lungiselela iThala leencwadi le-Olink® ukulungiselela ulandelelwano
Ngeli nyathelo, i-NaOH kunye ne-Tris-HCl i-dilutions ilungiselelwe, kwaye i-Library ye-Olink ehlanjululweyo kunye nekhwalithi ihlanjululwe kwaye ihlanjululwe ngamanyathelo alandelelanayo.
Lungiselela i-NaOH dilution
I-NaOH dilution isetyenziselwa ukuguqulela ithala leencwadi.
Lungisa ibhentshi
- 1 N NaOH isitokhwe
- MilliQ amanzi
- I-1x Microcentrifuge ityhubhu (1.5 mL)
- Ipayipi yesandla (10-100 μL)
- Iingcebiso ze-pipette zokucoca
Ngaphambi kokuba uqale
Phawula ityhubhu ye-microcentrifuge "NaOH".
Imiyalelo
- Lungiselela umxube we-0.2 N NaOH kwiThubhu ye-NaOH ngokweTheyibhile yesi-2.
- Vortex i-NaOH Tube ngokucokisekileyo kwaye ujikeleze ezantsi. Sebenzisa kwiiyure ze-12.
Itheyibhile 2. 0.2 AKUKHO I-NaOH dilution
| I-Reagent | Umthamo (μL) |
| MilliQ amanzi | 80 |
| 1 N NaOH isitokhwe | 20 |
2.4.2 Lungiselela i-Tris-HCl dilution
I-Tris-HCl dilution isetyenziselwa ukwenza iThala leeNcwadi lingasebenzi.
Lungisa ibhentshi
- I-1 M Tris-HCl pH 7.0 isitokhwe (isisombululo se-Trizma® hydrochloride)
- MilliQ amanzi
- I-1x Microcentrifuge ityhubhu (1.5 mL)
- Ipayipi yesandla (10-100 μL)
- Iingcebiso ze-pipette zokucoca
Ngaphambi kokuba uqale
- Phawula ityhubhu ye-microcentrifuge "Tris-HCl"
Imiyalelo
- Lungiselela i-200 mM ye-Tris-HCl dilution kwi-Tris-HCl Tube ngokweTheyibhuli yesi-3.
- Vortex i-Tris-HCl Tube ngokucokisekileyo kwaye uyijikelezise.
Uluhlu 3. 200 mM Tris-HCl dilution:
| I-Reagent | Umthamo (μL) |
| MilliQ amanzi | 80 |
| I-1M Tris-HCl pH 7.0 isitokhwe (isisombululo se-Trizma® hydrochloride) | 20 |
Nciphisa iilayibrari ze-Olink®
Ngeli nyathelo, iLayibrari ye-Olink ehlanjululweyo kunye nomgangatho olawulwayo ihlanjululwe 1:33.
Lungisa ibhentshi
- I-Lib Tube, elungiselelwe ngokuhambelana ne-Olink yokuhlola i-Manual yoMsebenzisi
- MilliQ amanzi
- I-1x Microcentrifuge ityhubhu (1.5 mL)
- Iipayipi ezenziwe ngesandla (0.5-10 kunye ne-100-1000 μL)
- Iingcebiso ze-pipette zokucoca
Ngaphambi kokuba uqale
- Nyibilikisa iLib Tube ukuba ingumkhenkce.
- Phawula ityhubhu entsha ye-microcentrifuge: "Dil".
Imiyalelo
- Yongeza i-96 μL yamanzi e-MilliQ kwi-Dil Tube.
- Vortex iLib Tube kwaye uyijikeleze ngokufutshane.
- Ukutshintshela i-3 μL ukusuka kwiThubhu yeLib ukuya kwiThubhu yeDil.
- Vortex i-Dil Tube kwaye uyijikeleze ngokufutshane
Denature kwaye unciphise iThala leeNcwadi ye-Olink® ukuya kugxininiso lokugqibela lokulayisha
Ngeli nyathelo, iThala leencwadi le-Olink elihlanjululweyo lihlanjululwe kwaye lihlanjululwe ngakumbi kugxininiso lokugqibela lokulayisha.
Lungisa ibhentshi
- I-Dil Tube, elungiselelwe kwinqanaba langaphambili
- 0.2 NONE
- I-200 mM Tris-HCl (pH 7.0) dilution, ilungiswe kwisinyathelo sangaphambili
- I-Hybridization buffer 1 (HT1) ibandakanywe kwi-NextSeq™ Accessory Box v2
- I-2x Microcentrifuge tubes (1.5 mL kunye ne-2 mL)
- Iipayipi ezenziwe ngesandla (0.5-10 kunye ne-100-1000 μL)
- Iingcebiso ze-pipette zokucoca
Ngaphambi kokuba uqale
- Nyibilikisa isithinteli seHT1 esinomkhenkce kwiqondo lobushushu begumbi. Gcina ku +4 °C de usetyenziswe.
- Phawula ityhubhu entsha ye-1.5 mL ye-microcentrifuge: "I-Den" (yeThala leeNcwadi).
- Phawula ityhubhu entsha ye-2 mL microcentrifuge: "Seq" (ukulungele ukulayisha iThala leencwadi).
Imiyalelo
- Ukutshintshela i-5 μL ukusuka kwiThubhu yeDil ukuya kwiThubhu yeDen.
- Yongeza i-5 μL ye-0.2 N NAOH kwiThubhu ye-Den.
- Vortex i-Den Tube kwaye uyijikeleze ngokufutshane.
- Fudumeza iThubhu yomHlaba kangangemizuzu emi-5 kwiqondo lobushushu begumbi ukuguqula iThala leencwadi.
- Yongeza i-5 μL ye-200 mM Tris-HCl (pH 7.0) kwi-Den Tube ukuze uthintele ukusabela.
- Vortex i-Den Tube kwaye uyijikeleze ngokufutshane.
- Yongeza i-985 μL ye-HT1 esele iqatyiwe kwiTube yeDen.
- Vortex i-Den Tube kwaye uyijikeleze ngokufutshane. Umbhobho unokugcinwa kwi +4 °C de usetyenziswe (kwangolo suku lunye).
- Ukutshintshela i-205 μL ukusuka kwiThubhu ye-Den ukuya kwi-Seq Tube.
- Yongeza i-1095 μL ye-HT1 echetyiweyo kwi-Seq Tube.
- Guqula i-Seq Tube ukuxuba ii-reagents kwaye uyijikelezise phantsi ngokufutshane. Umthamo wokugqibela wokulayisha yi-1.3 mL.
- Ngokukhawuleza uqhubele phambili kwi-2.5 Yenza i-Olink® sequencing run.
Yenza i-Olink® yokulandelelanisa ukubaleka
Ngeli nyathelo, i-cartridge ye-buffer, i-flow cell kunye ne-cartridge ye-reagent elungiselelwe equkethe i-Olink Library ilayishwa kwi-NextSeq 550, kwaye ulandelelwano luqaliswe ngokusebenzisa iresiphi yesiko le-Olink.
Lungisa ibhentshi
- I-Seq Tube (esele ilungele ukulayisha iThala leencwadi), elungiselelwe kwinyathelo langaphambili
- I-1x i-NextSeq™ 500/550 ye-Reagent ephezulu ye-Reagent Cartridge v2, elungiselelwe kwinqanaba elidlulileyo
- 1x I-NextSeq™ 500/550 iSeli yokuPhuhlisa okuPhakamileyo v2.5, elungiselelwe kwinqanaba elidlulileyo
- 1x NextSeq™ 500/550 Buffer Cartridge v2 (imijikelo engama-75), kwiqondo lobushushu begumbi
Cwangcisa iiparamitha zokuqhuba ngokulandelelana
Ngeli nyathelo, iiparamitha zokuqhuba ezilandelelanayo zikhethwa kwi-NextSeq™ 550.
- Kwisikrini sasekhaya se-NextSeq™ 550, khetha uMfuniselo.
- Kwisikrini sokukhetha i-Assay, khetha Ulandelelwano.
- Kwiphepha lokuSebenza ngokuseta, khetha imowudi yokuqhuba ngesandla kwaye emva koko Okulandelayo.
- Seta iiparamitha zokuqhuba ngolu hlobo lulandelayo:
- Kwindawo ethi Qhuba Igama, ngenisa i-ID yovavanyo olulodwa.
- Kwindawo yeSazisi yeThala leencwadi, ngenisa i-ID yeThala leeNcwadi olenzayo (uyazikhethela).
- Kwi-Read Type field, khetha ukhetho lokuFunda okulodwa.
- Faka inani lemijikelo ngolu hlobo lulandelayo:
- Funda eyoku-1:24
- Isalathiso 1: 0
- Isalathiso 2: 0
- Funda eyoku-2:0
- Gcina ibhokisi yokukhangela yeeprimer zesiko elingakhethwanga.
- Cwangcisa indawo yesiqulathi seefayili yesiphumo sangoku sedatha ekrwada. Khetha Bhrawuza ukutshintsha indawo yesiqulathi seefayili.
- Musa ukuseta i-Sample Sheet.
- Khetha Khupha izinto ezisetyenziswayo kolu hambo.
- Khetha Okulandelayo.
Layisha iseli yokuhamba kwi-NextSeq™ 550
- Susa iseli esetyenzisiweyo ekuqhubeni kwangaphambili.
- Beka iflowu cell entsha elungisiweyo kwi-stage.
- Khetha Layisha. Ucango luvalwa ngokuzenzekelayo.
- Xa i-ID yeseli ehambayo ibonakala kwisikrini kwaye abenzi boluvo bajongiwe eluhlaza, khetha Okulandelayo
Khulula isikhongozeli se-reagent
ISILUMKISO: Le seti yee-reagents iqulethe iikhemikhali ezinokuba yingozi. Nxiba izixhobo ezifanelekileyo zokukhusela kwaye ulahle ii-reagents ezisetyenzisiweyo ngokuhambelana nemigangatho esebenzayo. Ngolwazi oluthe kratya, bhekisa kwiSikhokelo seNkqubo ye-Illumina NextSeq 550.
- Vula ucango lwekhompatimenti ye-buffer, susa isikhongozeli se-reagents esichithiweyo ukusuka kwigumbi elisezantsi, kwaye ulahle umxholo ngokuhambelana nemigangatho esebenzayo.
- Slayidela isikhongozeli se-reagent esingenanto ubuyele kwindawo esezantsi yebuffer. Ukucofa okuvakalayo kubonisa ukuba isikhongozeli sibekwe ngokuchanekileyo.
Layisha ikhatriji yesithinteli
- Susa ikhatriji yesithinteli esetyenzisiweyo ukusuka kwindawo ephezulu ye-buffer kwaye ulahle umxholo ngokuhambelana nemigangatho esebenzayo.
- Slayidela ikhatriji entsha yesithinteli kwindawo ephezulu yesithinteli. Ukucofa okuvakalayo kubonisa ukuba i-cartridge ibekwe ngokuchanekileyo. Qinisekisa ukuba i-ID yekhatriji yesithinteli ibonakala kwikhusi kwaye abenzi boluvo bakhangelwe eluhlaza.
- Vala ucango lwekhompatimenti yesithinteli kwaye ukhethe Okulandelayo.
Layisha ikhatriji yereagent
- Vula umnyango we-reagent compartment, susa i-cartridge ye-reagent esetyenzisiweyo, kwaye ulahle umxholo ongasetyenziswanga ngokuhambelana nemigangatho esebenzayo. Idama elikwindawo yesi-6 liyasuswa ukuze kube lula ukulahlwa ngokukhuselekileyo.
- Gcoba itywina lendawo yokugcina amanzi #10 ebhalwe “Layisha iThala leeNcwadi Apha” ngencam yepipette ecocekileyo eyi-1 mL.
- Layisha i-1.3 mL yeThala leeNcwadi le-Olink ukusuka kwi-Seq Tube kwi-reservoir #10 ebhalwe ngokuthi “Layisha iThala leencwadi Apha”.
- Slayida ikhatriji entsha ye-reagent kwi-reagent compartment kwaye uvale umnyango we-reagent compartment.
- Khetha Layisha kwaye ulinde i-~30 imizuzwana de i-ID ye-cartridge ye-reagent ivele kwisikrini kwaye abenzi boluvo bakhangelwe eluhlaza.
- Kuluhlu lokuhla lweResiphi, khetha i [Siko] “Olink_NSQ550_HighOutput_V1” iresiphi ukhetho.
- Khetha Okulandelayo.
Qalisa ulandelelwano baleka
- Qinisekisa iiparamitha zokuqhuba eziboniswe kwiReview isikrini. Ukuhlela naziphi na iiparamitha, cofa uBuyela ukuze ubuyele kwiSebenzisa ukuSeta isikrini.
- Khetha Okulandelayo. Ubaleko luqala emva kokukhangela okuzenzekelayo kwangaphambili. Ixesha lokuqhuba ngokulandelelana limalunga ne-7h30 min.
- Coca indawo yokusebenza.
PHAWULA: Xa ulandelelwano lugqityiwe, isoftware iqalisa ukuhlamba okuzenzekelayo emva kokubaleka kusetyenziswa izisombululo zokuhlamba ezibonelelwe kwikhatriji yesithinteli kunye ne-NaOCl enikezelwe kwikhatriji ye-reagent. Oku kuhlamba kuthatha malunga nemizuzu engama-90. Iqhosha laseKhaya liba lisebenze xa uhlamba lugqityiwe. Iikhatriji ezisetyenzisiweyo kunye ne-flow cell zingashiywa kwindawo kude kube kuqhutywe okulandelayo.
Beka esweni inkqubela phambili
U-Olink usebenzisa i-NGS njenge-readout ukulinganisa inani lolandelelwano olwaziwayo ukuze uqikelele ukuxinwa kweprotheni enikeziweyo kwi-s.amples (ngokunxulumene nezinye iziqenduampkancinci). Umgangatho wedatha ukusuka nganye Phonononga ulandelelwano oluqhutywayo lumiselwa ikakhulu yiparamitha ye-QC ekhethekileyo kwitekhnoloji ye-Olink. Ke ngoko, iimetriki zolawulo lomgangatho osemgangathweni osetyenziswa kwi-NGS eqhelekileyo, njenge-Q-score, ayibalulekanga kangako.
Imbali yohlaziyo
| Inguqulelo | Umhla | Inkcazo |
| 1.1 | 2021-12-13 | Iinguqu zokuhlela |
| 1.0 | 2021-12-01 | Entsha |
www.olink.com
Ukusetyenziswa koPhando kuphela. Ayinakusetyenziswa kwiiNkqubo zokuDinga.
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© Copyright 2021 Olink Proteomics AB. Zonke iimpawu zorhwebo zeqela lesithathu ziyipropati yabanini bazo. Olink Proteomics, Dag Hammarskjölds väg 52B , SE-752 37 Uppsala, Sweden
1192, v1.1, 2021-12-13
Amaxwebhu / Izibonelelo
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I-Olink Phonononga ulandelelwano usebenzisa i-NextSeq 550 [pdf] Incwadi yokusebenzisa Jonga ulandelelwano usebenzisa i-NextSeq 550, Phonononga ulandelelwano, usebenzisa i-NextSeq 550, i-NextSeq 550 |
