Olink Ongorora Sequencing uchishandisa NextSeq 550

Nhanganyaya

Chinangwa chekushandisa
Olink® Ongorora ndeye multiplex immunoassay chikuva chekuwanikwa kweprotein biomarker yevanhu. Chigadzirwa chacho chakagadzirirwa Kutsvaga Kushandisa Chete, uye kwete kushandiswa mukuongorora maitiro. Basa remurabhoritari richaitwa chete nevashandi vakadzidziswa. Kugadziriswa kwedata kuchaitwa chete nevashandi vakadzidziswa. Migumisiro inofanirwa kushandiswa nevatsvakurudzi pamwe chete nemamwe makiriniki kana marabhoritari zvakawanikwa.

Nezve bhuku rino
Iri Bhuku reMushandisi rinopa mirairo inodiwa kutevedzana Olink® Ongorora Maraibhurari paIllumina® NextSeq™ 550. Mirayiridzo inofanira kunyatsoteverwa uye zvakajeka. Chero kutsauka mumatanho ese emurabhoritari kunogona kukonzera kukanganisa data. Usati watanga kufambiswa kwerabhoritari, bvunza iyo Olink® Ongorora Pamusoroview Mushandisi Manual yekusuma papuratifomu, kusanganisira ruzivo nezve reagents, zvishandiso uye zvinyorwa zvinodiwa, pamusoroview yemafambiro ebasa, pamwe chete nemirayiridzo yerabhoritari. Kuti uwane mirairo yemaitirwo eOlink® Ongorora Reagent Kits, tarisa kune inoshanda Olink® Ongorora Mushandisi Manual. Kuti uwane data yekugadzirisa uye kuongororwa kweOlink® Ongorora kutevedzana mhedzisiro, tarisa iyo Olink® MyData Cloud User Guide. Ese maratidziro uye kodzero dzekodzero dziri muchinhu ichi zvinhu zveOlink® Proteomics AB, kunze kwekunge zvataurwa neimwe nzira.

Tsigiro yehunyanzvi
Kuti uwane rutsigiro rwehunyanzvi, bata Olink Proteomics pa support@olink.com.

Laboratory mirayiridzo

Ichi chitsauko chinopa mirairo yekuteedzera maraibhurari eOlink paNextSeq™ 550 uchishandisa NextSeq™ 500/550 High Output Kit v2.5 (75 Cycles). Iyo protocol inoshandiswa pakutevedzana ndeyekugadziriswa kweIllumina® standard NGS workflow yeIllumina® NextSeq™ 550. Usati waenderera mberi nekugadzirisa, iva nechokwadi chokuti hutano hweRibhurari yakacheneswa yakasimbiswa. Tarisa kune inoshanda Olink Ongorora Mushandisi Manual kune mirairo nezve kutonga kwemhando.

Ronga kutevedzana kumhanya
Imwe Olink Raibhurari inogona kutevedzana neNextSeq™ 550 Yakakwira Output inoyerera sero uye nekumhanya. Huwandu hweMasero ekuyerera kweKubuda Kwepamusoro uye kumhanya kunodiwa kuteedzera akasiyana Olink Ongorora Reagent Kits inotsanangurwa muTebhu 1. Kana kupfuura kumhanya kumwe kuchidiwa, dzokorora mirairo inotsanangurwa mubhuku rino.

Table 1.Sequencing run planning:

Olink® Ongorora Reagent Kit	Nhamba yeOlink Libraries	Nhamba yemasero ekuyerera uye kumhanya (s)
Olink® Ongorora 384 Reagent Kit	1	1
Olink® Ongorora 4 x 384 Reagent Kit	4	4
Olink® Ongorora 1536 Reagent Kit	4	4
Olink® Ongorora Kuwedzera Reagent Kit	4	4
Olink® Ongorora 3072 Reagent Kit	8	8

Isa Olink® tsika yekubika
Munguva iyi, iyo Olink® tsika yekubika inoiswa paNextSeq™ 550. Iyi nhanho inongoda kuitwa kamwe chete, Olink isati yaitwa sequencing run kekutanga.
CHERECHEDZA: Iyo Olink tsika yekubika inongoshanda neNextSeq™ 500/550 Yakakwira Output Kits uye iyo NextSeq™ Kudzora Software 4.0.

1. Unzip uye isa iyo Olink tsika resipi Olink_NSQ550_HighOutput_V1 mune inotevera folda yeNextSeq™ 550 chiridzwa: C:\Chirongwa Files\Illumina\NextSeq Kudzora Software\Recipe\Custom\High\.
2. Pasi peSystem Customization> Manage Instrument, gonesa Tsika Recipes. Kana isina kusarudzwa, iyo tsika yekubika sarudzo haizooneke panguva yekumhanyisa kuseta.

CHERECHEDZA: MuNCS 4.0 software vhezheni, iyo sarudzo yekusarudza tsika yekubikira ichaitika chete mushure mekunge reagent cartridge yaiswa, kwete mune yekutanga setup peji.
CHERECHEDZA: Kumhanya kunofanirwa kuiswa mune yemanyorero modhi kubvumidza tsika dzekubika.

Gadzirira sequencing reagents

Munguva iyi, iyo reagent cartridge ine clustering uye sequencing reagents inonyungudutswa uye sero rekuyerera rinogadzirwa.

Gadzirira reagent cartridge
YAMBIRO: Iyo reagent cartridge ine makemikari anogona kuve ane njodzi. Pfeka midziyo yekudzivirira yakakwana uye urase mareagents akashandiswa zvinoenderana nezviyero zvinoshanda. Kuti uwane rumwe ruzivo, tarisa kuIllumina NextSeq 550 System Guide (gwaro #15069765).

Gadzirira bhenji

• 1x NextSeq™ 500/550 Yakakwira Kubuda Reagent Cartridge v2 (75 mitsetse).

Mirayiridzo

1. Isa chando reagent cartridge hafu-yakanyura mukamuri yemvura yakadzikama uye rega inyunguduke kweawa imwe. Ita shuwa kuti ese reagent reservoirs emakaturiji akanyungudika zvachose.
   CHERECHEDZA: Kuti zvive nyore, nyungudutsa cartridge zuva rakapfuura uye uichengete usiku hwose pa4 ° C. Pakupisa uku, ma reagents anogadzikana kusvika vhiki imwe.
2. Pukuta chigadziko checartridge zvakakwana nejira repepa uye bvisa zvisimbiso zvefoiri zviome nemucheka usina lint kana zvichidikanwa.
3. Pindura katuriji kagumi kuti unyatso kusanganisa ma reagents akanyungudika mukati.
4. Tora katuriji zvinyoro nyoro pabhenji kuti ubvise mabubble emhepo. Chengetedza cartridge patembiricha yekamuri kana ichizoshandiswa mukati memaawa mana.

Gadzirira kuyerera sero

Gadzirira bhenji

• 1x NextSeq™ 500/550 Yakakwira Kubuda Kuyerera Sero v2.5.

Mirayiridzo

1.  Uya nefiriji inoyerera sero kune tembiricha yekamuri kwemaminitsi makumi matatu.
2.  Pfeka hupfu hutsva husina magirovhosi (kudzivisa kusvibisa girazi pamusoro pesero rekuyerera).
3.  Kana wagadzirira kurodha kuyerera sero muchiridzwa, bvisa kuyerera sero kubva pasuru uye epurasitiki clamshell.
4.  Ongorora kuyerera sero. Kana marara kana guruva richionekwa pane chero nzvimbo yegirazi, chenesa nzvimbo inoshanda neine lint-isina isopropyl doro pukuta uye woiomesa neinodzika-lint lab tissue.

Gadzirira Olink® Library yekutevedzana

Munguva iyi nhanho, NaOH uye Tris-HCl dilutions inogadzirwa, uye iyo yakacheneswa uye inodzorwa mhando Olink Library inonatswa uye denatured mumatanho anoteedzana.

Gadzirira NaOH dilution
Iyo NaOH dilution inoshandiswa kuratidza maLibraries.

Gadzirira bhenji

• 1 N NaOH stock
• MilliQ mvura
• 1x Microcentrifuge chubhu (1.5 mL)
• Chinyorwa pipette (10-100 μL)
• Sefa mazano epipette

Usati watanga
Maka iyo microcentrifuge chubhu "NaOH".

Mirayiridzo

1. Gadzirira dilution ye0.2 N NaOH muNaOH Tube zvinoenderana neTafura 2.
2. Vortex iyo NaOH Tube zvakakwana uye tenderera pasi. Shandisa mukati maawa gumi nemaviri.

Tafura 2. 0.2 N NaOH dilution

Reagent	Vhoriyamu (μL)
MilliQ mvura	80
1 N NaOH stock	20

2.4.2 Gadzirira Tris-HCl dilution
Iyo Tris-HCl dilution inoshandiswa kumisa iyo denaturated Library.

Gadzirira bhenji

• 1 M Tris-HCl pH 7.0 stock (Trizma® hydrochloride solution)
• MilliQ mvura
• 1x Microcentrifuge chubhu (1.5 mL)
• Chinyorwa pipette (10-100 μL)
• Sefa mazano epipette

Usati watanga

• Maka iyo microcentrifuge chubhu "Tris-HCl"

Mirayiridzo

1. Gadzirira 200 mM Tris-HCl dilution muTris-HCl Tube maererano neTafura 3.
2. Vortex iyo Tris-HCl Tube zvakakwana uye itenderere pasi.

Tafura 3. 200 mM Tris-HCl dilution:

Reagent	Vhoriyamu (μL)
MilliQ mvura	80
1M Tris-HCl pH 7.0 stock (Trizma® hydrochloride solution)	20

Dilute Olink® Libraries
Munguva iyi nhanho, iyo yakacheneswa uye yemhando inodzorwa Olink Library inonatswa 1:33.

Gadzirira bhenji

• Lib Tube, yakagadzirirwa zvinoenderana neiyo inoshanda Olink Ongorora Mushandisi Manual
• MilliQ mvura
• 1x Microcentrifuge chubhu (1.5 mL)
• Manual pipettes (0.5-10 uye 100-1000 μL)
• Sefa mazano epipette

Usati watanga

• Nyora iyo Lib Tube kana yakaoma.
• Maka iyo itsva microcentrifuge chubhu: "Dil".

Mirayiridzo

1. Wedzera 96 ​​μL yeMilliQ mvura kune Dil Tube.
2. Vortex iyo Lib Tube uye itenderedze pasi muchidimbu.
3. Chinja 3 μL kubva kuLib Tube kuenda kuDil Tube.
4. Vortex iyo Dil Tube uye itenderedze pasi muchidimbu

Denature uye dilute Olink® Raibhurari kune yekupedzisira yekurodha yevasungwa
Pakati peichi nhanho, iyo yakanyungudutswa Olink Raibhurari inodzorwa uye inonatswazve kune yekupedzisira kurodha yekumisikidza.

Gadzirira bhenji

• Dil Tube, yakagadzirirwa mune yapfuura nhanho
• 0.2 N NaOH dilution, ichangogadzirwa munhanho yapfuura
• 200 mM Tris-HCl (pH 7.0) dilution, yakagadzirirwa mudanho rapfuura
• Hybridization buffer 1 (HT1) inosanganisirwa muNextSeq™ Accessory Bhokisi v2
• 2x Microcentrifuge tubes (1.5 mL uye 2 mL)
• Manual pipettes (0.5-10 uye 100-1000 μL)
• Sefa mazano epipette

Usati watanga

• Chengetedza HT1 yakaomeswa nechando patembiricha yekamuri. Chengetedza pa +4 °C kusvikira washandiswa.
• Maka iyo nyowani 1.5 mL microcentrifuge chubhu: "Den" (yeiyo denaturated Library).
• Maka iyo itsva 2 mL microcentrifuge chubhu: "Seq" (yekugadzirira kurodha Raibhurari).

Mirayiridzo

1. Chinja 5 μL kubva kuDil Tube kuenda kuDen Tube.
2. Wedzera 5 μL ye0.2 N NaOH kuDen Tube.
3. Vortex iyo Den Tube uye itenderere pasi muchidimbu.
4. Isa iyo Den Tube kwemaminetsi mashanu pane tembiricha yekudziya kuti utore Raibhurari.
5. Wedzera 5 μL ye 200 mM Tris-HCl (pH 7.0) kuDen Tube kuti urege kuita.
6. Vortex iyo Den Tube uye itenderere pasi muchidimbu.
7. Wedzera 985 μL ye prechilled HT1 kuDen Tube.
8. Vortex iyo Den Tube uye itenderere pasi muchidimbu. Iyo chubhu inogona kuchengetwa pa +4 ° C kusvika yashandiswa (zuva rimwe chetero).
9. Chinja 205 μL kubva kuDen Tube kuenda kuSeq Tube.
10. Wedzera 1095 μL ye prechilled HT1 kuSeq Tube.
11. Pindura iyo Seq Tube kusanganisa ma reagents uye nekuitenderedza pasi muchidimbu. Yekupedzisira kurodha vhoriyamu ndeye 1.3 mL.
12. Pakarepo enderera kune 2.5 Ita Olink® sequencing run.

Ita Olink® sequencing run

Munguva iyi, iyo buffer cartridge, iyo inoyerera sero uye yakagadzirwa reagent cartridge ine Olink Library inoiswa muNextSeq 550, uye kutevedzana kumhanya kunotanga uchishandisa Olink tsika resipi.

Gadzirira bhenji

• Seq Tube (ine yakagadzirira kurodha Raibhurari), yakagadzirirwa mune yapfuura nhanho
• 1x NextSeq™ 500/550 Yakakwira Kubuda Reagent Cartridge v2, yakagadzirirwa mudanho rapfuura.
• 1x NextSeq™ 500/550 Yakakwira Kubuda Kuyerera Sero v2.5, yakagadzirirwa mudanho rapfuura
• 1x NextSeq™ 500/550 Buffer Cartridge v2 (75 kutenderera), pamhepo tembiricha

Gadzirisa sequencing run parameters
Pakati peichi nhanho, kuteedzera run paramita inosarudzwa paNextSeq™ 550.

1. PaNextSeq™ 550 Home skrini, sarudza Edza.
2. Pane Sarudza Assay skrini, sarudza Sequence.
3. Mu Run Setup peji, sarudza Manual run mode uyezve Next.
4. Gadzira iyo run parameters sezvinotevera:
   • MuRun Name ndima, isa yakasarudzika ID yekuyedza.
   • Muchikamu cheLibhurari ID, isa ID yeLibhurari yauri kumhanyisa (sarudzo).
   • Muchikamu cheKuverenga Rudzi, sarudza iyo Single Read sarudzo.
   • Isa nhamba yema cycles sezvinotevera:
     • Verenga 1:24
     • Index 1:0
     • Index 2:0
     • Verenga 2:0
   • Chengetedza bhokisi rekutarisa kune zvekutanga zvetsika zvisina kusarudzwa.
   • Seta iyo inobuda folda nzvimbo yeyazvino mhanyisa data raw. Sarudza Bhurawuza kuti uchinje inobuda folda nzvimbo.
5. Usagadzira Sample Sheet.
6. Sarudza Purge zvinobatika pakuita uku.
7. Sarudza Next.

Rodha kuyerera sero muNextSeq™ 550

1. Bvisa sero rinoyerera rakashandiswa kubva pakumhanya kwekare.
2. Isa mutsva wakagadzirirwa kuyerera sero pane stage.
3. Sarudza Mutoro. Gonhi rinobva ravharwa.
4. Kana iyo yekuyerera cell ID ikabuda pachiratidziro uye masensa akatariswa mugirini, sarudza Next

Bvisa mudziyo we reagent
YAMBIRO: Iyi seti yemareagents ine makemikari anogona kuva nengozi. Pfeka midziyo yekudzivirira yakakwana uye urase mareagents akashandiswa zvinoenderana nezviyero zvinoshanda. Kuti uwane rumwe ruzivo, tarisa kuIllumina NextSeq 550 System Guide.

1. Vhura musuwo webuffer compartment, bvisa yakashandiswa reagents mudziyo kubva mukamuri rezasi, uye bvisa zvirimo zvinoenderana nezvinodiwa.
2. Svetera mudziyo usina chinhu reagent udzokere mukamuri yepasi buffer. Kudzvanya kunonzwika kunoratidza kuti mudziyo wakaiswa zvakanaka.

Rodha buffer cartridge

1. Bvisa iyo buffer cartridge yakashandiswa kubva kumusoro buffer compartment uye bvisa zvirimo zvinoenderana nezvinodiwa.
2. Svetera katuriji nyowani yebuffer mune yepamusoro buffer compartment. Kudzvanya kunonzwika kunoratidza kuti cartridge yakaiswa nemazvo. Ita shuwa kuti iyo buffer cartridge ID inoonekwa pachiratidziri uye kuti ma sensor anotariswa mugirini.
3. Vhara musuwo webuffer compartment uye sarudza Next.

Rodha reagent cartridge

1. Vhura musuwo weiyo reagent compartment, bvisa yakashandiswa reagent cartridge, uye bvisa izvo zvisina kushandiswa zvinoenderana nezviyero zvinoshanda. Dziva riri pachinhanho chechitanhatu rinobvisika kuti rifambise kuraswa kwakachengeteka.
2. Baya chisimbiso chedhamu #10 yakanyorwa kuti "Road Library Pano" ine yakachena 1 mL pipette tip.
3. Rodha 1.3 mL iyo Olink Raibhurari kubva kuSeq Tube mudura #10 yakanyorwa se "Road Library Pano".
4. Svetera iyo itsva reagent cartridge mukati meiyo reagent compartment uye vhara iyo reagent compartment gonhi.
5. Sarudza Mutoro uye mirira ~ 30 masekondi kusvika reagent cartridge ID yaonekwa pachiratidziro uye ma sensors akatariswa mugirini.
6. Kubva pane Recipe yekudonhedza pasi, sarudza iyo [Custom] "Olink_NSQ550_HighOutput_V1" nzira yekubika.
7. Sarudza Next.

Tanga kutevedzana kumhanya

1. Simbisa iyo inomhanya paramita inoratidzwa paReview skrini. Kuti ugadzirise chero ma paramita, tinya Kudzokera kuti udzokere kuRun Setup skrini.
2. Sarudza Next. Kumhanya kunotanga mushure me otomatiki pre-run cheki. Iyo yekutevedzana yekumhanya nguva ingangoita 7h30 min.
3. Chenesa nzvimbo yebasa.

CHERECHEDZA: Kana iyo yekutevedzana yapera, software inotanga otomatiki post-run kugeza uchishandisa wacha mhinduro dzakapihwa mubuffer cartridge uye NaOCl yakapihwa mu reagent cartridge. Kugeza uku kunotora maminetsi angangoita makumi mapfumbamwe. Bhatani reKumba rinotanga kushanda kana kugeza kwapera. Akashandiswa cartridges uye kuyerera sero anogona kusiiwa munzvimbo kusvikira kunotevera kumhanya.

Monitor kumhanya kufambira mberi
Olink inoshandisa NGS sekuverenga kuverenga kuti iverenge huwandu hwekutevedzana kunozivikanwa kuitira kufungidzira kuwanda kweprotein yakapihwa mu s.amples (zvinoenderana nemamwe sampzvishoma). Hunhu hwedhata kubva kune yega yega Ongorora kuteedzera kumhanya inonyanya kutsanangurwa neQC paramita yakasarudzika kune Olink tekinoroji. Nekudaro, akajairwa emhando yekudzora metrics anoshandiswa mune akajairwa NGS, senge Q-chibodzwa, haana kushomeka.

Nhoroondo yekudzokorora

Version	Date	Tsanangudzo
1.1	2021-12-13	Shanduko dzekunyora
1.0	2021-12-01	New

www.olink.com
Zvekutsvaga Shandisa Chete. Kwete Yekushandisa muDiagnostic Procedus.
Ichi chigadzirwa chinosanganisira rezinesi rekushandisa zvisiri zvekutengesa zveOlink zvigadzirwa. Vashandisi vezvokutengeserana vangada mamwe marezinesi. Ndokumbira ubate Olink
Proteomics AB yeruzivo. Iko hakuna waranti, inoratidzwa kana kurehwa, iyo inopfuurira kupfuura iyi tsananguro. Olink Proteomics AB haina mhosva yekukuvadzwa kwepfuma, kukuvara kwemunhu, kana kurasikirwa kwehupfumi kunokonzerwa nechigadzirwa ichi.
Chiratidzo chinotevera ndeche Olink Proteomics AB: Olink®.
Ichi chigadzirwa chakafukidzwa nematendi akati wandei uye patent application inowanikwa pa https://www.olink.com/patents/.
© Copyright 2021 Olink Proteomics AB. Ese echitatu bato trademark zvinhu zvevaridzi vazvo. Olink Proteomics, Dag Hammarskjölds väg 52B , SE-752 37 Uppsala, Sweden
1192, v1.1, 2021-12-13

Zvinyorwa / Zvishandiso

Olink Ongorora Sequencing uchishandisa NextSeq 550 [pdf] User Manual Ongorora Sequencing uchishandisa NextSeq 550, Ongorora Sequencing, uchishandisa NextSeq 550, NextSeq 550

References

• User Manual