UKULANDELANA KWE-UNIVERSAL TELL-Seq Ukuthuthukisa Ithagethi

Imininingwane:

• Igama Lomkhiqizo: TELL-SeqTM Target Enrichment
• Ngokuba: Sebenzisa Ucwaningo Kuphela, hhayi izinqubo zokuxilonga
• Umkhiqizi: I-Universal Sequencing Technology Corporation
• I-Genomic DNA edingekayo: 5ng
• Izimo Zesitoreji: Ibhafa ye-Tris pH 7.5-8.0 noma ibhafa ye-TE ephansi

Imiyalo yokusetshenziswa komkhiqizo

Izincomo Zokufaka kwe-Genomic DNA:
Qinisekisa ukuthi okungenani une-5ng ye-genomic DNA yephrothokholi. I-DNA yesisindo samangqamuzana aphezulu ibalulekile ekulandeleni okuphumelelayo. Linganisa i-DNA usebenzisa indlela esekelwe ku-fluorometric efana ne-Qubit dsDNA BR Assay Kit noma i-HS Kit. Nciphisa i-DNA egxilile ekugxiliseni ukusebenza (0.4ng/l kuya ku-1ng/l) kubhafa ye-Tris ngaphambi kokulinganisa. Gcina i-genomic DNA ku-Tris buffer ene-pH 7.5-8.0 noma i-TE buffer ephansi.

Okuqukethwe kwekhithi:

TELL-SeqTM Library Prep Kit, Usayizi Ojwayelekile uqukethe okubili amabhokisi:

• Ibhokisi 1: Iqukethe ama-reagents ahlukahlukene afaka i-Barcoding Enzyme, i-Exonuclease, nokuningi.
• Ibhokisi 2: Iqukethe i-TELL Bead Plexb, i-Wash Solution, kanye ne-Stop Solution.

Qaphela: Ungaqandi futhi uncibilikise ama-reagents e-Box 1 izikhathi ezingaphezu kwezingu-6.

I-FAQ:

1. Umbuzo: Ngingasebenzisa ezinye izindlela ngaphandle kwalezo ezisekelwe ku-fluorometric ukuze thola i-DNA?
   A: Kunconywa ukusebenzisa indlela esekelwe ku-fluorometric efana ne-Qubit dsDNA BR Assay Kit noma i-HS Kit ukuze uthole isilinganiso esinembile. Gwema izindlela ezikala kuphela ingqikithi ye-nucleic acid.
2. Q: Kufanele ngiyigcine kanjani i-genomic DNA ukuze ngithole imiphumela emihle?
   A: I-Genomic DNA kufanele igcinwe ku-Tris buffer ene-pH esukela ku-7.5 – 8.0 noma i-TE buffer ephansi (10mM Tris-HCl, 0.1 mM EDTA, pH 8.0) ukuze uthole imiphumela engcono kakhulu.

Okokusetshenziswa Kocwaningo Kuphela. Ayisetshenziselwa izinqubo zokuxilonga.
Idokhumenti # 100032-USG v4.0
Agasti 2024
Le dokhumenti ingeye-Universal Sequencing Technology Corporation futhi ihloselwe ukusetshenziswa ikhasimende layo kuphela ngokuphathelene nokusetshenziswa kwemikhiqizo echazwe lapha ngaphandle kwezinye izinjongo. Imiyalo ekulo mbhalo kufanele ilandelwe kahle ngabasebenzi abaqeqeshwe kahle ukuze kuqinisekiswe ukusetshenziswa okufanele nokuphephile kwekhithi ye-TELL-Seq™.
I-UNIVERSAL SEQUENCING TECHNOLOGY CORPORATION AYICABANGA NGANOMA YISIPHI ISIBOPHO ESIYENZEKA EMVA KOKUSETSHENZISWA OKUNGEMBILE KWEKHITHI YE-TELL-SEQ™.
©2023 I-Universal Sequencing Technology Corporation. Wonke Amalungelo Agodliwe. I-TELL-Seq™ wuphawu lokuthengisa lwe-Universal Sequencing Technology Corporation. Wonke amanye amagama, amalogo nezinye izimpawu zokuthengisa ziyimpahla yabanikazi bazo.

Umlando Wokubuyekeza

Idokhumenti #100032-USG v1.0	Novemba 2021	Ukukhishwa Kwasekuqaleni
Idokhumenti #100032-USG v2.0	Agasti 2022	Ukubuyekezwa kwephrothokholi ukuze kusebenze nekhithi ebuyekeziwe ye-TELL- Seq™ Library Prep V1 ene-Suspension Buffer EZ kanye nenketho ye-TELL Bead Plex
Idokhumenti # 100032-USG v3.0	Agasti 2023	Kukhishwe inketho ye-TELL Bead. TELL Bead Plex kuphela esetshenziswa ukuya phambili. Kwengezwe Inothi kanye nesithombe esinamasistimu okuxuba anconyiwe sesinyathelo esibalulekile sokuzungezisa amashubhu okufanele ngesikhathi senqubo yokubhala ibhakhodi ukuze kulondolozwe izici ze-DNA ezinesisindo esikhulu samangqamuzana.
Idokhumenti # 100032-USG v4.0	Agasti 2024	Umhlahlandlela womsebenzisi oshintshile wokucebisa okuqondisiwe okuvamile. Kwengezwe ulwazi olwengeziwe mayelana nokusebenzisa i-TELL-Seq™ Library Prep namanye amasistimu Okuthuthukisa Ithagethi

Isingeniso

Le phrothokholi ichaza ukuthi ingalungiswa kanjani ithagethi ethuthukisiwe yamitapo yolwazi ebhangqiwe ye-TELL-Seq™ ngokuthwebula ngokuxubile kusetshenziswa inhlanganisela ye-TELL-Seq™ Library Prep Kit kanye ne-Agilent® SureSelect Target Enrichment System. Uhlelo lwe-TELL-Seq™ Library Prep Kit luphinde luhambisane nezinye izinhlelo zokucebisa eziqondiwe ezifana ne-IDT ne-Twist Biosciences.
Ikhithi yokulungiselela umtapo wezincwadi we-TELL-Seq™ isebenzisa ubuchwepheshe obusha be-Transposase Enzyme Linked Long-read Sequencing (TELL-Seq™)† ukuze ilungiselele umtapo wolwazi obhanqiwe ukuze ukhiqize ukufundwa kwebhakhodi exhunywe ohlelweni lokulandelana kwe-Illumina®. I-Agilent® SureSelect Target Enrichment System ivumela ukunothiswa kwezifunda ezihlosiwe kusetshenziswa ama-probe okuthwebula akhethekile. I-Target Enrichment System ngayinye inephaneli ehlukile yama-probe evumela izifunda ezithile ukuthi zithwetshulwe ehambisana nemitapo yolwazi ye-TELL-Seq™.

Izincomo Zokufaka kwe-Genomic DNA
I-5ng Genomic DNA iyadingeka kule phrothokholi. I-high molecular weight (HMW) DNA ibalulekile ekulandeleni okuyimpumelelo kwe-TELL-Seq™.

• Kufuzo lomuntu, ubuncane be-sampUsayizi we-DNA kufanele ube mkhulu kuno-40Kb.
•  I-HMW DNA esukela ku-100Kb kuya ku-300Kb iyimpahla efanelekile yokusetshenziswa kwezigaba komuntu okuhle kakhulu.
• Gwema ukuphula i-HMW DNA ngesikhathi sokuphatha. Susa i-DNA yesisindo se-molecule ephansi (ekhonjwe njenge-smear engaphansi kuka-10Kb kujeli)ample uma bethula ingxenye ebalulekile ku-DNA sample.

Sebenzisa indlela esekelwe ku-fluorometric ukuze ulinganise i-DNA yokufaka. Uma usebenzisa i-Qubit dsDNA BR Assay Kit noma i-HS Kit, sebenzisa okungenani u-2 µL we-DNA ngayinye.ample ngesilinganiso. Gwema izindlela ezikala kuphela ingqikithi ye-nucleic acid, njenge-NanoDrop noma ezinye izindlela zokumunca i-UV. Ukuze uthole ukulinganisa okunembile kokugxiliswa kwe-HMW DNA, hlambulula i-DNA egxilile ekugxiliseni okusebenzayo (0.4ng/µl kuya ku-1ng/µl) kusibhafa se-Tris (pH 7.5-8.0) amahora ambalwa ngosuku ngaphambi kwesilinganiso sokugxilisa ingqondo nokulungiselelwa komtapo. I-Genomic DNA kufanele igcinwe ku-Tris buffer ene-pH esukela ku-7.5 – 8.0 noma i-TE buffer ephansi (10mM Tris-HCl, 0.1 mM EDTA, pH 8.0). Ukuze kuhlolwe ubumsulwa be-DNA sample, isilinganiso sesilinganiso sokumunca ku-260 nm kuya kokumunca ku-280 nm singasetshenziswa. Le protocol ithuthukiselwe i-DNA enamanani esilinganiso sokumunca angu-1.8–2.0. Uma kune-RNA eyeqile ku-DNA sample, kufanele isuswe ngokwelashwa kwe-RNase.

Okuqukethwe Kwekhithi

TELL-Seq™ Library Prep Kit, Usayizi Ojwayelekile (2 Amabhokisi)

Ibhokisi 1 kwangu-2: I-TELL-Seq™ Library Reagent Box 1 V1 (PN 100035)

QAPHELA: Ungaqandi futhi uncibilikise ama-reagents e-Box 1 izikhathi ezingaphezu kwezi-6.

a Okusetshenziswa ne-10× Primer II ku-TELL-Seq™ Library Multiplex Primer Kit ndawonye emtatsheni wezincwadi ampukuqinisa.

Ibhokisi lesi-2 kwangu-2: I-TELL-Seq™ Library Reagent Box 2 V1 (PN 100036)

Igama lengxenye	Umbala Wekhephu	Ivolumu (mL)	Izinga Lokushisa Lesitoreji
TSHELA I-Bead Plexb	CAP Orange	76	2°C kuya ku-8°C
Geza Isixazululo	ICAP Emhlophe	4500	2°C kuya ku-8°C
Misa Isixazululoc	CAP Natural	960	2°C kuya ku-25°C

b TELL Bead Plex isebenza kahle kukho kokubili i-Illumina kanye ne-non-Illumina Sequencing Systems.
c Ngaphambi kokusebenzisa, uma i-Stop Solution ingacacile noma inezimvula ezimhlophe, fudumeza ishubhu libe ngu-37°C. I-Vortex yokuncibilikisa noma iyiphi imvula. Ngemuva kokusetshenziswa kokuqala, gcina i-Stop Solution emiswe kabusha kuzinga lokushisa elilingana negumbi ukuze uyisebenzise ngokuzayo.

ISEXWAYISO

 Ipayipi lepayipi le-TELL-Read v1.1 noma ngaphezulu liyadingeka ukuze kuhlaziywe idatha yokulandelana evela emitatsheni yolwazi ye-TELL-Seq™ elungiswe nge-TELL Bead Plex.

I-TELL-Seq™ Library Prep Kit, HT24 (2 Amabhokisi)
Ibhokisi loku-1 kwangu-2: I-TELL-Seq™ Library Reagent Box 1 V1, HT24 (PN 100037) 

QAPHELA: Ungaqandi futhi uncibilikise ama-reagents e-Box 1 izikhathi ezingaphezu kwezi-6. 

a Okusetshenziswa ne-10× Primer II ku-TELL-Seq™ Library Multiplex Primer Kit ndawonye emtatsheni wezincwadi ampukuqinisa.

Ibhokisi loku-2 kwangu-2: I-TELL-Seq™ Library Reagent Box 2 V1, HT24 (PN 100038)

Igama lengxenye	Umbala Wekhephu		Ivolumu	Izinga Lokushisa Lesitoreji
TSHELA I-Bead Plexb	iwolintshi		456 mL	2°C kuya ku-8°C
Geza Isixazululo		Okuluhlaza okwesibhakabhaka	28.5 mL	2°C kuya ku-8°C
Misa Isixazululoc		Emhlophe	5.76 mL	2°C kuya ku-25°C

b TELL Bead Plex isebenza kahle kukho kokubili i-Illumina kanye ne-non-Illumina Sequencing Systems.
c Ngaphambi kokusebenzisa, uma i-Stop Solution ingacacile noma inezimvula ezimhlophe, fudumeza ishubhu libe ngu-37 ºC. I-Vortex yokuncibilikisa noma iyiphi imvula. Ngemuva kokusetshenziswa kokuqala, gcina i-Stop Solution emiswe kabusha kumazinga okushisa asekamereni ukuze uyisebenzise ngokuzayo.

I-PRO ICEBISO: I-TELL-Seq™ Library Prep Kits AMABILI, i-HT24 ehlanganisa kokubili iBhokisi 1 neBhokisi 2 ingabhangqwa nanoma yimaphi amaKits e-TELL-Seq™ Library Multiplex Primer.

ISEXWAYISO 
Ipayipi lepayipi le-TELL-Read v1.1 noma ngaphezulu liyadingeka ukuze kuhlaziywe idatha yokulandelana evela emitatsheni yolwazi ye-TELL-Seq™ elungiswe nge-TELL Bead Plex.

Ikhithi ye-TELL-Seq™ Library Multiplex Primer (1-8) (PN 100003)

ICEBISO LOPRO: Ikhithi ye-One TELL-Seq™ Library Multiplex Primer (1-8) iqukethe iziqalo ezanele ezingasetshenziswa ne-FOUR TELL-Seq™ WGS Library Prep Kits.

Ikhithi ye-TELL-Seq™ Library Multiplex Primer (9-16) (PN 100009)

ICEBISO LOPRO: I-ONE TELL-Seq™ Library I-Multiplex Primer (9-16) Ikhithi iqukethe ama-primer anele angasetshenziswa namakhithi okulungisela umtapo OKUNE TELL-Seq™ WGS, Usayizi Ojwayelekile.

Ikhithi ye-TELL-Seq™ Library Multiplex Primer (17-24) (PN 100010)

ICEBISO LOPRO: I-ONE TELL-Seq™ Library I-Multiplex Primer (17-24) Ikhithi iqukethe ama-primer anele angasetshenziswa namakhithi okulungisela umtapo OKUNE TELL-Seq™ WGS, Usayizi Ojwayelekile.

I-TELL-Seq™ Illumina® Sequencing Primer Kit (PN 100004)

Igama lengxenye	Umbala Wekhephu	Ukugxila	Ivolumu (mL)	Izinga Lokushisa Lesitoreji
Funda iPrimer engu-1	Mnyama	100mM	50	-25°C kuya ku-15°C

Funda iPrimer engu-2	Emhlophe	100mM	50	-25°C kuya ku-15°C
Inkomba 1 Primer	Okubomvu	100mM	50	-25°C kuya ku-15°C
Inkomba 2 Primer	Okuphuzi	100mM	50	-25°C kuya ku-15°C

I-TELL-Seq™ Target Blocker (PN 100019)

Igama lengxenye	Umbala Wekhephu	Ivolumu (mL)	Izinga Lokushisa Lesitoreji
TELL-Seq™ Target Blocker	ICAP Emhlophe	40	-25°C kuya ku-15°C

Izinto ezisetshenziswayo nezisetshenziswa (azinikeziwe)

Izinto ezisetshenziswayo

Iyasebenziseka	Umphakeli
Ishubhu ye-PCR engu-0.2 mL noma ishubhu lesicucu	Umhlinzeki welebhu ojwayelekile
Ithiphu ye-pipette engu-20 mL (i-orifice evamile futhi ebanzi)	Umhlinzeki welebhu ojwayelekile
Ithiphu ye-pipette engu-200 mL (i-orifice evamile futhi ebanzi)	Umhlinzeki welebhu ojwayelekile
Ubufakazi be-Ethanol 200 (obuphelele) bebhayoloji yamangqamuzana (500 mL)	Sigma-Aldrich, # E7023
Amanzi angenayo i-nuclease	Umhlinzeki welebhu ojwayelekile
AMPawu XP	Beckman, # A63880
I-Agilent Bioanalyzer High Sensitivity DNA Analysis Kit*	I-Agilent, # 5067-4626
I-TapeStation High Sensitivity D5000 ScreenTape Assay*	I-Agilent, # 5067-5592, #5067-5593
I-Qubit dsDNA HS Assay Kit	I-Thermo Fisher Scientific, # Q32851 noma i-Q32854
I-Qubit Assay Tubes	I-Thermo Fisher Scientific, # Q32856
Dynabeads MyOne Streptavidin T1	I-Thermo Fisher Scientific, # 65601, 65602 noma 65603
I-Agilent SureKhetha i-XT HS Capture Library Human All Exon 7	I-Agilent, # G9704N, G9705N noma i-G9706N
I-Agilent SureSelect XT HS Target Enrichment Kit, ILM Hyb Module (Post PCR), 16 Rxn	I-Agilent, #G9916B
Ibhafa ye-TE, pH 8.0	Umhlinzeki welebhu ojwayelekile

*Kuya ngokuthi yiluphi uhlelo olutholakala endaweni yomsebenzisi.

Izisetshenziswa

Izisetshenziswa	Umphakeli
I-Thermo Cycler	I-Biosystems esetshenziswayo
I-Magnetic stand yamashubhu e-PCR angu-0.2 mL	Umhlinzeki welebhu ojwayelekile
I-Rotator ye-tube	Umhlinzeki welebhu ojwayelekile
I-Incubator (ngo-35°C)	Umhlinzeki welebhu ojwayelekile
I-Vortexer	Umhlinzeki welebhu ojwayelekile
I-Microcentrifuge	Umhlinzeki welebhu ojwayelekile
I-Agilent Bioanalyzer*	I-Agilent
I-Agilent TapeStation*	I-Agilent
I-Qubit® Fluorometer 3.0	I-Thermo Fisher Scientific, # Q33216, Q33217 noma Q33218
I-Ice Bucket	Umhlinzeki welebhu ojwayelekile
I-SpeedVac Vacuum Concentrators (Ongakukhetha)	I-Thermo Fisher Scientific

*Kuya ngokuthi yiluphi uhlelo olutholakala endaweni yomsebenzisi.

TELL-Seq™ Human Exome Capture Workflow

TELL-Seq™ WGS Library Prep

Iphrothokholi

TELL-Seq™ WGS Library Prep
Iphrothokholi elandelayo ichaza inqubo eguquliwe ye-TELL-Seq™ yokulungiselela umtapo wolwazi wokulandelanisa kwegenome ngekhithi ye-TELL-Seq™ Library Prep kusetshenziswa i-DNA yomuntu.ampLes. I-DNA ye-genomic ehlukileampIzinhlobo ze-le zingashintshwa ngokulandela inqubo efanayo. Imitapo yolwazi ye-TELL-Seq™ iyahambisana nezinye izinhlelo zokuThuthukiswa kweThagethi, kodwa i-Agilent SureSelectXT HS Target Enrichment System isebenzisa i-SureSelect Human All Exon V8 Capture probe isetshenziswa njenge-ex.ample ku-protocol. Ukuze usetshenziswe kwamanye Amasistimu Okunothisa Okuqondisiwe sicela ulandele umthetho olandelwayo we-TELL-Seq™ Library Prep (ikhasi 10-22) ukuze ukhiqize amalabhulali afanelekile. Imitapo yolwazi ye-TELL-Seq™ ingasetshenziswa Njengokufaka kwe-DNA ngokulandela umthetho olandelwayo Wohlelo ngalunye oluthile Lokucebisa Okuqondiwe kanye nezivimba-Target ze-TELL-Seq™ ezisetshenziswa ngaphezu kwezivimbeli ezithile.

Ibhakhodi DNA

Izinto ezisetshenziswayo
➢ Faka i-genomic DNA (Umsebenzisi)

Usayizi weGenome	Inani Lokufaka	Ivolumu yokusabela (mL)	Preps/Kit
Okukhulu (Umuntu)	5 ng	150	4

QAPHELA: 

1. I-Genomic DNA kufanele igcinwe futhi ihlanjululwe kubhafa ye-Tris ene-pH esukela ku-7.5 ukuya ku-8.0 noma i-TE buffer ephansi (10mM Tris-HCl, 0.1 mM EDTA, pH 8.0).

➢ 5× Isithameli Sesenzo (Ibhokisi Lekhithi 1, Okuluhlaza okwesibhakabhaka)
➢ I-Cofactor II (Kit Box 1, Inhlaka)
➢ I-Enzyme Yokufaka Ibhakhodi (Kit Box 1, Mnyama)
➢ TSHELA Ubuhlalu noma TELL Bead Plex (Kit Box 2, Iwolintshi)
➢ I-Spension Buffer EZ (Kit Box 1, Okwemvelo)
➢ Amanzi angenayo inukliya (Umsebenzisi)
➢ 0.2 mL PCR tube noma strip tube (Umsebenzisi)
➢20 µL kanye no-200 µL amathiphu wepayipi orifice ububanzi (Umsebenzisi)

Ukulungiselela

1. Lungiselela okulandelayo:
   

   Into	Isitoreji	Isiyalezo
   5× Isivimbeli sokusabela	-25°C kuya ku-15°C	Ncibilika ekamelweni lokushisa. I-Vortex ukuxuba, ke   centrifuge kafushane. Qhubeka eqhweni.
   I-Cofactor II	-25°C kuya ku-15°C	I-Vortex ezoxutshwa, bese i-centrifuge kafushane. Gcina ku izinga lokushisa ekamelweni ebumnyameni. Vala ithubhu   cap ngokuqinile ngemva kokusetshenziswa ngakunye.
   Ibhakhodi Enzyme	-25°C kuya ku-15°C	Gcoba ishubhu izikhathi ezi-4 kuya kwezi-5 ukuze uxube. I-Centrifuge kafushane. Qhubeka eqhweni.
   TSHELA I-Bead Plex	2°C kuya ku-8°C	Centrifuge kafushane. Qhubeka eqhweni. Vala i-tube cap  ngokuqinile ngemva kokusetshenziswa ngakunye ukugwema noma yikuphi ukuhwamuka.
   Ukumiswa Kwesigcinalwazi EZ	-25°C kuya ku-15°C	Ncibilika ekamelweni lokushisa. I-Vortex ezoxutshwa, bese i-centrifuge kafushane. Gcina ku izinga lokushisa lekamelo.
   Amanzi angenayo i-nuclease	Ukushisa Kwegumbi	Gcina ezingeni lokushisa legumbi.

2. Misa i-tube rotator ku-incubator engu-35 °C (bheka Isinyathelo sesi-7 sesigaba senqubo).

ISEXWAYISO
Sebenzisa amathiphu e-wide orifice pipette ukuze udlulise futhi uxube i-DNA ye-molecular weight genomic ukugwema ukwephula i-DNA. Uma amathiphu epayipi orifice ebanzi engatholakali, sika 2mm-3mm ususa phezulu kwephuzu lepayipi elivamile ngensingo ehlanzekile ngaphambi kokuyisebenzisa.

Inqubo

1. I-Vortex TELL Bead Plex ngamandla okungenani imizuzwana engama-30. I-Pulse spin (i-centrifuge engekho ngaphezu kwesekhondi elingu-1) ukwehlisa isisombululo sobuhlalu esikhona esivalweni noma emaceleni eshubhu. Ngaphambi nje kokusetshenziswa, cindezela i-TELL Bead Plex ngethiphu engu-200 µL phezulu naphansi izikhathi ezi-5 ukuze uqinisekise ukuthi bonke ubuhlalu bumiswe kabusha ngendlela efanele.
2. Kushubhu le-PCR elingu-0.2 mL, hlanganisa ukusabela ngakunye ngokulandelana okulandelayo.
   

   I-Reagent	Ivolumu ngokusabela ngakunye (mL)
   	I-Genome Enkulu (150 mL)

   5× Isivimbeli sokusabela	30
   Amanzi angenayo i-nuclease I-Cofactor II	20 - Z (Z ivolumu ye-DNA)30
   TSHELA I-Bead Plex (0.5M amabhakhodi/mL)	19

3. Xuba kahle ngokufaka amapayipi phezulu naphansi izikhathi ezingu-10 noma ukuvunguza ngamandla imizuzwana emi-5 kanye ne-pulse spin ukuze ulethe isisombululo phansi. Engeza Ibhakhodi Enzyme.
   

   I-Reagent	Ivolumu ngokusabela ngakunye (mL)
   	I-Genome enkulu
   Ibhakhodi Enzyme	6 mL

4. Hlanganisa kahle ngokufaka amapayipi phezulu naphansi izikhathi ezingu-8. Gwema ukwethula amabhamuza omoya lapho ufaka ipayipi ngokugcina ithiphu le-pipette liphansi kwesixazululo eshubhu.
5. Sebenzisa ithiphu elibanzi le-orifice pipette, engeza i-reagent elandelayo.
   QAPHELA: I-Suspension Buffer EZ ibukeka kakhulu. Sebenzisa ukuqapha kanye ne-pipette kancane ukuze uqinisekise ukuthi ivolumu elungile ilethwa.
6. Setha ivolumu ye-pipette ku-110 μL. Sebenzisa ithiphu elibanzi le-orifice pipette, xuba ngobumnene isisombululo ngokufaka amapayipi kancane kancane phezulu naphansi izikhathi ezingu-6-8. Gwema ukwethula amabhamuza amaningi omoya lapho ufaka ipayipi ngokugcina ithiphu le-pipette liphansi kwesisombululo ku-tube.
7. Beka i-sample tube ku-tube rotator ku-incubator engu-35 ° C futhi ujikeleze kancane (10-15 rpm) imizuzu engu-30.

Sampamashubhu abekwe ku-Tube Rotator ku-incubator engu-35°C.

Qaphela: Ukuzungezisa kahle kweshubhu kubalulekile ukuze kulondolozwe izakhiwo ze-HMW DNA kanye nokwenza lula inqubo efanele yokubhala ibhakhodi. Amasistimu okuxuba anconyiwe aboniswa ngezansi (uhlangothi lwesobunxele). Amasistimu okuxuba angazungezi noma akhiqiza ukunyakaziswa okunamandla awahambisani nokulondolozwa kwezakhiwo ze-HMW DNA kanye ne-TELL-Seq; ezinye zalezi zinhlelo nazo zikhonjisiwe ngezansi (uhlangothi lwesokudla).

Zinzisa i-DNA 

Izinto ezisetshenziswayo
➢ I-Stabilizer (Kit Box 1, I-Violet)

Ukulungiselela

1. Lungiselela okulandelayo:

Into	Isitoreji	Isiyalezo
Isiqinisi	-25°C kuya ku-15°C	Gcoba ishubhu izikhathi ezi-4 kuya kwezi-5 ukuze uxube. Centrifuge kafushane. Qhubeka eqhweni.

Inqubo

1. Buyisa i-sample tube kusuka ku-35°C incubator.
2. Engeza i-Stabilizer ku-tube.
   

   I-Reagent	Ivolumu ngokusabela ngakunye (mL)
   	I-Genome enkulu
   Isiqinisi	3

3. Setha ivolumu ye-pipette ku-110 μL. Sebenzisa ithiphu elibanzi le-orifice pipette, xuba ngobumnene isisombululo ngokufaka amapayipi kancane kancane phezulu naphansi izikhathi ezingu-6-8. Gwema ukudala amabhamuza amaningi.
4. Beka i-sampbuyisela ku-tube rotator ku-incubator engu-35 ° C futhi uyijikelezise kancane (10-15 rpm) imizuzu engu-30.

Tagi-DNA 

Izinto ezisetshenziswayo
➢ TagI-Enzyme ekhulayo (Kit Box 1, Okubomvu)
➢ I-Exonuclease (Kit Box 1, Okuphuzi)

Ukulungiselela

1. Lungiselela okulandelayo:
   

   Into	Isitoreji	Isiyalezo
   Tagi-enzyme	-25°C kuya ku-15°C	Gcoba ishubhu izikhathi ezi-4 kuya kwezi-5 ukuze uxube. I-Centrifuge kafushane. Qhubeka eqhweni.
   I-Exonuclease	-25°C kuya ku-15°C	Gcoba ishubhu izikhathi ezi-4 kuya kwezi-5 ukuze uxube. I-Centrifuge kafushane. Qhubeka eqhweni.

2. Sebenzisa i-tube rotator efanayo ku-incubator engu-35 °C.

Inqubo

1. Buyisa i-sample tube kusuka ku-35°C incubator.
2. Engeza Tagi-Exonuclease ne-Enzyme ephuma ku-tube.
   

   I-Reagent	Ivolumu ngokusabela ngakunye (mL)
   	I-Genome enkulu
   Tagi-enzyme	2
   I-Exonuclease	3

3. Setha ivolumu ye-pipette ku-110 μL. Sebenzisa ithiphu elibanzi le-orifice pipette, xuba ngobumnene isisombululo ngokufaka amapayipi kancane kancane uye phezulu naphansi izikhathi ezingu-8. Kulesi sinyathelo, ukuxuba kudinga ukucophelela kakhulu. Gwema ukudala amabhamuza amaningi.
4. Beka i-sampbuyisela ku-tube rotator ku-incubator engu-35°C bese uyizungezisa kancane imizuzu eyi-10. Uma kunesidingo, inani elihlukile le Tagi-ging Enzyme ingasetshenziswa ukulungisa usayizi womtapo wolwazi.
   QAPHELA: Uma kukhethwa umtapo wolwazi omude, inani elincane TagI-ging enzyme ingasetshenziswa ekuphenduleni. Ngakolunye uhlangothi, uma kukhethwa ilabhulali yokufaka emfushane, kufika ku-6µL TagAma-Enzymes angasetshenziswa ekuphenduleni.
5. Qhubekela esinyathelweni esilandelayo ngokushesha ngemva kokufukamela.

Geza Ubuhlalu

Izinto ezisetshenziswayo

• Stop Solution (Kit Box 2, Yemvelo noma egcinwe ekamelweni lokushisa ngemva kokusetshenziswa kokuqala)
• Isixazululo Sokugeza (Kit Box 2, Mhlophe)
• 0.2 mL PCR tube noma strip tube (Umsebenzisi)

Ukulungiselela

1. Lungiselela okulandelayo:
   

   Into	Isitoreji	Isiyalezo
   Misa Isixazululo	2°C kuya ku-25°C	Hlola noma yiziphi izimvula. Uma ikhona, fukamela isigcinalwazi ku-37°C imizuzu engu-10, bese uvuthuza zize zincibilike. Gcina ekamelweni lokushisa okwe  ukusetshenziswa kwesikhathi esizayo.
   Geza Isixazululo	2°C kuya ku-8°C	Letha ekamelweni lokushisa.

2. Setha i-thermo cycler ngohlelo olulandelayo:
   • Inketho yesivalo se-preheat ibe ngu-100°C
   • 63°C ingunaphakade

Inqubo

1. Beka i-sample tube ekumeni kazibuthe iminithi elingu-1 noma kuze kube yilapho isisombululo sesicacile.
2. Ngenkathi ishubhu lisendaweni kazibuthe, langazelela futhi ulahle amandla amakhulu ngaphandle kokuphazamisa ubuhlalu.
3. Susa ishubhu endaweni kazibuthe. Engeza isixazululo sokugeza esingu-120 µL ku-sample tube. Ipayipi lokumisa kabusha ubuhlalu. Uma kunesidingo, i-pulse spin ukuze ulethe isisombululo.
4. Beka i-sample tube emuva kokuma kazibuthe umzuzu ongu-1 noma kuze kube yilapho isisombululo sesicacile.
5. Ngenkathi ishubhu lisendaweni kazibuthe, langazelela futhi ulahle amandla amakhulu ngaphandle kokuphazamisa ubuhlalu.
6. Susa ishubhu endaweni kazibuthe. Engeza u-80 µL we-Stop Solution kushubhu.
7. Pipe izikhathi eziningana ukuze umise ubuhlalu. Uma kunesidingo, i-pulse spin ukuze ulethe isisombululo.
8. Faka i-tube endaweni yokushisa yasekamelweni imizuzu emi-5.
9. Beka i-sample tube emuva kokuma kazibuthe umzuzu ongu-1 noma kuze kube yilapho isisombululo sesicacile.
10. Ngenkathi ishubhu lisendaweni kazibuthe, langazelela futhi ulahle amandla amakhulu ngaphandle kokuphazamisa ubuhlalu.
11. Susa ishubhu endaweni kazibuthe. Engeza Isixazululo Sokugeza esingu-120 µL kushubhu ye-PCR. Ipayipi lokumisa kabusha ubuhlalu.
12. Dlulisa sonke isixazululo sobuhlalu kushubhu entsha ye-PCR engu-0.2ml.
13. Fukamela ishubhu ku-63 °C kumshini wokushisa we-PCR imizuzu emi-3.
14. Beka i-s entshaample tube ekumeni kazibuthe ekamelweni lokushisa umzuzu ongu-1 noma kuze kube yilapho ikhambi selicacile.
15. Ngenkathi ishubhu lisendaweni kazibuthe, langazelela futhi ulahle amandla amakhulu ngaphandle kokuphazamisa ubuhlalu.
16. Susa ishubhu endaweni kazibuthe. Engeza Isixazululo Sokugeza esingu-120 µL kushubhu ye-PCR. Ipayipi lokumisa kabusha ubuhlalu. Uma kunesidingo, i-pulse spin ukuze ulethe isisombululo.
17. Fukamela ishubhu ku-63 °C kumshini wokushisa we-PCR imizuzu emi-3.
18. Beka i-sample tube ekumeni kazibuthe ekamelweni lokushisa umzuzu ongu-1 noma kuze kube yilapho ikhambi selicacile.
19. Ngenkathi ishubhu lisendaweni kazibuthe, langazelela futhi ulahle amandla amakhulu ngaphandle kokuphazamisa ubuhlalu. Sebenzisa ipayipi le-P20 ukuze ususe noma iyiphi i-supernatant esele.
20. Susa ishubhu endaweni kazibuthe. Phinda umise ubuhlalu ku-20 µL we-Wash Solution.

QAPHELA
Leli IPHUZU LOKUMIMA OKUPHEPHILE. Ubuhlalu obugeziwe bungagcinwa ku-2°C kuya ku-8°C amasonto amabili.

AmpLify Library

Izinto ezisetshenziswayo

• 2× PCR Master Mix (Kit Box 1, Pinki)
• 10× Primer I (Kit Box 1,  Mhlophe)
• 10× Primer II, T50# (Multiplex Primer Kit)
• Amanzi angenayo i-nuclease (Umsebenzisi)
• 0.2 mL PCR tube noma strip tube (Umsebenzisi)

Ukulungiselela

1. Lungiselela okulandelayo:
   

   Into	Isitoreji	Isiyalezo
   2× PCR Master Mix	-25°C kuya ku-15°C	Ncibilika ekamelweni lokushisa. Gcoba i-tube 4 kuya ku-5  izikhathi zokuxuba, bese i-centrifuge kafushane. Qhubeka eqhweni.
   10 × I-Primer I	-25°C kuya ku-15°C	Ncibilika ekamelweni lokushisa. I-Vortex ukuxuba, ke centrifuge kafushane. Qhubeka eqhweni.
   10× Primer II, T50#	-25°C kuya ku-15°C	Ncibilika ekamelweni lokushisa. I-Vortex ukuxuba, ke  centrifuge kafushane. Qhubeka eqhweni.
   Isithuthukisi	-25°C kuya ku-15°C	Ncibilika ekamelweni lokushisa. I-Vortex ukuxuba, ke centrifuge kafushane. Gcina ezingeni lokushisa legumbi.
   Amanzi angenayo i-nuclease	Ukushisa Kwegumbi	Gcina ezingeni lokushisa legumbi.

2. Setha Ilabhulali AmpUhlelo lwe-liification (LAP) ku-thermo cycler kanjena:
   • 63°C 2 amaminithi
   • 72°C 2 amaminithi
   • 98°C imizuzwana engu-30
   • [98°C imizuzwana engu-15, 63°C imizuzwana engu-20, 72°C imizuzwana engu-30] x Inombolo Yomjikelezo
   • 72°C 3 amaminithi
   • 4°C ingunaphakade

QAPHELA:
Inombolo yomjikelezo iyavumelana nezimo ngokusekelwe ezinhlelweni zokusebenza nesidingo. Ncoma ukuqala ngemijikelezo engu-13. Inombolo yomjikelezo ephakeme izokhiqiza umtapo wezincwadi we-TELL-Seq owengeziwe njengokufakwayo kwe-Hybridization kanye ne-Capture, kodwa izilinganiso eziphakeme zokuphindaphinda zokuhlaziya kokugcina kokulandelana. Inombolo yomjikelezo ophansi ingase yehlise izinga lokuphindaphinda, kodwa okokufaka okuphansi kwe-DNA kungase kubangele ukwehla kokusebenza kahle kokuthwebula kanye nokunciphisa inkimbinkimbi yomtapo wolwazi. Sicela ubheke ekugcineni kwe-Qualify and Quantify Library for Hybridization and Capture ukuze uthole ukucatshangelwa okwengeziwe lapho unquma inombolo yomjikelezo efanele.

Usayizi weGenome	Ivolumu yobuhlalu Esetshenzisiwe (B) ye-PCR	Ivolumu ye-PCR	Inombolo yomjikelezo
Okukhulu	20 mL	75 mL	12-14

Inqubo

1. Ubuhlalu be-Vortex ngamandla imizuzwana engu-10 ukuze bumise ubuhlalu. I-Pulse spin ukuze ulethe isisombululo. Usebenzisa ithiphu engu-20 µL, faka ubuhlalu phezulu naphansi izikhathi ezi-5 ukuze uqiniseke ukuthi bonke ubuhlalu bumiswe ngendlela efanele. Ngokushesha dlulisa lonke inani lesixazululo sobuhlalu eshubhuni elisha le-PCR.
2. Beka ishubhu le-PCR endaweni kazibuthe iminithi elingu-1 noma kuze kube yilapho isisombululo sesicacile.
3. Ngenkathi ishubhu likusitendi sikazibuthe, susa u-20 µ L amandla angaphezu kwavamile ngaphandle kokuphazamisa ubuhlalu. Susa ishubhu le-PCR kumagnet.
4. Engeza ama-reagents alandelayo kushubhu ye-PCR.
   

   I-Reagent	Ivolumu ngokusabela ngakunye (µL)
   	I-Genome Enkulu (75 µL)
   Amanzi angenayo i-nuclease	16 mL
   2× PCR Master Mix	37.5 mL
   10 × I-Primer I	7.5 mL
   10× Primer II, T50#	7.5 mL
   Isithuthukisi Okuhlaza	4.5 mL

5. Hlanganisa kahle nge-vortexing noma ipayipi. I-Pulse spin ukuze ulethe isisombululo.
6. Beka ishubhu kumjikelezo oshisayo bese uqhuba uhlelo lwe-LAP (bheka ngenhla) ngenani elifanele lemijikelezo.
7. Ngemuva kwe-PCR amp, sebenzisa umkhiqizo we-PCR ongu-2 µL ukuze uhlole ikhwalithi ku-Bioanalyzer noma ku-TapeStation. Bheka isigaba se-Qualify and Quantify Library ukuze uthole imiyalelo.

I-PRO ICEBISO: Uma isheke le-QC libonisa ukuthi isivuno selabhulali siphansi kakhulu, buyisela ishubhu enomkhiqizo we-PCR osele ku-thermocycler futhi ampthola omunye umjikelezo owodwa noma emibili eyengeziwe ngaphambi kokuthuthela esigabeni Sokuhlanza Umtapowolwazi.

QAPHELA:
Leli IPHUZU LOKUMIMA OKUPHEPHILE. Umkhiqizo we-PCR ungagcinwa ku -25°C kuya ku -15°C inyanga eyodwa.

Hlanza Umtapowolwazi

Izinto ezisetshenziswayo

• AMPure XP (Umsebenzisi)
• Ubufakazi be-Ethanol 200 (obuphelele) bebhayoloji yamangqamuzana (Umsebenzisi)
• Amanzi angenayo i-nuclease (Umsebenzisi)
• 0.2 mL PCR tube noma strip tube (Umsebenzisi)

Ukulungiselela

1. Lungiselela okulandelayo:

Into	Isitoreji	Isiyalezo
I-ethanol entsha engu-75% (v/v).	Ukushisa Kwegumbi	Idinga 400 ml ngesekhondiample. Xuba i-Ethanol engu-1.5 ml (ubufakazi obungama-200) no-0.5 mL wamanzi angenayo i-Nuclease. I-Vortex yokuxuba futhi igcinwe ekamelweni lokushisa.
AMPawu XP	2°C kuya ku-8°C	Yilethe ekamelweni lokushisa okungenani imizuzu engu-20 kanye ne-vortex ngamandla ukuze umise ubuhlalu futhi ngaphambi kokusetshenziswa.
Amanzi angenayo i-nuclease	Ukushisa Kwegumbi	Gcina ezingeni lokushisa legumbi.
Ibhafa ye-TE, pH 8.0	Ukushisa Kwegumbi	Gcina ezingeni lokushisa legumbi.

Inqubo

1. Kafushane centrifuge the sample PCR tube ukwehlisa sonke isixazululo.
2. Beka ithubhu endaweni kazibuthe iminithi elingu-1 noma kuze kube yilapho isisombululo sesicacile.
3. Ngenkathi ishubhu lisesitendi sikazibuthe, dlulisela amandla amakhulu kushubhu entsha ye-PCR engu-0.2 mL ngaphandle kokuphazamisa ubuhlalu.
4. Linganisa ivolumu ye-supernatant edlulisiwe (umkhiqizo we-PCR) nge-pipette.
5. Engeza ama-reagents alandelayo kumkhiqizo we-PCR kuvolumu ephelele engu-100 µL.
   

   I-Reagent	Ivolumu ngokusabela ngakunye
   PCR umkhiqizo	75 mL
   Amanzi angenayo i-nuclease	Ukuze ugcine isamba se-100 ml

6. Vortex ngamandla ukumisa kabusha AMPure XP bese wengeza u-78 µL AMPfaka i-XP kumkhiqizo we-PCR we-100 µL.
7. Hlanganisa ngepayipi phezulu naphansi izikhathi ezi-10.
8. Fukamela ekamelweni lokushisa imizuzu emi-5.
9. Beka ithubhu endaweni kazibuthe iminithi elingu-1 noma kuze kube yilapho isisombululo sesicacile.
10. Aspirate futhi ulahle supernatant ngaphandle kokuphazamisa AMPure ubuhlalu.
11. Ngenkathi ugcina ishubhu endaweni kazibuthe, engeza u-200 µL osanda kulungiswa 75% ethanol eshubhuni. Ivumele ihlale imizuzwana engu-30.
12. Aspirate futhi ulahle supernatant ngaphandle kokuphazamisa ubuhlalu.
13. Phinda izinyathelo 11-12 kanye futhi, ugcine ithubhu ekumeni kazibuthe isikhathi sonke.
14. Gcina ishubhu endaweni kazibuthe enekepisi livuliwe futhi uvumele ishubhu ukuthi lome imizuzu engu-1-2 ukuze lihwamuke iminonjana ye-ethanol. UNGABAMBI ngokweqile ubuhlalu.
15. Susa ishubhu kusitendi sikazibuthe bese wengeza amanzi angenayo i-nuclease angu-20 µL kubuhlalu.
16. I-Pipette noma i-vortex yokumisa kabusha ubuhlalu. Yiyeke ihlale imizuzu emi-5.
17. Beka ithubhu endaweni kazibuthe iminithi elingu-1 noma kuze kube yilapho isisombululo sesicacile.
18. Phinda uthole u-18 µL we-supernatant kushubhu elisha. Qaphela ukuthi ungaphazamisi ubuhlalu.
19. I-supernatant iqukethe umtapo wezincwadi we-TELL-Seq™.

QAPHELA:
Leli IPHUZU LOKUMIMA OKUPHEPHILE. Umtapo wezincwadi we-TELL-Seq ohlanzekile ungagcinwa ku- -25°C kuya ku- -15°C inyanga yonke.

Ufanele futhi Ulinganise Umtapowolwazi Wokucebisa Okuhlosiwe

Izinto ezisetshenziswayo

• I-Agilent High Sensitivity DNA Kit noma i-TapeStation High Sensitivity D5000 ScreenTape Assay (Umsebenzisi)
• I-Qubit dsDNA HS Assay Kit (Umsebenzisi)
• Ibhafa ye-TE, pH 8.0 (Umsebenzisi)

QAPHELA:
Ukuhlolwa komthamo komtapo wezincwadi we-qPCR ojwayelekile wohlelo lwe-Illumina kusebenza kulabhulali ye-TELL-Seq, kodwa ayidingeki.

Ukulungiselela

1. Lungiselela izinto ezisetshenziswayo ezidingekayo njengoba kudingwa yi-Bioanalyzer noma i-TapeStation ne-Qubit.

Inqubo

1. Sebenzisa u-1 µL welabhulali ye-Agilent High Sensitivity DNA Kit noma 2 µL welabhulali ye-TapeStation High Sensitivity D5000 ScreenTape Assay.
2. Hlola umkhiqizo we-PCR ongahlanzekile olondoloziwe kusukela ku- Amplify isigaba seLabhulali ngasikhathi sinye. Umkhiqizo we-PCR ongahlanzekile ungase ube nezinga eliphezulu le-primer dimer ne-adaptha dimer. Idinga ukuh
3. Umtapo wolwazi onosayizi omuhle kufanele ube nezingcezu eziningi zelabhulali ezingaphansi kuka-1000 bp (Umfanekiso 1).Umfanekiso 1. Example yokuhlanza umtapo wolwazifile kusukela ekuhlolweni kwe-TapeStation High Sensitivity D5000 Tape Tape.
4. Umtapo wolwazi ungagcinwa ku -25°C kuya ku -15°C.
5. Yenza umtapo wezincwadi we-TELL-Seq™ ohlanjululwe ka-10ample: hlanza u-2 µL welabhulali ye-TELL-Seq™ ngo-18 µL wamanzi angenayo i-nuclease. Sebenzisa umtapo wezincwadi ohlanjululwe ongu-4 µL ukuze uhlole ukugxilisa ingqondo nge-Qubit dsDNA HS Assay Kit.
6. Sebenzisa ukugxilisa ingqondo (ng/µL) nevolumu ukuze ubale isamba sesisindo se-TELL-Seq™ Library ngayinye engena kunqubo ye-SureSelectXT HS Target Enrichment System. 500 ng -1,000 ng TELL-Seq™ umtapo wezincwadi ngekampi-le inconywa ngemiphumela emihle kakhulu, kodwa okokufaka komtapo wezincwadi kuphansi njengo-250 ng ngesampkungenzeka nakuba ukusebenza kungaba nomthelela omubi.
   QAPHELA:
   Kunemikhawulo yevolumu ye-Hybridization kanye ne-Capture protocol. I-12 µL iyivolumu enkulu evunyelwe ukufaka i-DNA. Kufanele kucatshangelwe ngokucophelela ukuze kuqinisekiswe ukuthi umthamo womtapo wezincwadi we-DNA ungena ngaphakathi kwalolu hlu. Ngokufanelekile sebenzisa zonke izinto ezitholakalayo zomtapo wezincwadi we-TELL-Seq ngemva kwe-QC ku-Hybridization and Capture reaction.
7. (Ongakukhetha) Imitapo yolwazi ye-TELL-Seq™ ingagxiliswa kusetshenziswa i-SpeedVac Vacuum Concentrator. Uma i-SpeedVac izosetshenziselwa ukugxilisa umtapo wolwazi, ngemva kwalokho umtapo we-TELL-Seq™ AMPukuhlanzwa kwe-XP kungasuswa kubuhlalu be-XP okungenani ngamanzi angenayo i-nuclease angu-30 µL ukuze kutholakale kangcono. Ngemva kwe-QC, umtapo wezincwadi we-TELL-Seq™ ohlanzekile ungagxiliswa kuvolumu oyifunayo ye-Hybridization kanye ne-Capture.

QAPHELA:
Amanye Amasistimu Okunothisa Okuqondisiwe angasetshenziswa njenge-IDT ne-Twist Biosciences kusetshenziswa imitapo yolwazi ye-TELL-Seq™ edaliwe. Landela amaphrothokholi acacisiwe Wokuthuthukisa Ithagethi usebenzisa imitapo yolwazi ye-TELL-Seq™ njengokufaka kwe-DNA. Sebenzisa i-5ul ye-TELL-Seq™ TargetSeq Blocker ngaphezu kwezivimbeli ezinemininingwane kuphrothokholi ngayinye.

I-SureSelect Target Enothisayo
Iphrothokholi elandelayo iwukuguqulwa kwengxenye ye-Hybridization and Capture SureSelect XT HS Target Enrichment System ye-Illumina Paired-End Multiplexed Sequencing Library Protocol (Agilent, G9702-90000). Ukulungiswa kuvumela ukuhambisana kwe-TELL-Seq™ WGS Library Prep ne-Agilent SureSelect XT HS Target Enrichment System kanye nawo Wonke Ama-Exon V8 Capture Probes. Ama-reagents ahlakaniphile e-Hybridization kanye ne-Capture nje angathengwa ngokuhlukana ngefomethi ye-16-reaction (Agilent Part Number G9916B). Uma usebenzisa amanye Amasistimu Okunothisa Okuqondisiwe landela izimiso eziyisisekelo zesistimu ngayinye bese ufaka amalabhulali e-TELL-Seq™ njengokufaka kwe-DNA. I-5ul ye-TELL-Seq™ Target Blockers nayo iyadingeka ngaphezu kwezivimbe ezinemininingwane kuphrothokholi ngayinye Yokucebisa Okuhlosiwe

Hlanganisa i-TELL-Seq™ Library ibe yiphaneli yokuthwebula ye-Exome

Izinto ezisetshenziswayo

• I-SureSelect i-XT HS ne-XT Low Input Blocker Mix, 16 Rxn (Agilent, SureSelect XT HS Target Enrichment Kit, ILM Hyb Module, Box 2 (Post PCR), Okuluhlaza okwesibhakabhaka)
• SureSelect RNase Block, 16 Rxn (Agilent, SureSelect XT HS Target Enrichment Kit ILM Hyb Module, Box 2 (Post PCR), I-Violet)
• SureSelect Fast Hybridization Buffer, 16 Rxn (Agilent, SureSelect XT HS Target Enrichment Kit ILM Hyb Module, Box 2 (Post PCR), Bottle)
• TELL-Seq™ Target Blocker (UST, TELL-Seq™ Target Blocker Box, Mhlophe)
• I-SSel XT HS kanye ne-XT Low Input Human All Exon V7 (Agilent, SSel XT HS kanye ne-XT Low Input Human All Exon V7, Okubomvu)
• Amanzi angenayo i-nuclease (Umsebenzisi)
• 0.2 mL PCR tube noma strip tube (Umsebenzisi)
• TELL-Seq™ Library (Umsebenzisi)

Inani Lokufaka	Ivolumu yokusabela (mL)
500 -1,000 ng	Kufika ku-12 ml

Ukulungiselela

1. Lungiselela okulandelayo:
   

   Into	Isitoreji	Isiyalezo
   SureKhetha i-XT HS kanye ne-XT Low Input Blocker Mix	-25°C kuya ku-15°C	Thaw on ice. I-Vortex ezoxutshwa, bese i-centrifuge kafushane. Qhubeka eqhweni.
   SureSelect RNase Block	-25°C kuya ku-15°C	Thaw on ice. I-Vortex ezoxutshwa, bese i-centrifuge  kafushane. Qhubeka eqhweni.
   SureSelect Fast Hybridization Buffer	-25°C kuya ku-15°C	Ncibilikisa futhi ugcine ekamelweni lokushisa
   TELL-Seq™ I-TargetSeq Blocker	-25°C kuya ku-15°C	Ncibilika ekamelweni lokushisa. I-Vortex ezoxutshwa, bese i-centrifuge kafushane. Qhubeka eqhweni.
   TELL-Seq™ I-DNA Library	-25°C kuya ku-15°C	Ncibilika futhi uhlale eqhweni

   I-SSel XT HS kanye ne-XT Low Input Human All Exon V8

   -85°C kuya ku-75°C

   Thaw on ice. I-Vortex ezoxutshwa, bese i-centrifuge kafushane. Qhubeka eqhweni.

2. Setha iHybridization Programme (HP) kubhayisikili ye-thermo (enesivalo esishisayo IVULIWE) ngohlelo olungezansi. Qala uhlelo, bese ucindezela ngokushesha inkinobho ethi Misa Isikhashana, uvumele isivalo esishisayo sifinyelele izinga lokushisa ngenkathi usetha ukusabela.

Inombolo Yengxenye	Inombolo Yemijikelezo	Izinga lokushisa	Isikhathi
1	1	95°C	5 imizuzu
2	1	65°C	10 imizuzu
3	1	65°C	1 iminithi
4	60	65°C 37°C	1 iminithi imizuzwana engu-3
5	1	65°C	Bamba

Inqubo

1. Beka u-500–1000 ng womtapo wezincwadi we-TELL-Seq™ ngamunye olungisiweampfaka emithonjeni yamashubhu ehlutshiwe bese uletha ivolumu yokugcina emthonjeni ngamunye ku-12 µl usebenzisa amanzi angenayo i-nuclease uma kudingeka. Yenza isamba kahle ampI-DNA yelabhulali ye-TELL-Seq™ ehlanganisiwe, phakathi kwebanga le-500–1000 ng futhi isebenzise konke ukusabela kokuxutshwa.
2. Emtatsheni wezincwadi we-TELL-Seq™ ngamunyeampkahle, engeza u-5 µl we-SureSelect XT HS kanye ne-XT Low Input Blocker Mix bese wengeza u-5 µl we-TELL-Seq™ Target Blocker. Vala imithombo bese u-vortex ngesivinini esikhulu imizuzwana emi-5. Spina ishubhu yokuhlubula kafushane ukuze uqoqe uketshezi olukhipha noma yimaphi amabhamuza.
3. Dlulisela amashubhu kumjikelezo oshisayo bese ucindezela inkinobho ethi Dlala ukuze uqalise kabusha uhlelo lwe-HP olusethiwe.
   QAPHELA:
   Ibhayisikili elishisayo kufanele limiswe okwesikhashana ngesikhathi Sesegimenti 3 (bona i-HP) ukuze kuvunyelwe ama-reagents engeziwe ukuthi angezwe emithonjeni ye-Hybridization, njengoba kuchazwe esinyathelweni sesi-6. Phakathi Nezigaba 1 no-2 zohlelo lokuhamba ngebhayisikili elishisayo, qala ukulungisa ama-reagents engeziwe njengoba kuchazwe ku- isinyathelo 4 kanye nesinyathelo 5. Uma kudingeka, ungaqedela lezi zinyathelo zokulungiselela ngemva kokumisa isikhashana isithuthuthu esishisayo esigabeni sesi-3.
4. Lungiselela isixazululo esingu-25% se-SureSelect RNase Block (equkethe ivolumu engu-1 ye-RNase Block enomthamo wamanzi angu-3), ngokuvumelana nethebula elingezansi. Lungiselela inani elidingekayo ngenani lokusabela kwe-hybridization ekuhambeni, kanye nokweqile.
   

   I-Reagent

   Ivolumu ngokusabela ngakunye (mL)

   Ivolumu yokusabela oku-1		Ivolumu yokusabela okungu-8 (kufaka phakathi okweqile)	Ivolumu yokusabela okungu-24 (kufaka phakathi okweqile)
   SureSelect RNase Block	0.5 mL	4.5 mL	12.5 mL
   Amanzi angenayo i-nuclease	1.5 mL	13.5 mL	37.5 mL

5. Lungiselela i-Capture Library Hybridization Mix ngendlela elandelayo.
   

   I-Reagent	Ivolumu ngokusabela ngakunye (mL)
   	Ivolumu yokusabela oku-1	Ivolumu yokusabela okungu-8 (kufaka phakathi okweqile)	Ivolumu yokusabela okungu-24 (kufaka phakathi okweqile)
   Isixazululo esingu-25% se-RNase Block	2 mL	18 mL	50 mL
   I-SSel XT HS kanye ne-XT Okokufaka Okuphansi I-Human All Exon V7	5 mL	45 mL	125 mL
   SureSelect Fast Hybridization Buffer	6 mL	54 mL	150 mL

   Hlanganisa ama-reagents afakwe ohlwini ekamelweni lokushisa. Xuba kahle ngokuvukuza ngesivinini esikhulu imizuzwana emi-5 bese uphotha phansi isikhashana. Qhubeka ngokushesha esinyathelweni sesi-6.

6. Uma umjikelezo oshisayo uqala Ingxenye 3 ye-HP (umzuzu ongu-1 ku-65°C), cindezela inkinobho ethi Misa Isikhashana. Njengoba umjikelezo umisiwe, futhi ngenkathi ugcina i-DNA + Blocker sampLes kubhayisikili, dlulisa u-13 µl wezinga lokushisa legumbi Capture Library Hybridization Mix ukusuka esinyathelweni sesi-6 uye kumzuzwana ngamunyeampkahle le. Hlanganisa kahle ngokufaka amapayipi phezulu naphansi kancane kancane izikhathi ezingu-8 kuya kwezingu-10. Imithombo yokusabela kwe-hybridization manje iqukethe cishe u-35 µl
7. Qinisekisa ukuthi yonke imithombo ivalwe ngokuphelele. I-Vortex kafushane, bese u-spin strip tube kafushane ukuze ususe noma yimaphi amabhamuza kusukela phansi kwemithombo. Ngokushesha buyisela ishubhu yokuhlubula kumjikelezo oshisayo.
8. Cindezela inkinobho Dlala ukuze uqalise kabusha uhlelo lokuhamba ngebhayisikili elishisayo ukuze uvumele ukuxutshwa kwama-DNA alungisiweampLes ku-Capture Library.

Lungiselela ubuhlalu beMagnetic obuhlanganiswe ne-Streptavidin

Izinto ezisetshenziswayo

• I-Dynabeads MyOne Streptavidin T1 (Umsebenzisi)
• SureSelect Binding Buffer (Agilent, SureSelect XT HS Target Enrichment Kit ILM Hyb Module, Box 1 (Post PCR), CAP)

Ukulungiselela

Lungiselela okulandelayo:

Into	Isitoreji	Isiyalezo
Dynabeads MyOne Streptavidin T1	2°C kuya ku-8°C	Centrifuge ngamandla. Gcina ezingeni lokushisa legumbi.
SureSelect Binding Buffer,	Ukushisa Kwegumbi	Gcina ezingeni lokushisa legumbi.

Inqubo

1. Buyisa ngamandla ubuhlalu obuzibuthe be-Dynabeads MyOne Streptavidin T1 kumxube we-vortex. Ubuhlalu kazibuthe buhlala ngesikhathi sokugcinwa.
2. Nge-hybridization ngayinye sample, engeza u-50 µl wobuhlalu obumisiwe emithonjeni yepuleti elisha le-PCR noma ishubhu lokuhluthula.
3. Geza ubuhlalu ngokwengeza u-200 µl we-SureSelect Binding Buffer. Hlanganisa ngokufaka amapayipi phezulu naphansi izikhathi ezingu-20 noma uvale imithombo kanye ne-vortex ngesivinini esikhulu imizuzwana engu-5-10.
4. Faka ipuleti noma ishubhu lokuhlubula edivayisini yesihlukanisi kazibuthe.
5. Linda okungenani imizuzu emi-5 noma kuze kube yilapho ikhambi selicacile, bese ususa futhi ulahle amandla amakhulu.
6. Phinda Izinyathelo 3-5 izikhathi ezimbili ngaphezulu ngesamba sokugeza oku-3.
7. Misa kabusha ubuhlalu ku-200 µl we-SureSelect Binding Buffer.

Thatha i-Hybridized DNA usebenzisa ubuhlalu obuboshwe yi-Streptavidin

Izinto ezisetshenziswayo

• I-SureSelect Wash Buffer 1, 16 Rxn (Agilent, SureSelect XT HS Target Enrichment Kit ILM Hyb Module, Box 1 (Post PCR), )
• SureKhetha Geza I-Buffer 2, 16 Rxn (Agilent, SureSelect XT HS Target Enrichment Kit ILM Hyb Module, Box 1 (Post PCR), )

Ukulungiselela

Lungiselela okulandelayo:

Into	Isitoreji	Isiyalezo
SureSelect Wash Buffer 1,	Ukushisa Kwegumbi	Gcina ezingeni lokushisa legumbi.
SureSelect Wash Buffer 2,	Ukushisa Kwegumbi	Shisisa ama-aliquots angu-200 µl ku-70°C. Bheka Isinyathelo 4

Inqubo

1. Ngemuva kokuthi isinyathelo se-hybridization sesiqediwe futhi isithuthuthu esishisayo sifinyelele isinyathelo sokubamba esingu-65°C, dluliselaamples ekamelweni lokushisa.
2. Ngokushesha dlulisa ivolumu yonke (cishe u-30 µl) wengxube ngayinye yokuhlanganisa emithonjeni equkethe u-200 µl wobuhlalu be-streptavidin obugeziwe usebenzisa i-pipette yamashaneli amaningi. Pipette phezulu naphansi izikhathi 5-8 ukuxuba.
3. Gxumeka ishubhu lesiqeshana sokuthwebula ku-96- well plate mixer, uxube ngamandla (ku-1400–1800 rpm) noma i-rotator, imizuzu engu-30 ekamelweni lokushisa. Qiniseka ukuthi i-sampama-les axubana kahle emithonjeni.
4. Qhubekela esinyathelweni esilandelayo ngokushesha ngemva kokufukamela. Phakathi nokufukamela kwemizuzu engu-30 ukuze kubanjwe, ngaphambi kokuwashisa SureSelect Wash Buffer 2 at 70°C njengoba kuchazwe ngezansi. Faka ama-aliquots angu-200 µl we-Wash Buffer 2 emithonjeni yepuleti lemithombo engama-96 noma amashubhu okuqhafaza. I-Aliquot 6 imithombo ye-buffer ye-DNA ngayinyeample ekubalekeni. Vala imithombo bese ufukamela kumjikelezo oshisayo, nesivalo esishisayo IVULIWE, esibanjwe ku-70°C size sisetshenziswe
5. Uma isikhathi sokufukamela esiyimizuzu engama-30 esiqaliswe kuSinyathelo sesi-3 sesiphelile, phenya umugqaampkancane ukuqoqa uketshezi.
6. Faka ishubhu lokuhluthula kusihlukanisi kazibuthe ukuze uqoqe ubuhlalu. Linda kuze kube yilapho isisombululo sesicacile, bese ususa futhi ulahle amandla amakhulu.
7. Phinda umise ubuhlalu ku-200 µl we-SureSelect Wash Buffer 1. Hlanganisa ngokufaka amapayipi phezulu naphansi izikhathi ezingu-15–20, kuze kube yilapho ubuhlalu buphinde bumiswa ngokugcwele.
8. Faka ishubhu lesihluthulelo kusihlukanisi kazibuthe. Lindela ukuthi ikhambi lisule (cishe iminithi elingu-1), bese ususa futhi ulahle amandla amakhulu.
9. Susa amashubhu e-strip kusihlukanisi kazibuthe futhi uwadlulisele endaweni yokubeka emazingeni okushisa asekamelweni. Phinda umise ubuhlalu ku-200 µl ka-70°C olungiselelwe ngaphambilini Wash Buffer 2. Pipette phezulu naphansi izikhathi ezingu-15–20, kuze kube yilapho ubuhlalu buphinde bumiswa ngokugcwele. Vala imithombo ngamakepisi amasha bese u-vortex ngesivinini esikhulu imizuzwana engu-8. Spina ipuleti noma ishubhu lokuhlubula kafushane ukuze uqoqe uketshezi ngaphandle kokupelitha ubuhlalu.
10. Fukamisa i-sampLehlisa imizuzu emi-5 ku-70°C kumjikelezo oshisayo onesivalo esishisayo.
11. Faka ishubhu lesihluthulelo kusihlukanisi sikazibuthe ekamelweni lokushisa. Linda iminithi elingu-1 ukuze ikhambi lisule, bese ususa futhi ulahle amandla amakhulu.
12. Phinda Izinyathelo 9-11 izikhathi ezinhlanu ngaphezulu, ngesamba sokugeza oku-6.
13. Ngemva kokuqinisekisa ukuthi yonke i-wash buffer isusiwe, engeza u-25 µl wamanzi angenayo i-nuclease esigabeni ngasinye.ampkahle le. Pipette phezulu naphansi izikhathi ezingu-8 ukuze umise ubuhlalu futhi. Samples ingagcinwa eqhweni ngaphambili ampukuqinisa

AmpLify Umtapo Wezincwadi Othunjiwe

Izinto ezisetshenziswayo

• 5× Herculase II Reaction Buffer (Agilent, SureSelect XT HS Target Enrichment Kit, ILM Hyb Module Box 2 (Post PCR), CAP Clear)
• I-Herculase II Fusion DNA Polymerase (I-Agilent, SureSelect XT HS Target Enrichment Kit, ILM Hyb Module Box 2 (Post PCR), CAP Red)
• 100 mM dNTP Mix, (Agilent, SureSelect XT HS Target Enrichment Kit, ILM Hyb Module Box 2 (Post PCR), CAP Green)
• SureSelect Post-Capture Primer Mix (Agilent, SureSelect XT HS Target Enrichment Kit, ILM Hyb Module Box 2 (Post PCR), CAP Clear)

Ukulungiselela

1. Lungiselela okulandelayo:
   

   Into	Isitoreji	Isiyalezo
   5× I-Herculase II Reaction Buffer	-25°C kuya ku-15°C	Ncibilika ekamelweni lokushisa. Vortex ukuze hlanganisa, bese u-centrifuge kafushane. Qhubeka eqhweni.
   I-Herculase II Fusion DNA Polymerase	-25°C kuya ku-15°C	Centrifuge kafushane. Qhubeka eqhweni.
   100 mM dNTP Mix	-25°C kuya ku-15°C	Ncibilika ekamelweni lokushisa. Vortex ukuze  hlanganisa, bese u-centrifuge kafushane. Qhubeka eqhweni.
   SureSelect Post-Capture Primer Mix	-25°C kuya ku-15°C	Ncibilika ekamelweni lokushisa. I-Vortex ezoxutshwa, bese i-centrifuge kafushane. Qhubeka eqhweni.

2. Setha uhlelo olulandelayo ku-thermo cycler njengokulandelayo:
   • 98°C 2 amaminithi
   • [98°C imizuzwana engu-30, 63°C imizuzwana engu-30, 72°C 1 iminithi] x 9
   • 72°C 5 amaminithi
   • 4°C ingunaphakade

Inqubo

1. Lungiselela ivolumu efanelekile yengxube yokusabela kwe-PCR. Engeza ama-reagents alandelayo kushubhu ye-PCR ngokusekelwe enanini lokusabela.
   

   I-Reagent	Ivolumu ngokusabela ngakunye (µL)
   	Ivolumu yokusabela oku-1	Ivolumu yokusabela okungu-8 (kufaka phakathi okweqile)	Ivolumu yokusabela okungu-24 (kufaka phakathi okweqile)
   Amanzi angenayo i-nuclease	12.5µL	112.5µL	312.5µL
   5× I-Herculase II Reaction Buffer	10µL	90µL	250µL
   I-Herculase II Fusion DNA Polymerase	1µL	9µL	25µL
   100 mM dNTP Mix	0.5µL	4.5µL	12.5µL
   SureSelect Post-Capture Primer Mix	1µL	9µL	25µL

2. Lungiselela ivolumu efanelekile yengxube yokusabela kwe-PCR. Engeza ama-reagents alandelayo kushubhu ye-PCR ngokusekelwe enanini lokusabela. Engeza u-25 µl wengxube yokusabela ye-PCR elungiswe kuThebula 29 kumzuzwana ngamunyeampi-le equkethe ama-25 µl we-DNA enothiselwe eboshwe ngobuhlalu (ilungiselelwe ekhasini lama-26 futhi igcinwe eqhweni).
3. Hlanganisa kahle ukusabela kwe-PCR ngokufaka amapayipi phezulu naphansi kuze kube yilapho ukumiswa kobuhlalu kufana. Gwema ukuchaphaza samples phezu kwezindonga zomthombo; ungapholi i-sampkancane kulesi sinyathelo.
4. Beka ishubhu kumjikelezo oshisayo bese uqhuba uhlelo (bheka ngenhla) ngenani elifanele lemijikelezo.
5. Lapho i-PCR ampUhlelo lwe-lification luqediwe, phenya i-strip tube kafushane. Khipha ubuhlalu obunamekwe nge-streptavidin ngokubeka ipuleti noma ishubhu lokuhlubula endaweni kazibuthe ekamelweni lokushisa. Linda imizuzu emi-2 ukuze ikhambi lisule, bese ukhipha amandla angaphezu kwavamile (cishe ama-50 µl) uwayise emithonjeni yeshubhu lokuhluthula.

Hlanza Ilabhulali Ethunjiwe 

Izinto ezisetshenziswayo

• AMPure XP (Umsebenzisi)
• Ubufakazi be-Ethanol 200 (obuphelele) bebhayoloji yamangqamuzana (Umsebenzisi)
• Amanzi angenayo i-nuclease (Umsebenzisi)
• Ibhafa ye-TE, pH 8.0 (Umsebenzisi)
• 0.2 mL PCR tube noma strip tube (Umsebenzisi)

Ukulungiselela

Lungiselela okulandelayo:

Into	Isitoreji	Isiyalezo
I-ethanol entsha engu-75% (v/v).	Ukushisa Kwegumbi	Idinga 400 ml ngesekhondiample. Xuba i-Ethanol engu-1.5 ml (ubufakazi obungama-200) no-0.5 mL wamanzi angenayo i-Nuclease. I-Vortex yokuxuba futhi igcinwe ekamelweni lokushisa.
AMPawu XP	2°C kuya ku-8°C	Yilethe ekamelweni lokushisa okungenani imizuzu engu-20 kanye ne-vortex ngamandla ukuze umise ubuhlalu futhi ngaphambi kokusetshenziswa.
Amanzi angenayo i-nuclease	Ukushisa Kwegumbi	Gcina ezingeni lokushisa legumbi.
Ibhafa ye-TE, pH 8.0	Ukushisa Kwegumbi	Gcina ezingeni lokushisa legumbi.

Inqubo

1. Yehlisa isixazululo ngokujikeleza kwesekhondi elingu-~1 ku-centrifuge.
2. Vortex ngamandla ukumisa kabusha AMPure XP bese wengeza u-50 µL AMPusebenzisa i-XP kumkhiqizo ngamunye we-PCR.
3. Hlanganisa ngepayipi phezulu naphansi izikhathi ezi-10.
4. Fukamela ekamelweni lokushisa imizuzu emi-5.
5. Beka ithubhu endaweni kazibuthe iminithi elingu-1 noma kuze kube yilapho isisombululo sesicacile.
6. Aspirate futhi ulahle supernatant ngaphandle kokuphazamisa AMPure ubuhlalu.
7. Ngenkathi ugcina ishubhu endaweni kazibuthe, engeza u-200 µL osanda kulungiswa 75% ethanol eshubhuni. Ivumele ihlale imizuzwana engu-30.
8. Aspirate futhi ulahle supernatant ngaphandle kokuphazamisa ubuhlalu.
9. Phinda izinyathelo 7-8 kanye futhi, ugcine ithubhu ekumeni kazibuthe isikhathi sonke.
10. Shiya ishubhu endaweni kazibuthe enekepisi elivuliwe futhi uvumele ishubhu ukuthi lome imizuzu engu-1-2 ukuze lihwamuke iminonjana ye-ethanol. UNGABAMBI ngokweqile ubuhlalu.
11. Susa ishubhu kusitendi sikazibuthe bese wengeza ibhafa engu-25 µL TE kubuhlalu.
12. I-Pipette noma i-vortex yokumisa kabusha ubuhlalu. Yiyeke ihlale imizuzu emi-5.
13. Beka ithubhu endaweni kazibuthe iminithi elingu-1 noma kuze kube yilapho isisombululo sesicacile.
14. Phinda uthole u-23 µL we-supernatant kushubhu elisha. Qaphela ukuthi ungaphazamisi ubuhlalu.
15. I-supernatant iqukethe ilabhulali ye-TELL-Seq™ ethwetshiwe.

QAPHELA:
Leli IPHUZU LOKUMIMA OKUPHEPHILE. Umtapo wezincwadi othathiwe we-TELL-Seq ungagcinwa ku- -25°C kuya ku -15°C izinyanga eziyisithupha.

Qondanisa futhi Ulinganise Ilabhulali Ethathiwe ukuze ilandelelane

Izinto ezisetshenziswayo

• I-Agilent Bioanalyzer High Sensitivity DNA Kit noma i-TapeStation High Sensitivity D5000 ScreenTape Assay (Umsebenzisi)
• I-Qubit dsDNA HS Assay Kit (Umsebenzisi)
• Ibhafa ye-TE, pH 8.0 (Umsebenzisi)

Ukulungiselela

1. Lungiselela izinto ezisetshenziswayo ezidingekayo njengoba kudingwa yi-Bioanalyzer noma i-TapeStation ne-Qubit.

Inqubo

1. Sebenzisa u-1 µL welabhulali ye-Agilent Bioanalyzer High Sensitivity DNA Kit noma u-2 µL welabhulali ye-TapeStation High Sensitivity D5000 ScreenTape Assay.
2. Ukuze unqume ukugxiliswa komtapo wolwazi, setha iSifunda ku-Bioanalyzer noma isofthiwe yokuhlaziya ye-TapeStation ukusuka ku-150 bp ukuya ku-1000 bp. Qopha sample Concentration (nM) yalesi sifunda (bona Umfanekiso 2). Ukunquma usayizi womtapo wolwazi, setha iSifunda sisuka ku-150 bp siye ku-3000 bp. Qopha sample Usayizi Omaphakathi (bp) njengosayizi Welabhulali.
   ISEXWAYISO
   Ukufundwa kokugxilisa ingqondo okuvela ku-Bioanalyzer (noma i-TapeStation) kufanele kusetshenziswe njengesiqalo ukuze kwenziwe ukuhlanjululwa okudingekayo noma ukuhlanganisa umtapo wolwazi ukuze ulandelelane. Qinisekisa ukuqoqwa komtapo wolwazi wokugcina ohlanjululiwe wokulandelana kwezincwadi noma ichibi lomtapo ngekhithi ye-Qubit dsDNA HS Assay (bona Isinyathelo 6).Umfanekiso 2. ExampI-le of exome ithunjwe umtapo wolwazifile kusukela ku-Tape Station High Sensitivity D5000 Screen Tape assay.
3. Umtapo wezincwadi ungalandelana ngokushesha noma ugcinwe ku -25°C kuya ku -15°C.
4. Lapho ulandelanisa, nciphisa umtapo wolwazi usebenzisa i-TE buffer ekugxiliseni okunconywe uhlelo ngalunye lokulandelana kwe-Illumina®. Yenza i-pool yelabhulali ehlanjululwe ukuze uhleleke uma umtapo wolwazi engaphezu kweyodwa uzolandelana ngesikhathi esifanayo.
5. Linganisa ukugxiliswa komtapo wezincwadi nge-Qubit dsDNA HS Assay Kit. Sebenzisa inani likasayizi Omaphakathi elisuka esilinganisweni se-Bioanalyzer (noma i-TapeStation) njengosayizi welabhulali ukuze kuguqulwe ukugxiliswa kwesisindo ku-molar concentration (nM).

A = Ukugxila Okukhulu (ng/µL)
S = Usayizi Wezincwadi (bp)
I-Molar Concentration (nM) = (A*1,000,000)/(S*650)
Lungisa ivolumu edingekayo ekulungiseleleni ukulandelana uma ukugxiliswa komtapo wolwazi okukalwe nge-Qubit kuhlukile ekugxiliseni okunconyiwe okungaphezu kuka-10%.

Amadokhumenti / Izinsiza

UKULANDELANA KWE-UNIVERSAL TELL-Seq Ukuthuthukisa Ithagethi [pdf] Umhlahlandlela Womsebenzisi TELL-Seq Ukuthuthukisa Ithagethi, TELL-Seq, Ukucebisa Okuhlosiwe, Ukucebisa

Izithenjwa

• Imaniwali yosebenzisayo