I-TIANSeq iKhithi yeThala leeNcwadi ngqo kwi-Library (i-illumina)

Okwangoku, itekhnoloji yokulandelelana okuphezulu kokusebenza ikakhulu isekwe kwitekhnoloji yokulandelelana kwesizukulwana esilandelayo. Njengokuba ubude bokufunda kwitekhnoloji yokulandelelanisa isizukulwana esilandelayo bunqunyelwe, kufuneka siphule ulandelelwano olupheleleyo lube ngamathala eencwadi amancinci ngokulandelelana. Ngokweemfuno zovavanyo ezahlukeneyo zokulandelelana, sihlala sikhetha ukulandelelana okuphelileyo okanye ukulandelelana okuphela kabini. Okwangoku iziqwenga ze-DNA zelayibrari yokulandelelana kwesizukulwana esilandelayo ngokubanzi zihanjiswa kuluhlu lwama-200-800 bp.

a) I-DNA iphantsi ngokomgangatho kwaye ine-inhibitors. Sebenzisa iisampulu ezikumgangatho ophezulu zeDNA ukunqanda ukuthintelwa kwemisebenzi ye-enzyme.

b) Ixabiso lesampulu ye-DNA alonelanga xa usebenzisa indlela engenasimahla ye-PCR ukwakha ithala leencwadi le-DNA. Xa igalelo le-DNA eyahluliweyo lidlula i-50 ng, ukuhamba kwe-PCR-yasimahla kunokwenziwa ngexesha lenkqubo yokwakha ithala leencwadi. Ukuba inani lekopi yethala leencwadi liphantsi kakhulu ukuba linokulandelelana ngokuthe ngqo, ilayibrari ye-DNA inokunyuswa yi-PCR emva kokubopha iadaptha.

c) Ungcoliseko lwe-RNA lukhokelela kungcaciso engachanekanga yokuqala ye-DNA yokuchaneka kwe-RNA inokubakho kwinkqubo yokuhlanjululwa kwe-genomic DNA, enokuthi ikhokelele kubungakanani be-DNA engachanekanga kunye ne-DNA engonelanga yokulayisha ngexesha lokwakhiwa kwethala leencwadi. I-RNA ingasuswa ngokunyanga nge-RNase.

A-1

a) Amaqhekeza amancinci (60 bp-120 bp) avela amaqhekeza amancinci ahlala esiqhekeza iadaptha okanye iadimers ezenziwe ziadaptha. Ukucoca ngeAgencourt AMPure XP ubuhlalu bemagnethi kungazisusa ngokufanelekileyo ezi ziqhekeza zeadaptha kwaye kuqinisekiswe ukulandelelana komgangatho.

b) Amaqhekeza amakhulu avela kwilayibrari emva kokukhulisa kwe-PCR Ubungakanani beqhekeza leDNA yethala leencwadi liya kunyuka nge-120 bp emva kokuba iadaptha ibotshwe ngemigudu. Ukuba isiqwenga se-DNA sonyuka ngaphezulu kwe-120 bp emva kokubanjwa kweadaptha, kunokubangelwa kukuqhekeka kwesiqwengana esingaqhelekanga sokukhulisa i-PCR ngokugqithileyo. Ukunciphisa inani lemijikelezo ye-PCR kunokuthintela imeko.

c) Ubungakanani obungaqhelekanga bamaqhekeza eDNA elayibrari emva kokubopha iadaptha Ubude beadaptha kule khithi ngama-60 bp. Xa iziphelo ezibini zesiqwengana zibotshelelwe kwiiadaptha, ubude buya kunyuka nge-120 bp. Xa usebenzisa iadaptha ngaphandle kwale inikwe yile khithi, nceda unxibelelane nomthengisi ukuze anikezele ngolwazi olufanelekileyo njengobude beadaptha. Nceda uqinisekise ukuba ukuhamba komsebenzi kunye nokusebenza kulandela amanyathelo achazwe kule ncwadana.

d) Ubungakanani beqhekeza leDNA elingaqhelekanga phambi kokubanjwa kweadaptha Unobangela wale ngxaki unokubangelwa ziimeko zokuphendula ngendlela engeyiyo ngexesha lokuqhekeka kwe-DNA. Amaxesha okusabela ahlukeneyo kufuneka asetyenziselwe ukufaka okwahlukileyo kwe-DNA. Ukuba igalelo le-DNA lingaphezulu kwe-10 ng, sicebisa ukuba sikhethe ixesha lokuphendula le-12 min njengexesha lokuqala lokwenza izinto, kwaye ubungakanani beqhekeza obuveliswe ngeli xesha ikakhulu kuluhlu lwe-300-500 bp. Abasebenzisi banokonyusa okanye banciphise ubude beziqwengana ze-DNA kangange-2-4 min ngokweemfuno zabo zokuphucula iziqwenga zeDNA ngobungakanani obufunekayo.

A-2

a) Ixesha lokuqhekeka alilungiswanga Ukuba iDNA eyahluliweyo incinci kakhulu okanye inkulu kakhulu, nceda ujonge kwiZikhokelo zoKhetho lweXesha lokuQhekeka ezinikezwe kumyalelo wokumisela ixesha lokuphendula, kwaye usebenzise eli xesha njengendawo yokulawula, ukongeza ukuseta Inkqubo yokuphendula ukuze wandise okanye unciphise imizuzu emi-3 ukwenza uhlengahlengiso oluchanekileyo ngexesha lokuqhekeka.

A-3

Ukuhanjiswa kobungakanani ngokungaqhelekanga kwe-DNA emva konyango lokuqhekeka

a) Indlela engalunganga yokunyibilika yokuqhekeka kwe reagent, okanye reagent ayixutywanga ngokupheleleyo emva kokunyibilika. Thaw i-5 × Fragmentation Enzyme Mix reagent kwiqhwa. Nje ukuba unyibilikisiwe, xuba i-reagent ngokulinganayo ngokucofa ngobunono emazantsi etyhubhu. Musa ukwenza i-vortex reagent!

b) Isampulu yegalelo le-DNA iqulethe i-EDTA okanye ezinye izinto ezingcolisayo Ukupheliswa kweeon zetyuwa kunye neearhente zokukhohlisa kwinyathelo lokucoca i-DNA kubaluleke kakhulu kwimpumelelo yolingelo. Ukuba iDNA inyityilikisiwe kwi-1 × TE, sebenzisa indlela ebonelelweyo kwimiyalelo yokwenza ukwahlukana. Ukuba uxinizelelo lwe-EDTA kwisisombululo aluqinisekanga, kuyacetyiswa ukuba kuhlanjululwe i-DNA kwaye kuyinyibilikise emanzini angenanto yokuphendula okulandelayo.

c) Ukuchaneka kwe-DNA engachanekanga ubungakanani be-DNA eqhekeziweyo inxulumene ngokusondeleyo nesixa segalelo le-DNA. Phambi kokwahlulwa kwonyango, ubungakanani obuchanekileyo be-DNA kusetyenziswa iQubit, iPicogreen kunye nezinye iindlela kubalulekile ukumisela inani elichanekileyo le-DNA kwinkqubo yokuphendula.

 d) Ukulungiswa kwenkqubo yokuphendula akuhambisani nomyalelo. Ukuqinisekisa ukusebenza kakuhle, zonke izinto zokuphendula kufuneka zibekwe kumkhenkce kunye nokulungiswa kwenkqubo yokuphendula kufuneka yenziwe emva kokupholisa ngokupheleleyo. Emva kokuba amalungiselelo egqityiwe, nceda ucofe okanye umbhobho ukuze uxubeke kakuhle. Sukuvortex!

Indlela yokuxuba engafanelekanga (i-vortex, i-oscillation enobundlobongela, njl. Ke ngoko, xa ulungiselela isisombululo seMixutyana yokusabela, nceda ngobunono umbhobho phezulu naphantsi ukudibanisa, okanye sebenzisa incam yomnwe ukuze ucofe kwaye udibanise ngokulinganayo. Lumka ungaxubeki ne-vortex.

2. Kufuneka kusetyenziswe ubunyulu beDNA kulwakhiwo lwamathala eencwadi

■ Ukunyaniseka kwe-DNA elungileyo: Ibhendi ye-electrophoresis ingaphezulu kwama-30 kb, ngaphandle kokuyila

■ OD260 / 230:> 1.5

■ OD260 / 280: 1.7-1.9

3. Igalelo le-DNA kufuneka lichanekile Kucetyiswa ukuba kusetyenziswe iindlela zeQubit kunye nePicoGreen ukulinganisa iDNA, endaweni yeNanodrop.

4. Umxholo we-EDTA kwisisombululo se-DNA kufuneka umiselwe i-EDTA inefuthe elikhulu kwimpendulo yokusasazeka. Ukuba umxholo we-EDTA uphezulu, ukucocwa kwe-DNA kufuneka kwenziwe ngaphambi kovavanyo olulandelayo.

5. Isisombululo sokuqhekeka kwesiqhekeza kufuneka silungiswe emkhenkceni Inkqubo yokuqhekeka inovakalelo kwiqondo lobushushu kunye nexesha (ngakumbi emva kokudibanisa). Ukuqinisekisa ukuchaneka kwexesha lokuphendula, nceda ulungiselele inkqubo yokuphendula kumkhenkce.

6. Ixesha lokusabela kokuqhekeka kufuneka lichaneke Ixesha lokuphendula kwenyathelo lokuqhekeka liya kuchaphazela ngokuthe ngqo ubungakanani beemveliso zamaqhekeza, oko ke kuchaphazele ubungakanani bokusasazwa kwamaqhekeza e-DNA elayibrari.

1. Loluphi uhlobo lwesampulu olusebenzayo kule khithi?

Uhlobo lwesampulu olusebenzayo lwale khithi lunokuba yi-RNA iyonke okanye i-mRNA ecociweyo enesidima seRNA. Ukuba i-RNA iyonke isetyenziselwa ukwakha ithala leencwadi, kuyacetyiswa ukuba kusetyenziswe ikhithi yokuphelisa iRRNA (Ikati # 4992363/4992364/4992391) ukususa iRRNA kuqala.

2. Ngaba iisampulu ze-FFPE zingasetyenziselwa ukwakha ithala leencwadi ngale khithi?

I-mRNA kwiisampulu ze-FFPE iyakuthotywa isidima ukuya kuthi ga kwinqanaba elithile, ngentembeko engalunganga. Xa usebenzisa le khithi kulwakhiwo lwethala leencwadi, kuyacetyiswa ukuba ukwandise ixesha lokuqhekeka (mfutshane ixesha lokuqhekeka okanye ungenzi ukwahlulwa).

3. Usebenzisa inyathelo lokukhetha ubungakanani elinikezwe kwincwadana yemveliso, yintoni enokubangela ukuba icandelo elifakiweyo libonakale linxaxha kancinci?

Ukukhethwa kobungakanani kuya kwenziwa ngokungqinelana nenqanaba lokukhetha ubungakanani kule ncwadana yemveliso. Ukuba kukho ukuphambuka, isizathu sinokuba amaso emagnethi awalungelelananga kubushushu begumbi okanye awadityaniswanga ngokupheleleyo, ipipette ayichanekanga okanye ulwelo luhlala kwincam. Kuyacetyiswa ukuba usebenzise iingcebiso ngentengiso ephantsi kulingo.

4. Ukukhethwa kwee-adapters kulwakhiwo lwamathala eencwadi

Ikhithi yokwakha ilayibrari ayiqulathanga reagent yeadaptha, kwaye kuyacetyiswa ukuba kusetyenziswe le khithi kunye ne-TIANSeq Adapter-Index Adapter (Illumina) (4992641/4992642/4992378).

5. QC yethala leencwadi

Ukufunyanwa kobungakanani bethala leencwadi: I-Qubit kunye ne-qPCR zisetyenziselwa ukumisela ubuninzi boxinzelelo kunye nokuxinana kwe-molar kwithala leencwadi ngokulandelelana. Umsebenzi uhambelana ngokungqinelana nencwadana yemveliso. Ukuxinana kwethala leencwadi kuyakufezekisa iimfuno zokulandelelana kwe-NGS. Ukuchongwa koluhlu losasazo lwethala leencwadi: Kusetyenziswa i-Agilent 2100 Bioanalyzer ukufumana uluhlu lokuhanjiswa kwethala leencwadi.

6. Ukukhethwa kwenani lomjikelo wokukhulisa

Ngokwemiyalelo, inani lemijikelezo ye-PCR yi-6-12, kwaye inani lemijikelezo ye-PCR efunekayo kufuneka ikhethwe ngokwesampulu yegalelo. Kwiilayibrari ezinesivuno esikhulu, ngaphezulu kokukhulisa ngokwesiqhelo kwenzeka ngokwamanqanaba awahlukeneyo, abonakaliswa yincopho enkulu ethe kratya emva kwencopho yoluhlu ekujoliswe kulo ekufumaneni i-Agilent 2100 Bioanalyzer, okanye uxinzelelo olufunyenweyo lweQubit lusezantsi kunolo lwe-qPCR. Ubumnene ngaphezulu kokukhulisa yinto eqhelekileyo, engachaphazeli ukulandelelana kwethala leencwadi kunye nohlalutyo lwedatha olulandelayo.

7. I-spikes ibonakala kwiprofayili yokufumanisa i-Agilent 2100 Bioanalyzer

Ukuvela kweepikhi ekufumaneni kwe-Agilent 2100 Bioanalyzer kungenxa yokuqhekeka okungalinganiyo kweesampulu, apho kuya kubakho iziqwenga ezingaphezulu kubungakanani obuthile, kwaye oku kuyakucaca ngakumbi emva kokutyebiswa kwePCR. Kule meko, kuyacetyiswa ukuba ungenzi ukhetho lobungakanani, okt ukuseta imeko yokuqhekeka iye kwi-94 ° C kangange-15 min efukamileyo, apho ulwabiwo lwesiqwengana lincinci kwaye lujolise, kwaye i-homogeneity inokuphuculwa.