Iimveliso

Iilayibrari ezenziwe kwangaphambili ze-Illumina

Itekhnoloji yokulandelelanisa i-Illumina, esekelwe kwi-Sequencing by Synthesis (SBS), yindlela entsha ye-NGS eyamkelweyo kwihlabathi liphela, enoxanduva lokuvelisa ngaphezulu kwe-90% yedatha yokulandelelanisa yehlabathi. Umgaqo we-SBS ubandakanya ukufota ii-terminators eziguquguqukayo ezibhalwe nge-fluorescent njengoko i-dNTP nganye yongezwa, kwaye emva koko ziqhekeke ukuvumela ukufakwa kwesiseko esilandelayo. Njengoko zonke ii-dNTP ezine eziguqulweyo ze-terminator-bound zikhona kumjikelo ngamnye wokulandelelanisa, ukhuphiswano lwendalo lunciphisa ukukhetha kokufakwa. Le teknoloji iguquguqukayo ixhasa zombini iilayibrari ezifundwayo kunye nezimbini, ilungiselela uluhlu lwezicelo ze-genomic. Amandla aphezulu okusebenzisa i-Illumina sequencing kunye nokuchaneka kwayo kuyibeka njengesiseko sophando lwe-genomics, inika amandla izazinzulu ukuba zityhile ubunzima bee-genomes ngeenkcukacha ezingenakuthelekiswa nanto kunye nokusebenza kakuhle.

Inkonzo yethu yokulandelelanisa iilayibrari eyenziwe kwangaphambili ivumela abathengi ukuba balungiselele iilayibrari zokulandelelanisa ezivela kwimithombo eyahlukeneyo (i-mRNA, i-genome epheleleyo, i-amplicon, iilayibrari ezili-10x, phakathi kwezinye). Emva koko, ezi layibrari zingathunyelwa kumaziko ethu okulandelelanisa ukuze kulawulwe umgangatho kunye nokulandelelanisa kwiiplatifomu ze-Illumina.

Qhagamshelana nathi

Iinkcukacha zeNkonzo

Isiphumo seDemo

Iimbonakalo

●Amaqonga:I-Illumina NovaSeq 6000 kunye neNovaSeq X Plus

●Iindlela zokulandelelanisa:I-PE150 kunye ne-PE250

●Ulawulo lomgangatho weelayibrari ngaphambi kokulandelelana

●Ukulandelelanisa idatha ye-QC kunye nokuhanjiswa kwayo:ukuhanjiswa kwengxelo ye-QC kunye nedatha eluhlaza kwifomathi ye-fastq emva kokususa izinto ezininzi kunye nokucoca ii-Q30 reads

Iingenelo zeNkonzo

●Ukuguquguquka kweenkonzo zokulandelelanisa:umthengi angakhetha ukulandelana ngomgca, iseli yokuhamba, okanye ngobungakanani bedatha efunekayo (ulandelelwano lwendlela engaphelelanga).

●Amava amaninzi kwiqonga lokulandelelana kwe-Illumina:kunye namawaka eeprojekthi ezivaliweyo ezineentlobo ngeentlobo.

●Ukuhanjiswa kwengxelo ye-QC yokulandelelana:ngemilinganiselo yomgangatho, ukuchaneka kwedatha kunye nokusebenza ngokubanzi kweprojekthi yokulandelelana.

●Inkqubo yokulandelelanisa abantu abadala:ngexesha elifutshane lokubuyela umva.

●Ulawulo Oluqinileyo Lomgangatho: Sisebenzisa iimfuno ze-QC ezingqongqo ukuqinisekisa ukunikezelwa kweziphumo ezisemgangathweni ophezulu rhoqo.

Iiplatfomu zeSampulu

Iqonga	Iseli Yokuhamba	Imo yokulandelelanisa	Iyunithi	Imveliso eqikelelweyo
I-NovaSeq X	10B (iindlela ezi-8)	PE150	Indlela enye Indlela Engaphelelanga	375Gb / Indlela
I-NovaSeq X	25B (iindlela ezi-8)	PE150	Indlela enye Indlela Engaphelelanga	1000 Gb/Umgca
I-NovaSeq 6000	Iseli ye-SP Flow (iindlela ezi-2)	PE250	Iseli Yokuhamba Indlela enye Indlela Engaphelelanga	Ufundo lwe-325-400 M / Indlela
I-NovaSeq 6000	Iseli ye-S4 Flow (iindlela ezi-4)	PE150	Iseli Yokuhamba Indlela enye Indlela Engaphelelanga	~800 Gb / Indlela

Iimfuno zeSampuli

	Isixa seDatha (Gb)	I-Conc. (qPCR) PE150	I-Conc. (qPCR) PE250	Umqulu
Indlela Engaphelelanga	G ≤ 10	≥ 1 nM	≥ 2nM	≥ 25 μl
	10 < G ≤ 50	≥ 2 nM	≥ 3nM	≥ 25 μl
	50 < G ≤ 100	≥ 3 nM	≥ 4nM	≥ 25 μl
	G > 100	≥ 4 nM	≥ 4nM	≥ 25 μl
Indlela enye	NgokweNdlela nganye	≥ 1.5 nM/Idama lokubhukuda leThala leeNcwadi	≥ 2 nM/Idama lokubhukuda leThala leeNcwadi	≥ 25 µL/Idama lokubhukuda leThala leeNcwadi

Ukongeza kuxinzelelo kunye nobungakanani obupheleleyo, kufuneka iphethini efanelekileyo yokuphakama.

Qaphela: Ukulandelelana kweendlela zeelayibrari ezinazo iintlobo ngeentlobo kufuna iPhiX inyuswe ukuze kuqinisekiswe ukuba isiseko sisebenza kakuhle.

Sicebisa ukuba kungeniswe iilayibrari ezidityaniswe kwangaphambili njengeesampuli zokulandelelanisa iindlela. Ukuba ufuna i-BMKGENE yenze i-library pooling, nceda ujonge iimfuno zelayibrari zokulandelelanisa iindlela ezingaphelelanga.

Ubungakanani beThala leeNcwadi (Imephu yeNcam)

Kwi-PE150 sequencing, ubungakanani bokufakwa kufuneka bube yi-300–400 bp (ngaphandle kwee-adapters); kwi-PE250 sequencing, ubungakanani bokufakwa kufuneka bube yi-150–500 bp (ngaphandle kwee-adapters).
Amathala eencwadi kufuneka abe ne-peak enye ephambili, kungabikho ungcoliseko lwe-adaptha kwaye kungabikho zi-primer dimers.

Nceda uqhagamshelane nathi ukuba iisampulu zakho azihlangabezani neemfuno zezinto zokuqala.

Qhagamshelana nathi

Ukuhamba komsebenzi wenkonzo

Ulawulo lomgangatho weThala leeNcwadi

Ukulandelelana

Ulawulo lomgangatho wedatha

Ukuhanjiswa kweprojekthi

Ngaphambili: Ulandelelwano lwe-DNA/RNA -PacBio Sequencer

Okulandelayo: Ulandelelwano lweDNA/RNA – Ulandelelwano lweNanopore

Ingxelo yeQC yeThala leeNcwadi

Ingxelo ngomgangatho wethala leencwadi inikwa ngaphambi kokuba kulandelelaniswe, kuhlolwe ubungakanani bethala leencwadi, kwaye kuqhekeke.

Ingxelo ye-QC elandelelanayo

Itheyibhile 1. Izibalo malunga nedatha yokulandelelana.

Isazisi seSampuli	I-BMKID	Ukufunda okungavuthwanga	Idatha eluhlaza (bp)	Ukufundwa okucocekileyo (%)	Q20(%)	Q30(%)	I-GC(%)
C_01	BMK_01	22,870,120	6,861,036,000	96.48	99.14	94.85	36.67
C_02	BMK_02	14,717,867	4,415,360,100	96.00	98.95	93.89	37.08