Imikhiqizo

Imitapo yolwazi eyenziwe kusengaphambili ye-Illumina

Amalabhulali enziwe kusengaphambili e-Illumina Isithombe Esivelele

Ubuchwepheshe bokulandelana kwe-Illumina, obusekelwe ku-Sequencing by Synthesis (SBS), buyindlela entsha ye-NGS eyamukelwa emhlabeni wonke, ephethe ukukhiqiza idatha yokulandelana engaphezu kuka-90% emhlabeni. Isimiso se-SBS sihilela ukuthwebula izithombe ze-terminator eziguquguqukayo ezibhalwe nge-fluorescent njengoba i-dNTP ngayinye ingezwa, bese ihlukaniswa ukuze kuvunyelwe ukufakwa kwesisekelo esilandelayo. Njengoba wonke ama-dNTP amane aguquguqukayo aboshwe yi-terminator akhona kumjikelezo ngamunye wokulandelana, ukuncintisana kwemvelo kunciphisa ukubandlulula kokufakwa. Lobu buchwepheshe obuguquguqukayo busekela kokubili imitapo yolwazi efundwayo kanye neyamabili, bunakekela izinhlelo zokusebenza ezahlukahlukene ze-genomic. Amakhono aphezulu okusebenzisa i-Illumina sequencing kanye nokunemba kuyibeka njengetshe lesisekelo ocwaningweni lwe-genomics, okunika amandla ososayensi ukuthi bahlukanise ubunzima be-genomes ngemininingwane engenakuqhathaniswa kanye nokusebenza kahle.

Insizakalo yethu yokulandelelanisa amalabhulali eyenziwe ngaphambilini ivumela amakhasimende ukuthi alungiselele amalabhulali okulandelelanisa amalabhulali avela emithonjeni eyahlukahlukene (i-mRNA, i-genome ephelele, i-amplicon, amalabhulali ayi-10x, phakathi kwabanye). Ngemva kwalokho, lawa malabhulali angathunyelwa ezikhungweni zethu zokulandelelanisa amalabhulali ukuze kulawulwe ikhwalithi kanye nokulandelelanisa amapulatifomu e-Illumina.

Xhumana nathi

Imininingwane Yesevisi

Umphumela Wedemo

Izici

●Amapulatifomu:I-Illumina NovaSeq 6000 kanye ne-NovaSeq X Plus

●Izindlela zokulandelanisa:I-PE150 kanye ne-PE250

●Ukulawulwa kwekhwalithi yemitapo yolwazi ngaphambi kokulandelana

●Ukulandelanisa idatha ye-QC kanye nokulethwa:ukulethwa kombiko we-QC kanye nedatha eluhlaza ngefomethi ye-fastq ngemuva kokuhlukanisa izinto eziningi nokuhlunga ukufundwa kwe-Q30

Izinzuzo Zesevisi

●Ukuguquguquka kwezinsizakalo zokulandelela:ikhasimende lingakhetha ukulandelana ngomzila, iseli lokugeleza, noma ngenani ledatha elidingekayo (ukulandelana kwemizila engaphelele).

●Ulwazi oluningi ngeplatifomu yokulandelelana kwe-Illumina:nezinkulungwane zamaphrojekthi avaliwe anezinhlobo ezahlukene.

●Ukulethwa kombiko we-QC wokulandelana:ngezilinganiso zekhwalithi, ukunemba kwedatha kanye nokusebenza okuphelele kwephrojekthi yokulandelana.

●Inqubo yokulandelana kwabantu abadala:ngesikhathi esifushane sokujika.

●Ukulawulwa Kwekhwalithi Okuqinile: sisebenzisa izidingo ze-QC eziqinile ukuqinisekisa ukulethwa kwemiphumela esezingeni eliphezulu njalo.

Amapulatifomu Esampula

Ipulatifomu	Iseli Lokugeleza	Imodi yokulandelana	Iyunithi	Umphumela olinganiselwe
I-NovaSeq X	10B (imizila engu-8)	I-PE150	Indlela Eyodwa Indlela Engaphelele	375Gb / Umzila
I-NovaSeq X	25B (imizila engu-8)	I-PE150	Indlela Eyodwa Indlela Engaphelele	1000 Gb/Umzila
I-NovaSeq 6000	Iseli le-SP Flow (imizila emi-2)	I-PE250	Iseli Lokugeleza Indlela Eyodwa Indlela Engaphelele	Ukufundwa okungu-325-400 M / Umzila
I-NovaSeq 6000	Iseli lokugeleza le-S4 (imizila emi-4)	I-PE150	Iseli Lokugeleza Indlela Eyodwa Indlela Engaphelele	~800 Gb / Umzila

Izidingo Zesampula

	Inani Ledatha (Gb)	I-Conc. (qPCR) PE150	I-Conc. (qPCR) PE250	Ivolumu
Indlela Engaphelele	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 Eyodwa	Ngomzila Ngamunye	≥ 1.5 nM/Ichibi lomtapo wolwazi	≥ 2 nM/Ichibi lomtapo wolwazi	≥ 25 µL/Ichibi lokubhukuda lomtapo wolwazi

Ngaphezu kokuhlushwa kanye nenani eliphelele, iphethini efanele yokuphakama nayo iyadingeka.

Qaphela: Ukulandelana kwemizila yemitapo yolwazi ehlukahlukene kudinga ukukhushulwa kwe-PhiX ukuqinisekisa ukubizwa okuqinile kwesisekelo.

Sincoma ukuhambisa amalabhulali ahlanganiswe kusengaphambili njengezibonelo zokulandelana kwemizila. Uma udinga i-BMKGENE ukuthi yenze i-library pooling, sicela ubheke izidingo zelabhulali zokulandelana kwemizila engaphelele.

Usayizi Welabhulali (Imephu Ephakeme)

Kokulandelana kwe-PE150, usayizi wokufaka kufanele ube ngu-300–400 bp (ngaphandle kwama-adapter); kokulandelana kwe-PE250, usayizi wokufaka kufanele ube ngu-150–500 bp (ngaphandle kwama-adapter).
Amalabhulali kufanele abe ne-peak eyodwa enkulu, angabi nakungcola kwe-adaptha futhi angabi nama-primer dimers.

Sicela usithinte uma amasampula akho engahlangabezani nezidingo zezinto zokuqala.

Xhumana nathi

Ukuhamba Komsebenzi Wesevisi

Ukulawulwa kwekhwalithi yelabhulali

Ukulandelana

Ukulawulwa kwekhwalithi yedatha

Ukulethwa kwephrojekthi

Okwedlule: Ukulandelana kwe-DNA/RNA - I-PacBio Sequencer

Olandelayo: Ukulandelana kwe-DNA/RNA – I-Nanopore Sequencer

Umbiko we-QC weLabhulali

Umbiko ngekhwalithi yomtapo wolwazi unikezwa ngaphambi kokulandelana, ukuhlola inani lomtapo wolwazi, kanye nokuqhekeka kwawo.

Ukulandelanisa umbiko we-QC

Ithebula 1. Izibalo mayelana nedatha yokulandelana.

I-ID Yesampula	I-BMKID	Ukufunda okungavuthiwe	Idatha Engavuthiwe (bp)	Ukufunda okuhlanzekile (%)	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