Imikhiqizo

Ukulandelana kwe-DNA/RNA -I-PacBio Sequencer

Iplathifomu yokulandelana ye-PacBio iyinkundla yokulandelana efunde isikhathi eside, eyaziwa nangokuthi obunye bobuchwepheshe bokulandelana kwesizukulwane sesithathu (TGS).Ubuchwepheshe obuyisisekelo, i-single-molecule real-time(SMRT), inika amandla ukukhiqizwa kokufunda ngamashumi ama-kilo-base ubude.Ngesisekelo se-"Sequencing-by-Synthesis", ukulungiswa kwe-nucleotide eyodwa kufinyelelwa yi-Zero-mode waveguide(ZMW), lapho kuphela ivolumu elinganiselwe ezansi(indawo yokuhlanganiswa kwe-molecule), ikhanyisiwe.Ukwengeza, ukulandelana kwe-SMRT kugwema kakhulu ukulandelana okuqondile ohlelweni lwe-NGS, ngokuthi izinyathelo eziningi zokukhulisa i-PCR azidingeki kunqubo yokwakha umtapo wolwazi.

I-Platform: I-Sequel II, i-Revio

Xhumana nathi

Imininingwane Yesevisi

Imiphumela yedemo

Izici

Izindlela ezimbili zokulandelanisa ku-PacBio sequencer: I-Continuous Long Read (CLR) kanye ne-Circular Consensus Read (CCS)

Imodi yokulandelanisa

Usayizi Wezincwadi

Idatha YetiyethaYield (Ngeseli)

Isisekelo esisodwaUkunemba

Izinhlelo zokusebenza

I-CLR

20Kb, 30Kb, njll.

80 Gb ukuze 130 Gb

Cishe.85%

De novo, ukushaya kwe-SV, njll.

I-CCS

15-20 KB

14 kuya ku-40 Gb/Cell (Sequel II)

70 kuya ku-110 Gb/Cell (Revio)

Kuya ngamasampula

Cishe.99%

De novo, SNP/Indel/SV ukushaya, Iso-Seq,

Ukuqhathaniswa kokusebenza nezici ze-Revio ne-Sequel II

Imigomo	Isistimu ye-Sequel II	Isistimu ye-Revio	Khulisa
Ukuminyana okuphezulu	8 million ZMWs	25 million ZMWs	3x
Izigaba ezizimele	1	4	4x
Izikhathi zokugijima ezimfushane	Amahora angu-30	Amahora angu-24	1.25x
I-30X HiFi i-genome yomuntu / ngonyaka	88	1,300	15x ngokuphelele

Izinzuzo Zesevisi

● Isipiliyoni seminyaka engaphezu kwengu-8 kuplathifomu yokulandelana ye-PacBio enezinkulungwane zamaphrojekthi avaliwe anezinhlobo ezihlukahlukene.

● Ihlome ngokugcwele ngezinkundla zakamuva ze-PacBio Sequencing, i-Revio ukuze iqinisekise ukulandelana okwanele kokuphumayo.

● Isikhathi sokushintsha ngokushesha, isivuno esiphezulu sedatha nedatha enembe kakhudlwana.

● Kunikelwe emakhulwini okushicilelwe okusekelwe ku-PacBio okunomthelela ophezulu.

Izidingo Zesampula

Uhlobo lwesampula

Inani

I-Concentration (Qubit®)

Ivolumu

Ubumsulwa

Abanye

I-Genomic DNA

Kuya Ngesidingo Sedatha

≥50 ng/μl

≥15μl

OD260/280=1.7-2.2;
OD260/230=1.8-2.5;
Sula ukuphakama okungu-260 nm,akukho ukungcola

Ukugxila kudinga ukulinganiswa nge-Qubit ne-Qubit/Nanopore = 0.8-2.5

Isamba se-RNA

≥1.2μg

≥120 ng/μl

≥15μl

OD260/280=1.7-2.5;
OD260/230=0.5-2.5;akukho ukungcola

I-RIN inani ≥7.5

5≥28S/18S≥1

Uhlelo lokusebenza lwesevisi

Ukulungiselela isampula

Ukwakhiwa komtapo wolwazi

Ukulandelanisa

Ukuhlaziywa kwedatha

Ukulethwa kwephrojekthi

Okwedlule: Umkhiqizi we-OEM uMrna Sequencing Service - I-Illumina ende engafaki ikhodi - i-Biomarker

Olandelayo: I-Illumina ne-BGI

1.In-house Data Yield

Idatha ekhiqizwe kumaseli e-CCS angama-63 (kusukela ezinhlotsheni ezingama-26)

IDATHA-PacBio-CCS-15 KB	Isilinganiso	Ubukhulu	Okuncane	I-Median
Yield - subreads (Gb)	421.12	544.27	221.38	426.58
I-Yiled - CCS(Gb)	25.93	38.59	10.86	25.43
I-Polymerase N50	145,651	175,430	118,118	144,689
Izifundo ezingaphansi kwe-N50	17,509	23,924	12,485	17,584
I-CCS N50	14,490	19,034	9,876	14,747
Isilinganiso sobude-Polymerase	67,995	89,379	49,664	66,433
Isilinganiso sobude-Iziqephu ezingezansi	15,866	21,036	11,657	16,012
Ubude obumaphakathi-CCS	14,489	19,074	8,575	14,655

Idatha ekhiqizwe kumaseli angu-16 e-CLR (kusukela ezinhlotsheni ezingama-76)

IDATHA-PacBio-CLR-30Kb	Isilinganiso	Ubukhulu	Okuncane	I-Median
Yield - subreads (Gb)	142.20	291.40	50.55	142.49
I-Polymerase N50	39,456	121,191	15,389	35,231
Izifundo ezingaphansi kwe-N50	28,490	41,012	14,430	29,063
Isilinganiso sobude-Polymerase	22,063	48,886	8,747	21,555
Isilinganiso sobude-Iziqephu ezingezansi	17,720	27,225	8,293	17,779

2.Idatha ye-QC – IdemoIzibalo ngesivuno sedatha

Isampula	ccs Ufunda Num	Isamba sezisekelo ze-ccs (bp)	I-ccs Ifunda i-N50 (bp)	ccs Mean Length (bp)	I-ccs efunde isikhathi eside (bp)	Izisekelo zokufunda ezingaphansi (bp)	isilinganiso se-ccs(%)
PB_BMKxxx	3,444,159	54,164,122,586	15,728	15,726	36,110	863,326,330,465	6.27