Zogulitsa

DNA / RNA sequencing -PacBio Sequencer

PacBio sequencing platform ndi nsanja yowerengera nthawi yayitali, yomwe imadziwikanso kuti ndi imodzi mwaukadaulo wa Third-Generation Sequencing (TGS).Ukadaulo wapakatikati, single-molecule real-time(SMRT), umapatsa mphamvu zowerengera ndi makumi a kilo-base kutalika.Pansi pa "Sequencing-by-Synthesis", kusamvana kwa nucleotide imodzi kumatheka ndi Zero-mode waveguide(ZMW), pomwe voliyumu yocheperako pansi (malo a molecule synthesis), imawunikiridwa.Kuphatikiza apo, kutsatizana kwa SMRT kumapewa kukondera kwanthawi yayitali mu dongosolo la NGS, chifukwa njira zambiri zokulira za PCR sizofunikira pakumanga laibulale.

Platform: Sequel II, Revio

Lumikizanani nafe

Tsatanetsatane wa Utumiki

Zotsatira za Demo

Mawonekedwe

Mitundu iwiri yotsatizana pa PacBio sequencer: Continuous Long Read (CLR) ndi Circular Consensus Read (CCS)

Njira Yotsatirira

Kukula kwa Library

Theoretical DataPerekani (Pa Cell)

Single-baseKulondola

Mapulogalamu

CLR

20Kb, 30Kb, etc.

80 Gb mpaka 130 Gb

Pafupifupi.85%

Pa novo, kuyimba kwa SV, etc.

Mtengo CCS

15-20 KB

14 mpaka 40 Gb/Cell (Sequel II)

70 mpaka 110 Gb/Cell (Revio)

Zimatengera zitsanzo

Pafupifupi.99%

Pa novo, SNP/Indel/SV kuyimba, Iso-Seq,

Kuyerekeza kwa magwiridwe antchito ndi mawonekedwe a Revio ndi Sequel II

Terms	Sequel II System	Revio dongosolo	Wonjezani
Kuchulukana kwakukulu	8 miliyoni ZMWs	25 miliyoni ZMWs	3x
Magawo odziyimira pawokha	1	4	4x
Nthawi zazifupi zothamanga	30 maola	24 maola	1.25x
30X HiFi ma genome amunthu / chaka	88	1,300	15x zonse

Ubwino wa Utumiki

● Kupitilira zaka 8 pa nsanja yotsatizana ya PacBio yokhala ndi ma projekiti masauzande otsekedwa okhala ndi mitundu yosiyanasiyana.

● Wokonzeka bwino ndi nsanja zaposachedwa za PacBio Sequencing, Revio kutsimikizira kutsatizana kokwanira.

● Nthawi yosinthira mwachangu, kuchulukirachulukira kwa data ndi data yolondola kwambiri.

● Zoperekedwa m'mabuku ambirimbiri okhudza PacBio.

Zitsanzo Zofunika

Mtundu Wachitsanzo

Ndalama

Kukhazikika (Qubit®)

Voliyumu

Chiyero

Ena

Genomic DNA

Zimatengera Zofunikira pa Data

≥50 ng/μl

≥15μl

OD260/280=1.7-2.2;
OD260/230=1.8-2.5;
Chotsani pachimake pa 260 nm,palibe kuipitsidwa

Kuyikirako kumafunika kuyezedwa ndi Qubit ndi Qubit/Nanopore = 0.8-2.5

Mtengo wa RNA

≥1.2μg

≥120 ng/μl

≥15μl

OD260/280=1.7-2.5;
OD260/230=0.5-2.5;palibe kuipitsidwa

Mtengo wa RIN ≥7.5

5≥28S/18S≥1

Njira ya utumiki

Kukonzekera kwachitsanzo

Kumanga laibulale

Kutsata

Kusanthula deta

Kutumiza kwa polojekiti

Zam'mbuyo: Wopanga OEM Mrna Sequencing Service - Illumina yayitali yosalemba zolemba - Biomarker

Ena: Illumina ndi BGI

1. Kutulutsa kwa data m'nyumba

Deta yopangidwa kuchokera ku 63 CCS maselo (kuchokera 26 mitundu)

DATA-PacBio-CCS-15 KB	Avereji	Max	Min	Wapakati
Zokolola - zowerengeka (Gb)	421.12	544.27	221.38	426.58
Yiled - CCS(Gb)	25.93	38.59	10.86	25.43
Polymerase N50	145,651	175,430	118,118	144,689
Zithunzi za N50	17,509	23,924	12,485	17,584
CCS N50	14,490	19,034	9,876	14,747
Utali wapakati-Polymerase	67,995	89,379	49,664	66,433
Avereji yautali-Magawo ang'onoang'ono	15,866	21,036	11,657	16,012
Utali wapakati-CCS	14,489	19,074	8,575	14,655

Deta yopangidwa kuchokera ku maselo 16 a CLR (kuchokera ku mitundu 76)

DATA-PacBio-CLR-30Kb	Avereji	Max	Min	Wapakati
Zokolola - zowerengeka (Gb)	142.20	291.40	50.55	142.49
Polymerase N50	39,456	121,191	15,389	35,231
Zithunzi za N50	28,490	41,012	14,430	29,063
Utali wapakati-Polymerase	22,063	48,886	8,747	21,555
Avereji yautali-Magawo ang'onoang'ono	17,720	27,225	8,293	17,779

2.Data QC - ChiwonetseroZiwerengero za kutulutsa kwa data

Chitsanzo	ccs Amawerenga Num	Mabasi onse a ccs (bp)	ccs Amawerenga N50 (bp)	ccs Kutalikirana kwa Utali (bp)	ccs Kuwerenga Kwambiri Kwambiri (bp)	magawo ang'onoang'ono (bp)	ccs mlingo (%)
PB_BMKxxx	3,444,159	54,164,122,586	15,728	15,726	36,110	863,326,330,465	6.27