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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


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 chitsanzo

Kukonzekera kwachitsanzo

Kukonzekera kwa Library

Kumanga laibulale

Kutsata

Kutsata

Kusanthula deta

Kusanthula deta

Chitsanzo cha QC

Kutumiza kwa polojekiti


  • Zam'mbuyo:
  • Ena:

  • 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

     

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