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DNA/RNA sequencing -PacBio Sequencer

PacBio sequencing platform is a long-read sequencing platform, which is also known as one of the Third-Generation Sequencing(TGS) technologies. The core technology, single-molecule real-time(SMRT), empowers the generation of reads with tens of kilo-base in length. On base of “Sequencing-by-Synthesis”, single nucleotide resolution is achieved by Zero-mode waveguide(ZMW), where only limited volume at the bottom(site of molecule synthesis), is illuminated. In addition, SMRT sequencing largely avoids sequence-specific bias in NGS system, in that most of PCR amplification steps are not required in library construction process.


Service Details

Demo Results

Features

Two sequencing modes on PacBio sequencer: Continuous Long Read (CLR) and Circular Consensus Read (CCS)

Sequencing Mode Library Size Theoretical Data Yield (Per Cell) Single-base Accuracy Applications
CLR 20Kb, 30Kb, etc. 80 Gb to 130 Gb Approx. 85% De novo, SV calling, etc.
CCS 15-20 Kb 14 to 40 Gb/cell (CCS) Approx. 99% De novo, SNP/Indel/SV calling, Iso-Seq,

Service Advantages

Ø Over 7 years experience on PacBio sequencing platform with thousands of closed projects with various species.

Ø Fully equipped with the latest PacBio Sequencing platforms, Sequel II to guarantee sufficient sequencing throughput

Ø Faster Turn-around time, higher data yield and more accurate data.

Ø Contributed in hundreds of high-impact PacBio-based publications

Sample Requirements


Sample Type Amount Concentration (Qubit®) Volume Purity Others
 Genomic DNA Depend on Data Requirement ≥20 ng/μl ≥15μl OD260/280=1.6-2.3;
OD260/230=1.5-2.5;
Clear peak at 260 nm,no contaminations
Concentration needs to be measured by Qubit and Qubit/Nanopore = 0.8-2.0
Total RNA ≥1.5μg ≥40 ng/μl ≥15μl OD260/280=1.7-2.5;
OD260/230=0.5-2.5;no contaminations
 RIN value ≥7.5

 

Service Workflow

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Sample preparation

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Library construction

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Sequencing

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Data analysis

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Project delivery


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  • 1.In-house Data Yield

    Data generated from 63 CCS cells (from 26 species)

    Data-Pacbio-CCS-15 Kb Average Max Min Median
    Yield - subreads (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
    Subreads N50 17,509 23,924 12,485 17,584
    CCS N50 14,490  19,034 9,876 14,747
    Average length-Polymerase 67,995 89,379 49,664 66,433
    Average length-Subreads 15,866 21,036 11,657 16,012
    Average length-CCS 14,489 19,074 8,575 14,655

    Data generated from 16 CLR cells (from 76 species)

    DATA-Pacbio-CLR-30Kb Average Max Min Median
    Yield - subreads (Gb) 142.20 291.40 50.55 142.49
    Polymerase N50 39,456 121,191 15,389 35,231
    Subreads N50 28,490  41,012 14,430 29,063
    Average length-Polymerase 22,063 48,886 8,747 21,555
    Average length-Subreads 17,720  27,225 8,293 17,779

    2.Data QC – Demo Statistics on data yield

    Sample

    ccs Reads Num

    Total ccs Bases (bp)

    ccs Reads N50 (bp)

    ccs Mean Length (bp)

    ccs Longest Read (bp)

    subreads Bases (bp)

    ccs rate(%)

    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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