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Full-length mRNA sequencing -PacBio

De novo full-length transcriptome sequencing, also known as De novo Iso-Seq takes the advantages of PacBio sequencer in read length, which enables sequencing of full-length cDNA molecules without any breaks. This completely avoids any errors generated in transcript assembly steps and constructs unigene sets with isoform-level resolution. This unigene sets provides powerful genetic information as “reference genome” at transcriptome-level. In addition, combining with next generation sequencing data, this service empowers an accurate quantification of isoform-level expression.

Platform: PacBio Sequel II
Library: SMRT bell library

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  • Service Details

    Demo Results

    Case Study

    Service Advantages


    ●  Direct read-out of full-length cDNA molecule from 3'- end to 5'- end

    ●  Iso-form level resolution in sequence structure

    ●  Transcripts with high accuracy and integrity

    ●  Highly compatible to vaiours species

    ●  Large sequencing capacity with 4 PacBio Sequel II sequencing platforms equipped

    ●  Highly experienced with over 700 Pacbio-based RNA sequencing projects

    ●  BMKCloud-based result delivery: Customized data-mining available on platform.

    ●  After-sale services valid for 3 months upon project completion

    Service Specifications

    Platform: PacBio Sequel II

    Sequencing library:  Poly A- enriched mRNA library

    Recommended data yield: 20 Gb/sample (Depending on species)

    FLNC(%): ≥75%

    *FLNC: Full-length non-chimeric transcipts

    Bioinformatics analyses

    ●  Raw data processing
    ●  Transcript identification
    ●  Sequence structure
    ●  Expression Quantification
    ●  Function Annotation

    full length pacbio

    Sample Requirements and Delivery

    Sample Requirements:



    Amount (μg)



    ≥ 120

    ≥ 0.6



    Limited or no protein or DNA contamination shown on gel.

         For plants: RIN≥7.5;

         For animals: RIN≥8.0;

         5.0≥ 28S/18S≥1.0;

         limited or no baseline elevation

    Tissue: Weight(dry): ≥1 g
    *For tissue smaller than 5 mg, we recommend to send flash frozen(in liquid nitrogen) tissue sample.

    Cell suspension: Cell count = 3×106 - 1×107
    *We recommend to ship frozen cell lysate. In case that cell counts smaller than 5×105, flash frozen in liquid nitrogen is recommended, which is preferable for micro extraction.

    Blood samples: Volume≥1 mL

    Microorganism: Mass ≥ 1 g

    Recommended Sample Delivery

    2 ml centrifuge tube (Tin foil is not recommended)
    Sample labeling: Group+replicate e.g. A1, A2, A3; B1, B2, B3... ...


    1. Dry-ice: Samples need to be packed in bags and buried in dry-ice.
    2. RNAstable tubes: RNA samples can be dried in RNA stabilization tube(e.g. RNAstable®) and shipped in room temperature.

    Service Work Flow

    Sample QC

    Experiment design

    sample delivery

    Sample delivery

    Pilot experiment

    RNA extraction

    Library Preparation

    Library construction



    Data analysis

    Data analysis

    After sale Services

    After-sale services

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  • 1.FLNC length distribution

    Length of full-length non-chimeric read(FLNC) indicates length of cDNA in library construction. FLNC length distribution is a crucial indicator in evaluating quality of library construction.


    FLNC read length distribution

    2.Complete ORF region length distribution

    We use TransDecoder to predict protein coding regions and corresponding amino acid sequences to generate unigene sets, which contains complete non-redundant transcript information in all samples.


    Complete ORF region length distribution

    3.KEGG pathway enrichment analysis

    Differentially expressed transcripts(DETs) can be identified by aligning NGS-based RNA sequencing data on full-length transcript sets generated by PacBio sequencing data. These DETs can further processed for various functional analysis, e.g. KEGG pathway enrichment analysis.


    DET KEGG pathway enrichment -Dot plot

    BMK Case

    The developmental dynamics of the Populus stem transcriptome

    Published: Plant Biotechnology Journal, 2019

    Sequencing strategy:
    Sample collection: stem regions: apex, first internode(IN1), second internode(IN2), third internode(IN3), internode(IN4) and internode(IN5) from Nanlin895
    NGS-sequence: RNA of 15 individuals were pooled as one biological sample. Three biological replicates of each points were processed for NGS sequence
    TGS-sequence: Stem regions were divided into three regions, i.e. apex, IN1-IN3 and IN4-IN5. Each region was processed for PacBio sequencing with four types of libraries: 0-1 kb, 1-2 kb, 2-3 kb and 3-10 kb.

    Key results

    1.A total of 87150 full-length transcripts were identified, in which, 2081 novel isoforms and 62058 novel alternative spliced isoforms were identified.
    2.1187 lncRNA and 356 fusion genes were identified.
    3.From primary growth to secondary growth, 15838 differentially expressed transcripts from 995 differentially expressed genes were identified. In all DEGs, 1216 were transcription factors, most of which has not yet been reported.
    4.GO enrichment analysis revealed importance of cell division and oxidation-reduction process in primary and secondary growth.

    • PB-full-length-RNA-Sequencing-case-study

      Alternative splicing events and different isoforms

    • PB-full-length-RNA-alternative-splicing

      WGCNA analysis on transcription factors


    Chao Q, Gao ZF, Zhang D, et al. The developmental dynamics of the Populus stem transcriptome. Plant Biotechnol J. 2019;17(1):206-219. doi:10.1111/pbi.12958

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