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Isoform Sequencing Service (Full-length Transcript Sequencing)

Overview

Isoform Sequencing Service(Iso-Seq), based on cutting-edge PacBio SMRT (Single Molecule, Real Time) technology, enables full-length reads being sequenced of entire transcript isoforms from 5’ UTR to 3’ polyadenylation without assembly required. Iso-seq is an ultra high-throughput method for characterizing gene fusion, alternative splicing and gene fusion events and improving annotations for genomes and discovery of novel transcripts by complementing for the potential error by the short reads.

Service Specifications

Applications

In Medical Research

  • Transcript annotation
  • Fusion gene exploration
  • Disease mechanism investigation

In Agricultural Research

  • Functional study
  • Fusion gene exploration
  • Development and stress study;
  • Collaboration for gene prediction and genome annotation

Advantages

  • Largest sequencing capacity: We have the largest Illumina and PacBio sequencing capacity in the world, allowing us to provide high quality data, fast turnaround, and affordable prices.
  • Comprehensive data analysis: We use industry standard software and mature in-house pipeline to discover novel transcripts, differential expressions, and function annotations.

Sample Requirements

 

Library Type Sample Type Amount RIN
(Agilent 2100)
Purity
(NanoDrop)
SMRTbell Library Total RNA ≥ 5 μg (Concentration ≥ 300ng/μl) ≥ 9, with smooth baseline;
28S/18S ≥ 1.5
OD260/280: 1.8-2.2;
OD260/230: ≥ 1.8;

Sequencing Parameter and Analysis

Platform PacBio Sequel system
Recommended Sequencing Depth ≥ 15 G bases pair per sample
Standard Data Analysis
For Species with Reference For Species without Reference
  • Data Quality Control
  • Identification, clustering and correction of full-length transcripts
  • Alternative Splicing Analysis
  • Prediction and annotation of novel genes and novel trasnscripts
  • Fusion Transcript Analysis
  • Alternative Polyadenylation
  • lncRNA prediction
  • Quantification and Differential Expression Analysis
  • Functional Enrichment Analysis
  • Data Quality Control
  • Identification, clustering and correction of full-length transcripts
  • Simple Sequence Repeat (SSR) Analysis
  • Functional annotation of transcripts
  • Quantification and Differential Expression Analysis
  • Functional Enrichment Analysis
  • Note: For detailed information, please refer to the Service Specifications and contact us for customized requests.

    Project Workflow

    Sampling:

    Fresh-frozen primary and metastatic tumors with paired normal tissue

    Sequencing Strategy:

    1. Illumina Technology: sequenced on an Illumina Hiseq X Ten platform to generate 125 bp paired-end reads.
    2. PacBio System: sequenced on a PacBio RS II small-molecule real-time (SMRT) sequencing platform by two SMRT cells.

    Figure 1. a. Correlation analysis on the numbers of transcriptomic events (ATSS, AS and APA) and detected numbers of isoforms for normal ovarian tissue, primary tumor, and distal metastasis. b Expression of genes with multiple isoforms was compared with those with a single isoform.

    Figure 2. Hierarchical clustering of isoform expression in normal tissue and ovarian tumors.

    Figure 3. Identified somatic genetic and transcriptomic aberrations in genes involved in proteostatic stress regulation. P Primary tumor, M Metastatic tumor.
    Conclusion:

    This study integrated second- and third-generation sequencing platforms to generate a multidimensional dataset on a patient affected by metastatic epithelial ovarian cancer. Besides, it reveals clinical application of the emerging long-read full-length analysis for improving molecular diagnostics is feasible and informative. An in-depth understanding of the tumor transcriptome complexity allowed by leveraging the hybrid sequencing approach lays the basis to reveal novel and valid therapeutic vulnerabilities in advanced ovarian malignancies.

    Self-Recognition of an Inducible Host lncRNA by RIG-I Feedback Restricts Innate Immune Response

    Background:

    Innate immune system can sense the invading pathogens via pattern recognition receptors (PRRs) to initiate efficient innate response for the elimination of the pathogens. As the most extensively studied PRR for recognition of RNA virus, retinoic acid-inducible gene-I (RIG-I) has been shown to recognize viral RNAs in the cytoplasm and trigger innate immune response through the production of type I interferons (IFNs) and proinflammatory cytokines. However, the biological significance and the underlying mechanisms for the interaction of lncRNAs with RBPs in the immunity and inflammation remain to be further investigated. The increasing evidence for the RBP-lncRNA interactions in association with protein functions inspired us to ask whether RIG-I can bind to ‘‘self’’ cellular lncRNAs, and if so, what is the biological function and importance of such self-recognition in maintaining immune homeostasis by feedback restricting or timely terminating RIG-I recognition of ‘‘non-self’’ RNA-induced innate inflammatory response.

    Sequencing Strategy:

    1. RIP-seq
    2. Pacific Bioscience RS II platform

    Figure 4. Location and read depth of cluster analysis of RIP-seq data mapped to Lsm3 loci using PacBio platform and Illumina Platform.
    Conclusion:

    In this study, the full-length transcriptome sequencing was used to identify the full-length sequence of cytoplasmic lnc-Lsm3b, and a self-recognition model of lncRNA-RIG-I to inhibit RIG-I activation was found. This approach prevents overexpression of IFN-I to maintain the body’s immune homeostasis. lncRNA was identified as an important regulatory element for nucleic acid innate immune recognition and inflammation regulation, and it also revealed the key functions of lncRNA in anti-virus, providing new ideas for the study of prevention and treatment of inflammatory diseases.

    A survey of the sorghum transcriptome using single-molecule long reads

    Background:

    Sorghum, a C4 crop plant used for food, feed, fibre and fuel, is one of the best-adapted cereals to drought and temperature; hence, used as a model system to investigate the molecular basis of adaptation to abiotic stresses. Although the genome sequence of several sorghum lines has been completed recently29,30, the transcriptome is not well annotated; the extent of alternative splicing (AS), the number of splice isoforms and transcriptome diversity due to alternative polyadenylation (APA) are largely unknown.

    Sampling:

    Sorghum (Sorghum bicolor L. Moench) seedlings under drought stress and control.

    Sequencing Strategy:

    1. Illumina Platform: Hiseq
    2. PacBio System: performed on a PacBio RS II instrument for a total of 28 SMRTcells.

    Figure 5. An example of a gene that produces 13 novel splice isoforms.

    Figure 6. PCR validation of alternative splicing events identified by Iso-Seq.
    Conclusion:

    In this study, full-length splice isoforms and APA sites of the sorghum transcriptome were sequenced and identified using Pacific Biosciences single-molecule real-time long-read isoform sequencing and developed a pipeline called TAPIS (Transcriptome Analysis Pipeline for Isoform Sequencing) to identify. The analysis results reveal transcriptome-wide full-length isoforms at an unprecedented scale and uncovered novel genes. These results greatly enhance sorghum gene annotations and aid in studying gene regulation in this important bioenergy crop.


    Figure 1 Transcript Classification


    Figure 2 Alternative Splicing (AS)


    Figure 3 Alternative Ployadenylation (APA)


    Figure 4 GO Enrichment


    Figure 5 lncRNA Prediction


    Figure 6 Transcription Factor