Iso-Seq/PacBio Transcriptome Sequencing

Service Description

RNA Sequencing (RNA-seq) has become the most frequently used method for the majority of researchers conducting gene expression profiling. However, it is difficult to obtain a complete picture of the transcriptome because short reads cannot accurately assemble complex transcripts.

 

Isoform Sequencing Technology

“Isoform Sequencing” (Iso-seq) developed by Pacific Biosciences (PacBio), is based on long-read sequencing technology. The unique long-read sequencing feature allows this method to identify new isoforms with extraordinary precision. The Iso-Seq application generates full-length cDNA sequences — from the 5’ end of transcripts to the poly-A tail — eliminating the need for transcriptome reconstruction using isoform-inference algorithms. The Iso-Seq method provides accurate information about alternatively spliced exons and transcriptional start sites. It also reveals information about poly-adenylation sites for transcripts across the full complement of isoforms within targeted genes or the entire transcriptome.

Project Workflow

We care for your project from the start through to reporting of results. Highly experienced laboratory professionals follow strict quality procedures to ensure the integrity of your results.

  • Sample QC
  • Library QC
  • Sequencing QC
  • Data QC
  • Sample Preparation
  • Library Construction
  • Sequencing
  • Raw Data Output
  • Bioinformatics Analysis
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Sequencing Service Specification

BGI Iso-Seq/PacBio transcriptome sequencing services are executed on the Sequel I/II platform.

  • Sample Preparation

    Sample Preparation

    • Library preparation - Standard Iso-Seq library / Multi- throughput Iso-Seq library/polyA Iso-Seq library
  • Data Analysis

    Data Analysis

    • Clean data, standard and customized data analysis options available
    • Data storage services and bioinformatics applications available upon request
  • Turnaround Time

    Turnaround Time

    • Typical 40 working days from sample QC acceptance to sequencing data availability

Sample Requirements

We can process your total RNA, whole blood, cell line, fresh frozen tissue samples, with the following general requirements:

Minimum Sample Volume: 15 µl

Total RNA Amount

Concentration, RIN, 28S/18S

Recommended

m≥3 µg

c≥300ng/µL
RIN≥8

Required

1µg≤m<3 µg

28S/18S(23S/16S)≥1.4

Data Analysis

Besides clean data, BGI offers a range of standard and customized bioinformatics options for your Iso-seq/PacBio transcriptome sequencing. Reports and output data files are delivered in industry-standard file formats: FASTQ, BAM, cout, .xls, .png

STANDARD ANALYSIS (WITHOUT REFERENCE)

  • Remove the low-quality reads and short reads

  • Identify the full-length, non-chimeric transcripts and non-full-length, non-chimeric transcripts

  • Build similarity graph using BLASR, get cluster consensus

  • Polish the consensus sequences and get high quality full-length, non-chimeric transcripts

  • Merge consensus sequences of all libraries and remove redundancy

  • Annotation of the full-length non-chimeric transcripts (Nr、Nt、Swissprot、KEGG、GO、COG and Interpro)

  • CDS prediction

  • SSR prediction

STANDARD ANALYSIS (WITH REFERENCE)

  • Remove the low-quality reads and short reads;

  • Identify the full-length, non-chimeric transcripts and non-full-length, non-chimeric transcripts

  • Build similarity graph using BLASR, get cluster consensus

  • Polish the consensus sequences and get high quality full-length, non-chimeric transcripts

  • Align to the reference genome with GMAP

  • Merge consensus sequences of all libraries and remove redundancy

  • Transcripts classification

  • Novel transcripts analysis

  • Long noncoding RNA prediction

  • Splicing site detection and annotation

  • Gene fusion detection and annotation

MULTI-THROUGHPUT ISO-SEQ

Analysis of Standard Iso-Seq + Gene/Transcripts quantification; differentially expressed gene detection and annotation

POLYA ISO-SEQ

Analysis of Standard Iso-Seq + Gene/Transcript quantification; differentially expressed gene detection and annotation; polyA length analysis

CUSTOMIZED ANALYSIS

  • Further customization of bioinformatics analysis to suit your needs is available

  • Please contact your BGI technical representative for details

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