Iso-Seq

CD Genomics provides Iso-Seq service, an end-to-end solution for direct sequencing and analysis of full-length transcript isoforms, avoiding the need for transcript reconstruction and inference, and enabling discovery of novel transcripts and genes, functional characterization of isoforms and alternative splicing (AS) events, and identification of fusion genes. Please contact our expert team to discuss how our Iso-Seq service can refine your NGS projects.

Overview

RNA Sequencing (RNA-seq) is by far the most frequently used approach for gene expression profiling. Traditional short-read next-generation sequencing (NGS) is difficult to obtain a complete picture of the transcriptome since computational reconstruction cannot perfectly assemble complex transcripts. Isoform Sequencing (Iso-Seq) based on cutting-edge Pacific Biosciences (PacBio)'s Single Molecule, Real-Time (SMRT) Sequencing technology provides unique long read capability for sequencing large amplicons and whole transcripts with extraordinary precision. It generates full-length cDNA strands from the 5' UTR to the 3' poly-A tail, eliminating errors that occur with short-read sequencing and complex transcript reconstruction using isoform-inference algorithms. The ultra high-throughput Iso-Seq method provides accurate information about transcriptional start sites, poly-adenylation sites, alternatively spliced exons, gene fusion, natural antisense transcripts (NAT), and circular RNAs (circRNAs) within targeted genes or the entire transcriptome, improving functional annotations for transcriptome and identification of novel transcripts by complementing for the potential error generated by NGS methods. CD Genomics provides Iso-Seq service on the PacBio Sequel II system with proven performance, delivering high quality results with superior sensitivity and accuracy.

Features

Flexibility Low Input Optimization Bioinformatics
We offer the flexibility to prepare libraries and analyze data according to your needs. Lower input requirements when compared to other methods. Optimized workflows to maximize output and accuracy. Industry standard software and in-house bioinformatics pipelines of proven performance.

Project Workflow

Sample Preparation

1. Sample Preparation

RNA purification; quality assessment and quantification

Library Preparation

2. Library Preparation

Standard Iso-Seq library/ Multi-throughput Iso-Seq library/

Sequencing

3. Sequencing

PacBio Sequel II, ≥ 15 G bases pair per sample.

Data Analysis

4. Data Analysis

Visualize and preprocess results, and perform custom bioinformatics analysis.

Bioinformatics Analysis Pipeline

Bioinformatics Analysis Pipeline

In-depth data analysis:

  • Build similarity graph using BLASR
  • Polish the consensus sequences
  • Align to the reference genome (if with reference)
  • Sequence merge and redundancy removal
  • Annotation of transcripts (Nr、Nt、Swissprot、KEGG、GO、COG and Interpro)
  • Transcripts classification
  • Novel transcripts identification
  • Splicing site detection and annotation
  • Gene fusion analysis

Sample Requirements

Sample storage: RNA can be dissolved in ethanol or RNA-free ultra-pure water and stored at -80°C. RNA should avoid repeated freezing and thawing.

Shipping Method: When shipping RNA samples, the RNA sample is stored in a 1.5 mL Eppendorf tube, sealed with sealing film. Shipments are generally recommended to contain 5-10 pounds of dry ice per 24 hours.

Deliverable: raw data as BAM files, coverage summary, QC report, custom bioinformatics analyses.

References:

  1. Beiki H, Liu H, Huang J, et al. Improved annotation of the domestic pig genome through integration of Iso-Seq and RNA-seq data. BMC genomics, 2019, 20(1): 344.
  2. Gao Y, Wang H, Zhang H, et al. PRAPI: post-transcriptional regulation analysis pipeline for Iso-Seq. Bioinformatics, 2018, 34(9): 1580-1582.
* For Research Use Only. Not for use in diagnostic procedures.


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