Iso Sequencing using PacBio

Introduction and Workflow

Iso-Seq, or Isoform Sequencing, leverages PCR free PacBio's Single Molecule, Real-Time (SMRT) sequencing technology to deliver detailed and accurate insights into RNA transcripts. PCR free SMRT technology produces long reads of upto 25Mbps length with 99.9% base level accuracy through circular consensus sequencing (CCS) mode.
PacBio's SMRT sequencing technology enables the reading of full-length cDNA molecules in a single pass, allowing for the accurate identification of various isoforms, including alternative splicing events and post-transcriptional modifications. This approach minimizes errors and provides a high-resolution map of transcript structures.
The process begins with the isolation of high-quality RNA, which is then converted into cDNA followed by sequencing using PacBio SMRT technology.
The bioinformatics analysis of Iso-Seq data involves assembling full-length transcripts, annotating them, and comparing them to reference genomes or existing databases. This detailed analysis reveals the complexity of gene expression, including novel isoforms and splice variants, and aids in understanding their functional roles in various biological processes.
Iso-Seq using PacBio is particularly valuable for exploring gene expression at a granular level, discovering novel transcripts, and enhancing annotations of complex genomes.

Unlike traditional methods, which may miss complex isoforms or splice variants, Iso-Seq captures full-length RNA sequences, enabling the identification of all transcript isoforms, including those with complex splicing and alternative start or end sites.
Provides high-fidelity data with long read lengths, reducing errors and missing information common in shorter-read sequencing methods.
Reduces challenges with transcript assembly and alignment by providing full-length reads that eliminate ambiguities and enhance transcriptome reconstruction accuracy. Improves genome annotations by precisely identifying gene structures, exons, introns, and regulatory elements, leading to more accurate functional insights.
Uncovers novel isoforms, splice variants, and post-transcriptional modifications, providing a comprehensive view of gene expression. This detailed approach is essential for functional genomics, transcriptomics, and alternative splicing studies, offering valuable insights into gene regulation and function.

Isoform sequencing is widely utilized across plants, animals, microbes, and humans.
Gene Annotation- Enhances the annotation of complex genomes by providing precise information on transcript structures, helping to accurately define gene boundaries and functions.
Alternative Splicing Analysis- Investigates alternative splicing events and isoform diversity, contributing to a deeper understanding of gene regulation and functional diversity.
Functional Genomics- Supports functional studies by linking isoform-specific expression profiles to phenotypic traits, disease mechanisms, and developmental processes.
Disease Research- Identifies aberrant isoforms associated with diseases, aiding in the discovery of biomarkers and therapeutic targets for conditions such as cancer and genetic disorders.
Plant and Crop Improvement- It allows the haplotype resolution of complex polyploids like those in plants. Explores transcript diversity in plants and crops, contributing to better understanding of stress responses, developmental processes, and traits relevant for agriculture.
Microbial Genomics- Studies microbial transcriptomes to uncover complex regulatory networks, metabolic pathways, and interactions within microbial communities, advancing our knowledge in microbiology and biotechnology.

Frozen cells in specific frozen lysates, High Quality RNA

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