ISOFORM SEQUENCING

ISOFORM SEQUENCING

Overview - What is transcript isoform sequencing?

Transcript isoforms are mRNAs that are encoded by the same DNA sequence but differ in their transcription start sites (TSSs), protein coding sequences or untranslated regions (UTRs). In higher eukaryotes, nearly all multi-exon genes produce transcript variants by regulatory processes such as alternative splicing, alternative promoters or alternative polyadenylation. These transcript isoforms could alter gene function, affect RNA stability and produce proteins that vary in structure, function or subcellular localisation. Deregulated generation of splice variants could lead to the development of numerous widespread diseases. A complete understanding of the full repertoire of mRNA isoforms is essential for the understanding of the biology of an organism. 

Despite the importance of alternative splicing, comprehensive profiling of transcript isoforms has been challenging. PacBio’s long read next-generation sequencing technology aims to address problems associated with transcript assembly by developing RNA sequencing workflows for full-length mRNA transcripts. PacBio’s isoform sequencing (Iso-SeqTM) protocols help to produce multi-kilobase-long reads that span the whole transcript from the 5’end to the polyA tail. These whole-length reads permit the identification of whole isoforms with exceptional precision. PacBio’s single-molecule sequencing platform provides a direct and unbiased observation of transcript diversity, either broadly or in a targeted fashion. The Iso-SeqTM approach helps researchers to gain unprecedented insights into alternative transcription and to improve genome annotation and gene discovery.  

Applications - What are the advantages of isoform sequencing?

Transcript isoform sequencing makes feasible RNA research that requires: 

  • Direct sequencing of full-length transcripts in any organism without any reference 
  • Genome-wide or targeted analysis of transcript diversity to explore frequency and types of alternative splicing events
  • Studies on allele-specific gene expression
  • Information on differential isoform expression between cells, tissues and particular developmental, health or stress states
  • Discovery of novel genes and isoforms
  • Identification of promoters and splice sites for better understanding of gene regulation
  • Accurate estimation of transcript abundance at gene or isoform level

Workflow - Methods & technologies for transcript isoform sequencing

RNA-Seq using long read technology involves reverse transcription of isolated RNA into full-length cDNA. This step is followed by large scale cDNA amplification and generation of size selected cDNA libraries. These libraries are then sequenced on a PacBio platform using single molecule real time (SMRT) technology. After the sequencing reaction, full-length cDNA reads are identified by the presence of both 5’ cDNA primers and polyA stretches preceding the 3’ primers. Reads generated from the same isoforms are clustered to generate an initial consensus sequence, which is error corrected against non-full-length reads to generate a final high-quality consensus sequence. These high-quality reads are aligned to a reference or de novo consensus isoforms are predicted with specifically designed algorithms.  

Scientific expertise: transcript isoform sequencing

GATC Biotech is one of the few providers of comprehensive full-length cDNA sequencing services. The company’s R&D team has successfully optimised the original Iso-Seq protocols from PacBio to produce long sequencing reads that require minimal re-construction and assembly efforts. GATC Biotech’s isoform sequencing service combines the team’s expertise and proprietary knowledge in full length cDNA library preparation, sequencing and BioIT data analysis to provide a complete solution for exploring the complexity of the entire transcriptome. 

Publications

Find here, a list of selected research articles supported by GATC Biotech’s sequencing products.

Related products to sequencing of transcript variants

Are you interested in profiling the transcript heterogeneity of your organism of interest? Are you studying regulatory mechanisms of gene expression like alternative splicing? Then take advantage of our full-service for genome-wide analysis of mRNA transcript variants. From library preparation to sequencing on the PacBio platform to expert BioIT analysis to a final comprehensive GATC Data Analysis report, we help to quickly and conveniently advance your RNA research. 

Discover our comprehensive service line for RNA research, INVIEW TRANSCRIPTOME, which is specifically designed to meet individual research needs. Use our INVIEW TRANSCRIPTOME ISOFORM DISCOVER to generate high quality isoforms in their whole length. Enjoy unprecedented access to the entire transcriptome or target particular regions of interest that are difficult to characterise with short read technology.

Contact us for more information.

Further reading on transcript isoform sequencing

Gonzalez-Garay, Manuel L et al. (2016) Introduction to Isoform Sequencing using Pacific Biosciences technology (Iso-Seq). Transcriptomics and Gene Regulation 9:141-160.

Tilgner H et al. (2014). Defining a personal, allele-specific, and single-molecule long-read transcriptome. PNAS 111(27):9869-9874.

ISO-SEQ™ is a registered trademark by Pacific Biosciences Inc., Menlo Park, USA.