Quality Statement

Pacific Biosciences is committed to providing high-quality products that meet customer expectations and comply with regulations. We will achieve these goals by adhering to and maintaining an effective quality-management system designed to ensure product quality, performance, and safety.


Image Use Agreement

By downloading, copying, or making any use of the images located on this website (“Site”) you acknowledge that you have read and understand, and agree to, the terms of this Image Usage Agreement, as well as the terms provided on the Legal Notices webpage, which together govern your use of the images as provided below. If you do not agree to such terms, do not download, copy or use the images in any way, unless you have written permission signed by an authorized Pacific Biosciences representative.

Subject to the terms of this Agreement and the terms provided on the Legal Notices webpage (to the extent they do not conflict with the terms of this Agreement), you may use the images on the Site solely for (a) editorial use by press and/or industry analysts, (b) in connection with a normal, peer-reviewed, scientific publication, book or presentation, or the like. You may not alter or modify any image, in whole or in part, for any reason. You may not use any image in a manner that misrepresents the associated Pacific Biosciences product, service or technology or any associated characteristics, data, or properties thereof. You also may not use any image in a manner that denotes some representation or warranty (express, implied or statutory) from Pacific Biosciences of the product, service or technology. The rights granted by this Agreement are personal to you and are not transferable by you to another party.

You, and not Pacific Biosciences, are responsible for your use of the images. You acknowledge and agree that any misuse of the images or breach of this Agreement will cause Pacific Biosciences irreparable harm. Pacific Biosciences is either an owner or licensee of the image, and not an agent for the owner. You agree to give Pacific Biosciences a credit line as follows: "Courtesy of Pacific Biosciences of California, Inc., Menlo Park, CA, USA" and also include any other credits or acknowledgments noted by Pacific Biosciences. You must include any copyright notice originally included with the images on all copies.


You agree that Pacific Biosciences may terminate your access to and use of the images located on the PacificBiosciences.com website at any time and without prior notice, if it considers you to have violated any of the terms of this Image Use Agreement. You agree to indemnify, defend and hold harmless Pacific Biosciences, its officers, directors, employees, agents, licensors, suppliers and any third party information providers to the Site from and against all losses, expenses, damages and costs, including reasonable attorneys' fees, resulting from any violation by you of the terms of this Image Use Agreement or Pacific Biosciences' termination of your access to or use of the Site. Termination will not affect Pacific Biosciences' rights or your obligations which accrued before the termination.

I have read and understand, and agree to, the Image Usage Agreement.

I disagree and would like to return to the Pacific Biosciences home page.

Pacific Biosciences

Sequencing 101: What’s the Value of Sequencing Full-length RNA Transcripts?

Thursday, July 23, 2020

The study of genomics has revolutionized our understanding of science, but the field of transcriptomics grew with the need to explore the functional impacts of genetic variation. While different tissues in an organism may share the same genomic DNA, they can differ greatly in what regions are transcribed into RNA and in their patterns of RNA processing. By reviewing the history of transcriptomics, we can see the advantages of RNA sequencing – using a full-length transcript approach – become clearer.

Reaching for the Transcriptome

Even before genome sequencing became commonplace, scientists were able to measure gene expression activity using hybridization approaches like Northern blots, or later, microarrays.  While these techniques provided a rigorous method for RNA measurement, they were limited in that they could only measure known transcripts. Additionally, many of these methods made the subtle differences between transcript isoforms difficult to decipher.

The RNA-seq approach yields reads that do not span multiple exon junctions.

As next-generation sequencing (NGS) grew, researchers had the ability to interrogate the activity of all expressed genes including those that were previously unknown. This was a huge leap forward in the ability to characterize the transcriptome in an organism. The approach, known as RNA-seq, quickly replaced microarrays in many labs due to the enormous amount of information it could generate.

Understanding gene activity — not just the genes encoded by the genome, but the ones that are turned on at a specific time or in a specific cell or tissue — is critical for elucidating biological consequences. But even as RNA-seq expanded, scientists grew concerned about the accuracy of results when NGS short reads had to be pieced back together computationally to create whole transcripts. This is especially difficult in complex eukaryotes where one gene can generate many isoforms that differ in their transcription start site and alternative RNA processing.

Transcriptome reconstruction with RNA-seq as presented by Korf, I. (2013) Genomics: the state of the art in RNA-seq analysis. Nature Methods.

In a Mendelspod interview, Stanford genomics expert Mike Snyder (@SnyderShot) described the RNA-seq process this way: “We take RNA, we blow it up into little fragments, and then we try and assemble them back together to see what the transcriptome looked like in the first place. … You can’t always figure out which parts of the puzzle belong together.”

A similar concept was presented by Ian Korf (@IanKorf) in a Nature Methods article, where he equated RNA-seq to reassembling magazine articles that had been put through a shredder. Suffice it to say, there have been concerns about the need for extensive computational inference when using an RNA-seq approach.

Capturing the Whole Transcriptome with the Advantages of Long Reads

What scientists really needed went beyond RNA-seq; for the most accurate view of biology possible, they had to have a way to capture isoforms in their entirety. The introduction of long-read sequencing answered this need.

With Single Molecule, Real-Time (SMRT) Sequencing, researchers can generate highly accurate long reads, or HiFi reads, that are tens of kilobases long. That’s enough to capture most transcripts in a single sequencing read, with no downstream bioinformatic assembly required. This became known as the Iso-Seq method because it enabled full-length isoform sequencing, which can be used to explore a transcriptome in its entirety or in a targeted fashion to examine individual genes.

HiFi reads enable sequencing of full-length transcripts that span all exon junctions so that transcript assembly is not needed.

As scientists began using SMRT Sequencing for transcriptome projects, a common theme emerged: long-read studies, even of the most well characterized organisms, routinely showed far more transcript diversity than previously observed. It quickly became clear that short-read NGS platforms greatly underestimated the diversity of expressed isoforms. Key published examples of the Iso-Seq method revealing previously unknown isoform diversity across different types of organisms include:

Beyond Alternative Splicing

In addition to alternative splicing, the Iso-Seq method is important for understanding alternative polyadenylation, genome annotation, fusion transcripts, isoform variant phasing, and long noncoding RNAs. Here are just a few examples of how the Iso-Seq method allowed for new transcriptome studies:

Alternative Polyadenylation

Connect alternative polyadenylation motifs to specific isoforms to determine their role in agronomically important traits.

The diversity of cleavage sites detected in a sorghum gene as identified with SMRT Sequencing.

Wang, B., et al. (2018) A comparative transcriptional landscape of maize and sorghum obtained by single-molecule sequencing. Genome Research, 28(6), 921-932.


Genome Annotation

Gain a complete view of the porcine transcriptome including the identification of genes missing in the assembly.

The Iso-Seq method verified known genes and rescued missing genes during annotation of the new pig genome assembly.

Warr, A., et al. (2020) An improved pig reference genome sequence to enable pig genetics and genomics research. GigaScience, 9, 1-14.


Fusion Transcripts

Discover novel fusion transcripts in clinically relevant genes to understand disease mechanisms.

The Iso-Seq method reveals a functional Igμ, a gene fusion event between IGH and DUX4, as well as an antisense DUX4 gene copy.

Tian, L., et al. (2019). Long-read sequencing unveils IGH-DUX4 translocation into the silenced IGH allele in B-cell acute lymphoblastic leukemia. Nature Communications, 10(1), 2789.


Isoform Variant Phasing

Detect and phase mutations in disease genes for clinically relevant insights.

Six patient samples were sequenced and phased, revealing double mutations in the PIK3CA gene located on the same allele (cis).

Vasan, N., al. (2019) Double PIK3CA mutations in cis increase oncogenicity and sensitivity to PI3Kα inhibitors. Science, 366(6466), 714–723.


Long Noncoding RNAs

Identify long noncoding RNA for a more comprehensive view of transcriptional and translation regulation

The length distribution of lncRNAs identified in C. breviculmis using the SMRT sequencing.

Teng, K., et al., (2019) PacBio single-molecule long-read sequencing shed new light on the complexity of the Carex breviculmis transcriptome. BMC Genomics, 20(1), 789.



Full-length transcript sequencing offers many benefits to scientists looking to interrogate transcriptomes. Whether exploring the human transcriptome to better understand health and disease or studying plants and animals to advance agricultural or conservation efforts.


Learn more about how discovering full-length transcripts could make a difference for your research and contact a PacBio scientist to get started with your next sequencing project.


Explore other posts in the Sequencing 101 series:

The Evolution of DNA Sequencing Tools

Introduction to PacBio Sequencing and the Sequel II System

From DNA to Discovery – The Steps of SMRT Sequencing

Why Are Long Reads Important for Studying Viral Genomes?

Looking Beyond the Single Reference Genome to a Pangenome for Every Species

Understanding Accuracy in DNA Sequencing

Ploidy, Haplotypes, and Phasing – How to Get More from Your Sequencing Data

DNA Extraction – Tips, Kits, & Protocols

VIDEO: Sequencing 101: How Long-Read Sequencing Improves Access to Genetic Information

Subscribe for blog updates:




Press Release

PacBio Grants Equity Incentive Award to New Employee

Friday, January 14, 2022