Full-Length Isoform Sequencing with PacBio Iso-Seq

Traditional RNA sequencing fragments your transcripts into short reads that must be computationally pieced back together. PacBio Iso-Seq changes the game by capturing complete transcripts from 5' to 3' in a single read.

At Admera Health, we partner with you on Iso-Seq projects to deliver high-quality sequencing and analysis with rigorous QC at every stage.

What Names Iso-seq Different than Traditional Bulk RNA-seq?

Iso-Seq is a long-read, full-length transcript sequencing approach that captures complete isoforms without assembly, enabling precise characterization of splicing, transcript structure, and gene regulation in eukaryotic systems.

Four Key Advantages:

• Full-Length Transcript Coverage (5' to 3')
  See exactly which exons combine in each isoform without the guesswork. You get the complete picture from cap to poly(A) tail.

• HiFi Accuracy (>99.9%)
  PacBio's High-Fidelity sequencing delivers exceptional per-base accuracy, eliminating the traditional trade-off between read length and quality. Confidently identify true biological isoforms vs. sequencing errors.

• Robust Detection of Alternative Splicing
  Discover novel splice junctions, rare isoforms, and tissue-specific variants that short-read methods miss. Perfect for characterizing complex splicing patterns.

• Accurate Isoform-Level Quantification
  Count actual molecules rather than inferring abundances from fragments. Get reliable estimates of how much of each isoform is expressed.

What Can You Study With Iso-seq?

• Novel isoform discovery in any tissue or condition

• Alternative splicing changes in disease, development, or treatment

• Fusion transcript identification in cancer samples

• Transcriptome annotation for non-model organisms

• Isoform switching where gene expression stays constant but ratios change

• Allele-specific expression when combined with genomic data

Sample Requirements

Iso-seq is designed for eukaryotic samples (animals, plants, fungi, protists).

What we need:

• Sample type

  • Extracted total RNA with QC documentation

• Amount

  • 1-2 µg (minimum 500 ng per sample)

• Integrity

  • RIN ≥ 7

• Purity

  • OD 260/280: 1.8-2.0
    OD 260/230: 2.0-2.2

Why These Metrics Matter

• RIN ≥ 7: Iso-Seq sequences full-length molecules. Degraded RNA = truncated transcripts and incomplete isoform information.

• Purity ratios: Contaminants inhibit the enzymes used in cDNA synthesis and library prep. Clean RNA = successful libraries.

• Sufficient quantity: Ensures adequate material for QC validation, library prep, and potential re-sequencing if needed.

• What to provide: Extracted RNA shipped on dry ice with QC documentation (Bioanalyzer/TapeStation trace, concentration data, purity ratios).

The Iso-seq Workflow

1. RNA Quality Control

• Independent QC validation upon receipt

• Verify RIN, concentration, and purity

• We communicate status of QC before proceeding with library preparation

• Timeline: 2-3 business days

2. cDNA Synthesis & Library Preparation

• Generate full-length cDNA using template-switching technology

• Construct SMRTbell libraries optimized for HiFi sequencing

• Rigorous QC: size distribution, concentration, ligation efficiency

• Timeline: 1-2 weeks

3. HiFi Sequencing on PacBio Revio

• State-of-the-art platform generates >99.9% accuracy reads

• Multiple passes over circular templates create high-quality consensus

• Sequencing depth tailored to your goals (discovery vs. quantification)

• Real-time performance monitoring

• Timeline: 1-2 weeks

4. Bioinformatics Pipeline

• HiFi read generation from raw data

• De-segmentation to remove concatemers

• Demultiplexing (if samples barcoded)

• Isoform clustering to group similar reads

• Mapping to reference genome

• Transcript classification: Known isoforms, novel combinations, or truly novel transcripts

5. Downstream Analysis (Optional)

• Differential expression at gene and isoform level

• GSEA (Gene Set Enrichment Analysis)

• GO enrichment (biological processes, molecular functions)

• KEGG pathway analysis

• Splicing variant analysis with visualizations

• Timeline: +2-3 weeks for full analysis

Total Timeline: 4-6 weeks (core processing) or 6-8 weeks (with full analysis)

How To Get Started

Step 1: Define Your Project

Contact us to discuss:

• Your organism and biological questions

• Number of samples and comparisons

• Timeline and budget
  

Step 2: Plan RNA Extraction & QC

• Decide: extract yourself or have us do it?

• Ensure samples meet requirements

• Prepare QC documentation
  

Step 3: Determine Sequencing Depth

50-60 million segmented reads are generated per REVIO cell. Up to 12 samples can be multiplexed in one library prep.‍ ‍If you’re unsure about what sequencing depth is needed for your project, our team will help to optimize depth for your goals and budget.

Step 4: Select Analysis Needed

Choose between standard Iso-seq analysis or customized pipelines.

Step 5: Ship Your Samples

• Ship RNA on dry ice with QC documentation

• We validate upon receipt before proceeding with clear communication at every step
  

Step 6: Receive Your Data

• Organized file structure with standard formats

• Comprehensive reports and visualizations

• Post-delivery consultation and support provided
  

Frequently Asked Questions

What organisms can you work with?
Any eukaryotic organism, model or non-model. Reference genomes help but aren't required. Not suitable for bacteria/prokaryotes.

What if my RNA is slightly degraded (RIN 6-7)?
Contact us. Borderline samples may work but will produce truncated reads. We can advise whether to proceed or obtain higher-quality RNA.

Can I multiplex many samples?
Yes, up to 12 samples can be multiplexed in one library to be sequenced on 1 to 2 REVIO cells.

How is Iso-Seq different from standard RNA-seq?
Standard RNA-seq fragments transcripts and requires assembly. Iso-Seq sequences complete isoforms in single reads, no assembly needed. Use Iso-Seq for isoform discovery; use standard RNA-seq for deep quantification across many samples.

Can I combine Iso-Seq with short-read RNA-seq?
Yes! Use Iso-Seq to discover/annotate isoforms, then short-read RNA-seq for cost-effective quantification. We can design integrated workflows.

Do you offer rush service?
Yes, expedited timelines available for additional fees. Contact us with your deadline.

What support do you provide after data delivery?
Post-delivery consultation to review results, email support for interpretation questions, and additional analysis services if needed.

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Why Choose Admera for Iso-seq?

Choosing Admera Health for your Iso-seq project means more than just accessing state-of-the-art PacBio long-read technology, it’s about partnering with a team that prioritizes scientific integrity and collaboration at every stage. We integrate rigorous QC protocols to protect your investment, offering the flexibility of tailored workflows and expert bioinformatic analysis designed to meet the needs of your research. From initial experimental design to final publication, our approach is defined by transparency and dedicated support, ensuring you have the clear communication and specialized project management needed to transform complex transcriptomic data into meaningful discovery.