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Updated: Jul 4, 2025

Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations
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Full-length isoform concatenation sequencing to resolve cancer transcriptome complexity.

Saranga Wijeratne1, Maria E Hernandez Gonzalez1, Kelli Roach1

  • 1The Steve and Cindy Rasmussen Institute for Genomic Medicine, Abigail Wexner Research Institute at Nationwide Children's Hospital, 575 Children's Crossroad, Columbus, OH, 43215, USA.

BMC Genomics
|January 29, 2024
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Summary

We developed PacBio Full-Length Isoform Concatemer Sequencing (PB_FLIC-Seq) to increase long-read sequencing output, improving detection of novel cancer isoforms. This method enhanced isoform discovery in a pediatric glioma, revealing significant extracellular matrix gene expression changes.

Keywords:
ConcatenationIsoform discoveryLong-read RNA sequencingTumor transcriptome

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Area of Science:

  • Genomics
  • Molecular Biology
  • Cancer Research

Background:

  • Cancer transcriptomes are complex, with aberrant splicing leading to differential isoform expression.
  • Short-read sequencing has limitations in accurately inferring full-length transcripts.
  • Long-read sequencing (Iso-Seq) offers full-length isoform resolution but has lower read output.

Purpose of the Study:

  • To develop a novel concatenation workflow, PacBio Full-Length Isoform Concatemer Sequencing (PB_FLIC-Seq), to increase unique long-reads.
  • To enhance the detection of moderate to low-level expressed isoforms and improve overall isoform discovery.
  • To apply PB_FLIC-Seq for profiling transcriptomes in cancer, specifically a pediatric diffuse midline glioma.

Main Methods:

  • Developed and implemented the PB_FLIC-Seq concatenation workflow for PacBio long-read RNA sequencing.
  • Sequenced a commercial reference (Spike-In RNA Variants; SIRV) to evaluate method performance.
  • Applied PB_FLIC-Seq to profile a pediatric diffuse midline glioma and adjacent non-malignant tissue.

Main Results:

  • PB_FLIC-Seq demonstrated a 3.4-fold increase in read output and improved SIRV recall compared to standard Iso-Seq.
  • Identified differential full-length isoform expression in the glioma, including an 11,676-fold increase in a Secreted Protein Acidic and Cysteine Rich (SPARC) isoform.
  • Detected several novel cancer-specific isoforms using the PB_FLIC-Seq method.

Conclusions:

  • The PB_FLIC-Seq methodology effectively increases sequenced full-length isoform reads on the PacBio platform.
  • This approach yields improved discovery of expressed isoforms, crucial for complex transcriptome characterization.
  • PB_FLIC-Seq application in a pediatric glioma highlights its utility in identifying cancer-specific isoform expression.