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Updated: Jul 27, 2025

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High-throughput RNA isoform sequencing using programmed cDNA concatenation.

Aziz M Al'Khafaji1, Jonathan T Smith2, Kiran V Garimella3

  • 1Broad Institute of MIT and Harvard, Cambridge, MA, USA. aalkhafa@broadinstitute.org.

Nature Biotechnology
|June 8, 2023
PubMed
Summary
This summary is machine-generated.

Multiplexed Arrays Isoform Sequencing (MAS-ISO-seq) boosts long-read sequencing throughput by over 15-fold. This advancement significantly enhances the discovery of differentially spliced genes in single-cell studies.

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Area of Science:

  • Molecular Biology
  • Genomics
  • Bioinformatics

Background:

  • Long-read RNA sequencing captures full transcript isoforms but suffers from low throughput.
  • Limited throughput hinders comprehensive analysis of transcriptomic complexity.

Purpose of the Study:

  • Introduce Multiplexed Arrays Isoform Sequencing (MAS-ISO-seq) to enhance long-read sequencing throughput.
  • Improve the efficiency of full-length transcript isoform sequencing.

Main Methods:

  • Developed MAS-ISO-seq for programmable concatenation of complementary DNAs (cDNAs).
  • Optimized cDNA molecules for high-throughput long-read sequencing on the Sequel IIe platform.
  • Applied MAS-ISO-seq to single-cell RNA sequencing of tumor-infiltrating T cells.

Main Results:

  • Achieved >15-fold throughput increase, generating nearly 40 million cDNA reads per run.
  • Demonstrated a 12- to 32-fold increase in discovering differentially spliced genes.
  • MAS-ISO-seq significantly expands the scope of isoform sequencing.

Conclusions:

  • MAS-ISO-seq overcomes throughput limitations of existing long-read RNA sequencing methods.
  • The technique substantially improves the discovery of transcript isoforms and alternative splicing events.
  • MAS-ISO-seq is a powerful tool for high-resolution transcriptomic analysis, particularly in complex biological systems.