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High-throughput RNA isoform sequencing using programmed cDNA concatenation


Full-length RNA-sequencing methods using long-read technologies can capture complete transcript isoforms, but their throughput is limited. We introduce multiplexed arrays isoform sequencing (MAS-ISO-seq), a technique for programmably concatenating complementary DNAs (cDNAs) into molecules optimal for long-read sequencing, increasing the throughput >15-fold to nearly 40 million cDNA reads per run on the Sequel IIe sequencer. When applied to single-cell RNA sequencing of tumor-infiltrating T cells, MAS-ISO-seq demonstrated a 12- to 32-fold increase in the discovery of differentially spliced genes.

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Fig. 1: MAS-ISO-seq workflow and experimental validation using synthetic RNA isoforms.
Fig. 2: Single-cell isoform-resolved sequencing of primary human CD8+ T cells with MAS-ISO-seq.

Data availability

Links to the datasets used in this study can be found at

Human tumor-infiltrating CD8+ T cells single-cell RNA-sequencing data are available from dbGAP with accession number phs003200.v1.p1.

Code availability

An online repository of code for the Longbow tool used in this study can be found at


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We thank W. Kretzschmar for the helpful discussions. This work was supported by Broad Institute SPARC awards 800353 (to A.M.A., K.V.G., N.H. and P.C.B.) and 800307 (to K.V.G.); National Institutes of Health grant (U19 AI082630 to N.H.), Adelson Medical Research Foundation (to N.H.), National Human Genome Research Institute grant (RM1HG006193 to N.H. and P.C.B.), with additional support from the Center for Cell Circuits at the Broad Institute (HG006193). M.A.S. is a Cancer Research Institute Irvington Fellow supported by the Cancer Research Institute (CRI award 4071).

Author information

Authors and Affiliations



A.M.A. conceived and developed the molecular workflow and designed and performed the experiments. K.V.G. developed the statistical annotation software with contributions from J.S. J.S. developed the data processing pipeline with contributions from V.P. and M.G. and performed bioinformatic analyses. M.B. performed Smart-seq3 and single-cell RNA-seq data analysis and statistical modeling and devised the isoform identification algorithm with contributions from J.S. V.P. developed the UMI and CBC error correction algorithms and conducted the bioinformatic analysis with contributions from A.M.A. S.S. aided through discussions and analysis. G.M.B., E.M.B. and M.S.F. consented patients, collected samples and processed and generated the 10× Genomics scRNA-seq data. M.A.S assisted with T-cell data analysis. M.C., A.D., T.B. and S.G. aided in the data generation and helped troubleshoot early iterations of the protocol. A.A.P. and E.B. provided access to cloud computing and other resources to facilitate data processing and analysis. A.M.A., K.V.G., J.S., M.B., V.P., P.C.B. and N.H. cowrote the manuscript.

Corresponding authors

Correspondence to Aziz M. Al’Khafaji, Kiran V. Garimella, Mehrtash Babadi, Victoria Popic, Paul C. Blainey or Nir Hacohen.

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Competing interests

The funding that contributed to the subject matter of this manuscript is described as follows: Broad Institute SPARC award, National Institutes of Health grant (U19 AI082630), Adelson Medical Research Foundation, National Human Genome Research Institute grant (RM1HG006193), support from the Center for Cell Circuits at the Broad Institute (HG006193) and Cancer Research Institute award (4071). A.M.A., K.V.G., J.S., M.B., P.C.B. and N.H. are inventors on a licensed, pending international patent application, having serial number PCT/US2021/037226, filed by Broad Institute of MIT and Havard, Massachusetts General Hospital and Massachusetts Institute of Technology, directed to certain subject matter related to the MAS-seq method described in this manuscript. Broad Institute of MIT and Harvard and Pacific Biosciences of California entered into a collaboration agreement relating to this research subsequent to the submission of this manuscript. A.A.P. is a venture partner and employee of GV. He has received funding from Verily, Microsoft, Illumina, Bayer, Pfizer, Biogen, Abbvie, Intel and IBM. M.S.F. receives funding from Bristol-Myers Squibb. G.M.B. has served on SAB and on the steering committee for Nektar Therapeutics. She has SRAs with Olink Proteomics and Palleon Pharmaceuticals. She served on SAB and as a speaker for Novartis. N.H. holds equity in BioNTech and is a founder and equity holder of Danger Bio. P.C.B. is a consultant to and/or holds equity in companies that develop or apply genomic or genome editing technologies: 10× Genomics, General Automation Lab Technologies/Isolation Bio, Celsius Therapeutics, Next Gen Diagnostics LLC, Cache DNA, Concerto Biosciences, Stately Bio, Ramona Optics, Bifrost Biosystems and Amber Bio. P.C.B.’s group receives research funding from industry for unrelated work. The remaining authors declare no competing interests.

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Nature Biotechnology thanks Omid Faridani and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Supplementary Figs. 1–21, Supplementary Table 1 and Supplementary Note.

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Al’Khafaji, A.M., Smith, J.T., Garimella, K.V. et al. High-throughput RNA isoform sequencing using programmed cDNA concatenation. Nat Biotechnol (2023).

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