Full-length RNA-sequencing methods using long-read technologies can capture complete transcript isoforms, but their throughput is limited.
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 , a technique for programmably concatenating complementary DNAs 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.References Hardwick, S. A., Joglekar, A., Flicek, P., Frankish, A. & Tilgner, H. U. Getting the entire message: progress in isoform sequencing.Volden, R. et al. Improving nanopore read accuracy with the R2C2 method enables the sequencing of highly multiplexed full-length single-cell cDNA.Schlecht, U., Mok, J., Dallett, C. & Berka, J. ConcatSeq: a method for increasing throughput of single molecule sequencing by concatenating short DNA fragments.Kanwar, N., Blanco, C., Chen, I. A. & Seelig, B. PacBio sequencing output increased through uniform and directional fivefold concatenation.Zhao, M., Lee, W.-P., Garrison, E. P. & Marth, G. T. SSW library: an SIMD Smith-Waterman C/C++ library for use in genomic applications.Tang, A. D. et al. Full-length transcript characterization of SF3B1 mutation in chronic lymphocytic leukemia reveals downregulation of retained introns.We thank W. Kretzschmar for the helpful discussions. This work was supported by Broad Institute SPARC awards 800353 and 800307 ; National Institutes of Health grant , Adelson Medical Research Foundation , National Human Genome Research Institute grant , with additional support from the Center for Cell Circuits at the Broad Institute . M.A.S. is a Cancer Research Institute Irvington Fellow supported by the Cancer Research Institute .Aziz M. Al’Khafaji, Jonathan T. Smith, Kiran V. Garimella, Mehrtash Babadi, Victoria Popic, Moshe Sade-Feldman, Michael Gatzen, Siranush Sarkizova, Marc A. Schwartz, Emily M. Blaum, Allyson Day, Maura Costello, Tera Bowers, Stacey Gabriel, Eric Banks, Anthony A. Philippakis, Paul C. Blainey & Nir HacohenMoshe Sade-Feldman, Emily M. Blaum & Nir HacohenA.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.The funding that contributed to the subject matter of this manuscript is described as follows: Broad Institute SPARC award, National Institutes of Health grant , Adelson Medical Research Foundation, National Human Genome Research Institute grant , support from the Center for Cell Circuits at the Broad Institute and Cancer Research Institute award . 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. Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author or other rightsholder; author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
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