High-throughput RNA isoform sequencing using programmed cDNA concatenation

doi:10.1038/s41587-023-01815-7

. 2024 Apr;42(4):582-586.

doi: 10.1038/s41587-023-01815-7. Epub 2023 Jun 8.

High-throughput RNA isoform sequencing using programmed cDNA concatenation

Aziz M Al'Khafaji^#¹, Jonathan T Smith^#², Kiran V Garimella^#³, Mehrtash Babadi^#⁴, Victoria Popic^#⁵, Moshe Sade-Feldman^{2

6}, Michael Gatzen², Siranush Sarkizova², Marc A Schwartz^{2

7

8

9}, Emily M Blaum^{2

6}, Allyson Day², Maura Costello², Tera Bowers², Stacey Gabriel², Eric Banks², Anthony A Philippakis², Genevieve M Boland¹⁰, Paul C Blainey^{11

12

13}, Nir Hacohen^{14

15

16

17}

Affiliations

¹ Broad Institute of MIT and Harvard, Cambridge, MA, USA. aalkhafa@broadinstitute.org.
² Broad Institute of MIT and Harvard, Cambridge, MA, USA.
³ Broad Institute of MIT and Harvard, Cambridge, MA, USA. kiran@broadinstitute.org.
⁴ Broad Institute of MIT and Harvard, Cambridge, MA, USA. mehrtash@broadinstitute.org.
⁵ Broad Institute of MIT and Harvard, Cambridge, MA, USA. vpopic@broadinstitute.org.
⁶ Department of Medicine, Center for Cancer Research, Massachusetts General Hospital, Boston, MA, USA.
⁷ Department of Pediatrics, Harvard Medical School, Boston, MA, USA.
⁸ Division of Hematology/Oncology, Boston Children's Hospital, Boston, MA, USA.
⁹ Department of Pediatric Oncology, Dana Farber Cancer Institute, Boston, MA, USA.
¹⁰ Division of Surgical Oncology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA.
¹¹ Broad Institute of MIT and Harvard, Cambridge, MA, USA. pblainey@broadinstitute.org.
¹² Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA. pblainey@broadinstitute.org.
¹³ Koch Institute for Integrative Cancer Research at the Massachusetts Institute of Technology, Cambridge, MA, USA. pblainey@broadinstitute.org.
¹⁴ Broad Institute of MIT and Harvard, Cambridge, MA, USA. nhacohen@broadinstitute.org.
¹⁵ Department of Medicine, Center for Cancer Research, Massachusetts General Hospital, Boston, MA, USA. nhacohen@broadinstitute.org.
¹⁶ Harvard Medical School, Boston, MA, USA. nhacohen@broadinstitute.org.
¹⁷ Center for Immunology and Inflammatory Diseases, Massachusetts General Hospital, Charlestown, MA, USA. nhacohen@broadinstitute.org.

^# Contributed equally.

PMID: 37291427
PMCID: PMC12236355
DOI: 10.1038/s41587-023-01815-7

High-throughput RNA isoform sequencing using programmed cDNA concatenation

Aziz M Al'Khafaji et al. Nat Biotechnol. 2024 Apr.

. 2024 Apr;42(4):582-586.

doi: 10.1038/s41587-023-01815-7. Epub 2023 Jun 8.

Authors

Affiliations

¹ Broad Institute of MIT and Harvard, Cambridge, MA, USA. aalkhafa@broadinstitute.org.
² Broad Institute of MIT and Harvard, Cambridge, MA, USA.
³ Broad Institute of MIT and Harvard, Cambridge, MA, USA. kiran@broadinstitute.org.
⁴ Broad Institute of MIT and Harvard, Cambridge, MA, USA. mehrtash@broadinstitute.org.
⁵ Broad Institute of MIT and Harvard, Cambridge, MA, USA. vpopic@broadinstitute.org.
⁶ Department of Medicine, Center for Cancer Research, Massachusetts General Hospital, Boston, MA, USA.
⁷ Department of Pediatrics, Harvard Medical School, Boston, MA, USA.
⁸ Division of Hematology/Oncology, Boston Children's Hospital, Boston, MA, USA.
⁹ Department of Pediatric Oncology, Dana Farber Cancer Institute, Boston, MA, USA.
¹⁰ Division of Surgical Oncology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA.
¹¹ Broad Institute of MIT and Harvard, Cambridge, MA, USA. pblainey@broadinstitute.org.
¹² Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA. pblainey@broadinstitute.org.
¹³ Koch Institute for Integrative Cancer Research at the Massachusetts Institute of Technology, Cambridge, MA, USA. pblainey@broadinstitute.org.
¹⁴ Broad Institute of MIT and Harvard, Cambridge, MA, USA. nhacohen@broadinstitute.org.
¹⁵ Department of Medicine, Center for Cancer Research, Massachusetts General Hospital, Boston, MA, USA. nhacohen@broadinstitute.org.
¹⁶ Harvard Medical School, Boston, MA, USA. nhacohen@broadinstitute.org.
¹⁷ Center for Immunology and Inflammatory Diseases, Massachusetts General Hospital, Charlestown, MA, USA. nhacohen@broadinstitute.org.

^# Contributed equally.

PMID: 37291427
PMCID: PMC12236355
DOI: 10.1038/s41587-023-01815-7

Abstract

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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Conflict of interest statement

Competing interests statement:

The authors declare the following competing interests:

The following provided funding that contributed to the subject matter of this manuscript: Broad Institute SPARC award, National Institutes of Health grants U19 AI082630, Adelson Medical Research Foundation, National Human Genome Research Institute grants 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 Inc. 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: 10X 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.

Figures

**Fig. 1:. MAS-ISO-seq workflow and experimental validation using synthetic RNA isoforms.**
**(a)** Schematic of the MAS-ISO-seq intramolecular cDNA multiplexing workflow. **(b)** Sankey diagram reporting MAS-ISO-seq run yield of sample 1 at various stages of processing. **(c)** Observed ERCC concentrations as measured in MAS-ISO-seq and Smart-seq3 experiments vs. reference concentrations (R-squared > 0.95 for both). **(d)** Log-ratio of observed to reference concentrations of short and long SIRV isoforms in SIRV-Set 4 vs. transcript length for Smart-seq3 and MAS-ISO-seq. **(e)** Isoform identification confusion matrix for SIRV isoforms as measured by Smart-seq3 reconstructions and MAS-ISO-seq observations.

**Fig. 2:. Single-cell isoform-resolved sequencing of primary human CD8+ T cells with MAS-ISO-seq.**
**(a)** UMAP embedding of single-cell gene expression of 5,270 CD8+ T cells from short or long-read analyses; the long-read UMAP is annotated with the cell identities determined from the short-read data. **(b)** Scatter plot of unique gene or transcript counts in cells vs. UMI counts per cell for short-read (Illumina) and long-read (MAS-ISO-seq). (c) CD45 (*PTPRC*) isoform analysis using either CITE-seq or MAS-ISO-seq (natural log raw counts). **(d)** Force directed graph of CD8+ T cells with insets depicting pseudotime progression and differential CD45 isoform expression along the pseudotime axis. **(e)** Levels of isoforms along pseudotime and in each cluster. **(f)** Expression of hnRNPLL along the pseudotime progression (log normalized counts); **(g)** Downsampling analysis of MAS-ISO-seq reads; (top) evolution of UMAP embedding vs. depth; (middle) adjusted Rand index (ARI) between short-reads reference annotations and downsampled long reads vs. depth; (bottom) number of statistically significant differentially spliced genes vs. depth. Typical Iso-Seq read depths shaded in gray.

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References

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[1] Hardwick SA, Joglekar A, Flicek P, Frankish A & Tilgner HU Getting the Entire Message: Progress in Isoform Sequencing. Front. Genet. 10, 709 (2019). - PMC - PubMed

[2] Hardwick SA, Joglekar A, Flicek P, Frankish A & Tilgner HU Getting the Entire Message: Progress in Isoform Sequencing. Front. Genet. 10, 709 (2019). - PMC - PubMed

[3] Baralle FE & Giudice J Alternative splicing as a regulator of development and tissue identity. Nat. Rev. Mol. Cell Biol. 18, 437–451 (2017). - PMC - PubMed

[4] Baralle FE & Giudice J Alternative splicing as a regulator of development and tissue identity. Nat. Rev. Mol. Cell Biol. 18, 437–451 (2017). - PMC - PubMed

[5] Scotti MM & Swanson MS RNA mis-splicing in disease. Nat. Rev. Genet. 17, 19–32 (2015). - PMC - PubMed

[6] Scotti MM & Swanson MS RNA mis-splicing in disease. Nat. Rev. Genet. 17, 19–32 (2015). - PMC - PubMed

[7] Dvinge H, Kim E, Abdel-Wahab O & Bradley RK RNA splicing factors as oncoproteins and tumour suppressors. Nat. Rev. Cancer 16, (2016). - PMC - PubMed

[8] Dvinge H, Kim E, Abdel-Wahab O & Bradley RK RNA splicing factors as oncoproteins and tumour suppressors. Nat. Rev. Cancer 16, (2016). - PMC - PubMed

[9] Kanitz A et al. Comparative assessment of methods for the computational inference of transcript isoform abundance from RNA-seq data. Genome Biol. 16, 1–26 (2015). - PMC - PubMed

[10] Kanitz A et al. Comparative assessment of methods for the computational inference of transcript isoform abundance from RNA-seq data. Genome Biol. 16, 1–26 (2015). - PMC - PubMed

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High-throughput RNA isoform sequencing using programmed cDNA concatenation

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High-throughput RNA isoform sequencing using programmed cDNA concatenation

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