Although messenger RNAs are key molecules for understanding life, until now, no method has existed to determine the full-length sequence of endogenous mRNAs including their poly(A) tails. Moreover, although non-A nucleotides can be incorporated in poly(A) tails, there also exists no method to accurately sequence them. Here, we present full-length poly(A) and mRNA sequencing (FLAM-seq), a rapid and simple method for high-quality sequencing of entire mRNAs. We report a complementary DNA library preparation method coupled to single-molecule sequencing to perform FLAM-seq. Using human cell lines, brain organoids and Caenorhabditis elegans we show that FLAM-seq delivers high-quality full-length mRNA sequences for thousands of different genes per sample. We find that 3′ untranslated region length is correlated with poly(A) tail length, that alternative polyadenylation sites and alternative promoters for the same gene are linked to different tail lengths, and that tails contain a substantial number of cytosines.
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The software used for data analysis is available at https://github.com/rajewsky-lab/FLAMAnalysis.
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We thank A. Rybak-Wolf for providing iPS cells and cerebral organoids samples, J. Froehlich for providing C. elegans samples, A. Boltengagen for cell culture, C. Quedenau, D. Yumi Sunaga-Franze and S. Sauer of the BIMSB Genomics platform for adapter ligation, sequencing runs and preprocessing of sequencing data, H. Lickert for providing the XMO01 iPS cell line and S. Formichetti from the laboratory of G. Macino for providing HeLa S3 cells. We also thank all the members of the N. Rajewsky laboratory for critical and useful discussions. I.L. is recipient of an EMBO Long Term Fellowship (no. ALTF 1235-2016). J.A. is member of the MDC–NYU exchange PhD program. N.K. was supported by the DFG Leibniz prize (N.R.) and grants DFG RA 838/8-2, HGF ExNet-0036 and DFG KA 5006/1-1.
I.L., J.A., N.K., S.A. and N.R. are named inventors on a patent application directed to genome-wide full-length mRNA and poly(A) tail sequencing. European Patent application EP18198248 has been filed with priority of October 2018.
Peer review information: Lei Tang was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
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Legnini, I., Alles, J., Karaiskos, N. et al. FLAM-seq: full-length mRNA sequencing reveals principles of poly(A) tail length control. Nat Methods 16, 879–886 (2019). https://doi.org/10.1038/s41592-019-0503-y
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