FLAM-seq: full-length mRNA sequencing reveals principles of poly(A) tail length control

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Abstract

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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Fig. 1: Full-length poly(A) mRNA sequencing (FLAM-seq).
Fig. 2: Validation of poly(A) length estimation by FLAM-seq.
Fig. 3: Poly(A) length profiles in human and C. elegans samples.
Fig. 4: Widespread tail length dependency on mRNA isoform.
Fig. 5: Nucleotide composition of poly(A) tails.

Data availability

All sequencing data have been deposited on NCBI GEO under the accession number GSE126465. Raw data used for each graphical representation in the main figures are available in the Supplementary Information and Datasets 1–5.

Code availability

The software used for data analysis is available at https://github.com/rajewsky-lab/FLAMAnalysis.

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Acknowledgements

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.

Author information

I.L., S.A. and J.A. conceived and optimized FLAM-seq. I.L., S.A. and J.A. performed all the experiments. J.A. and N.K. conceived and implemented the FLAM-seq analysis pipeline. J.A., N.K. and I.L. performed the computational analyses. N.R. contributed to both experimental and analyses design and supervised the whole project.

Correspondence to Nikolaus Rajewsky.

Ethics declarations

Competing interests

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.

Additional information

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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Supplementary information

Supplementary Information

Supplementary Notes 1 and 2 and Supplementary Figs. 1–5.

Reporting Summary

Supplementary Protocol

Supplementary protocol for Full-length poly(A) and mRNA sequencing (FLAM-seq).

Source data

Dataset 1

Source data for Fig. 1.

Dataset 2

Source data for Fig. 2.

Dataset 3

Source data for Fig. 3.

Dataset 4

Source data for Fig. 4.

Dataset 5

Source data for Fig. 5.

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