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

Nature Methods
Ivano LegniniNikolaus Rajewsky

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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Citations

Feb 7, 2020·Genome Research·Nathan P RoachJohn K Kim
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Methods Mentioned

BETA
PAL-seq
PCR
TAIL-seq
as
in vitro transcription

Software Mentioned

SMRT
SMRT Link browser
PAL
Bioanalyzer
BedTools Coverage
seq
Ensembl
Gviz
IDT
GENCODE

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