Fig. 5

a, Schematic overview of the standards used to assess the ability of Nano3P-seq to accurately quantify the base content of polyA tails. b, IGV snapshots of nucleotide composition in cDNA standard tails sequenced using Nano3P-seq. Gray regions indicate the mapped part of the reads, whereas colored letters indicate soft-clipped bases (unmapped), which are the base-called tails, after trimming the adapter. c, Probability of base composition (A, green; G, orange; C, blue; U, red) per position in the last 20 nucleotides of the cDNA standard tails. See also Supplementary Note 1. d, Percentage of reads belonging to groups classified on the basis of their polyA tail base composition. Some sequence examples belonging to different groups are illustrated below the bar plots. Samples in this analysis are embryonic mRNAs across zebrafish developmental stages (2, 4, and 6 h.p.f., shown in blue, green, and red, respectively) in three biological replicates and a control that includes sequin R1 and R2 groups of RNAs (gray). Statistical comparison of means was performed using the Kruskal–Wallis test. n = 3 biological replicates, data are presented as mean ± s.e.m. e, PolyA tail length estimation distributions of mRNA reads belonging to groups classified on the basis of their polyA tail base composition across zebrafish development stages (2, 4, and 6 h.p.f.). f, Left, IGV snapshots of reads mapping to zebrafish actb mRNA. Right, zoomed images of individual reads with different terminal bases (top, all-A reads; middle, Term-G reads; bottom, Term-U reads) are shown.

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