Evaluation of imprecision of end-to-end sequence capture at RNA 5′ ends. (A) Mechanisms, numbered by Roman numeral, of precise and imprecise template-jumping to the 3′rC adapter template. Column 1 illustrates two possible and one unlikely specificity of sequence-junction formation following the color legend of Figures 1 and 2A. Phosphodiester bonds are represented by circles: gray for chemically synthesized bonds, concentric double circle for BoMoC-polymerized, unfilled for input template RNA. Strand 3′ end symbol of a small open circle indicates 3′-OH. The red octagon indicates the presence of a 5′ fluorescent dye. Column 2 defines the NTA that would prime 3′rC adapter-template capture. Column 3 defines whether the mapped template 5′-end was precise. (B,C) Comparison of mean fraction of alignments with 5′ precision for libraries prepared with different 3′rC adapter template designs (Table 5). Significant differences in 5′ precision (denoted by asterisk) were determined by a paired, two-sided Student's t-test for each miRNA, with P-values adjusted by Bonferroni correction. A horizontal bar denotes replicate libraries. See complete summary statistics in Table 4.
