Figure 4:

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 one 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 symbols are a small black circle for dideoxynucleotide, a small open circle for 3′-OH, and a triangle for 3′-OH replacement with an unextendible carbon linker. The red octagon indicates the presence of a 5′ fluorescent dye. Column two defines the NTA that would prime 3′rC adapter-template capture. Column three 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. See complete summary statistic in Table 4.