Evaluating imprecision of end-to-end sequence capture at RNA 3′ ends. (A) Mechanisms, numbered by Roman numeral, of 3′-end capture in OTTR. Column 1 illustrates five specificities for capturing an input-template 3′ end, following the color legend from Figure 1. 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 2 specifies the labeling status of the input-template 3′ end. Column 3 identifies the primer-duplex 3′ overhang supporting a template jump. Column 4 details the impact on 3′ sequence coverage. Column 5 categorizes the 3′-end capture mechanism. (B) Library-wide CV of miRXplore miRNA counts and mean fraction of alignments with complete 3′-end coverage (3′ precision). BoMoC, yPAP (for 30 or 120 min), or no enzyme (unlabeled) was used for 3′ labeling. (C–E) Panel pairs depict 3′-end coverage with BoMoC (left) or no enzyme (right) for 3′ labeling. The miRNA sequence and name, and relative rank-order, are indicated at top. At bottom, position “0” is the miRNA 3′ end. Right panels have at bottom the miRNA sequence color-coded by alignment inclusion (pink) or exclusion (black). The Read2 (R2) primer sequence and adjacent +1 T overhang (+T) are in blue, and reference to the capture mechanism detailed in (A) is indicated. Input RNA 3′ sequence excluded by adapter trimming is indicated in black. The miRNA mmu-miR-33-5p is miRBase: MIMAT0000667, the miRNA hsa-miR-599 is miRBase: MIMAT0003267, and the miRNA mghv-miR-M1-3-3p is miRBase: MIMAT0001566.
