Impact of adapter template UMI sequence on complete 5′-end capture
| Fig. | 3′r/ddC adapter template sequence | 3′r/ddC adapter template # RNA | 3′r/ddC adapter template end | CV | Mean difference | CI 95% | Adjusted P-value | miRNA (n) | 5′ precision |
|---|---|---|---|---|---|---|---|---|---|
| 4B | NNNC-3′ | 6 | 3′-OH | 1.07 | 4.92% | (4.64%−5.21%) | 8×10−160 | 902 | 0.7 |
| 4B | NNNNNYC-3′ | 6 | 3′-OH | 0.65 | 3.3% | (2.99%−3.62%) | 2.8×10−74 | 903 | 0.68 |
| 4B | NNNNNAC-3′ | 6 | 3′-OH | 0.72 | 10.1% | (9.56%−10.65%) | 8×10−176 | 889 | 0.75 |
| 4B | NNNNNGC-3′ | 6 | 3′-OH | 0.73 | 10.45% | (9.98%–10.91%) | 2×10−221 | 883 | 0.75 |
| 4B | NNNNNRC-3′ | 6 | 3′-OH | 0.81 | 11.54% | (11.04%–12.03%) | 3×10−232 | 881 | 0.76 |
| 4C | NNNNNYC-3′ | 2 | 3′-OH | 0.66 | −3.4% | (−3.56% to −3.23%) | 4×10−202 | 904 | 0.66 |
| 4C | NNNNNYC-3′ | 2 | 3′-OH | 0.65 | −3.35% | (−3.51% to −3.18%) | 7×10−196 | 904 | 0.66 |
| 4C | NNNNNDC-3′ | 1 | 3′-H | 0.68 | 2.95% | (2.73%–3.17%) | 1×10−112 | 904 | 0.72 |
| 4C | NNNNNDC-3′ | 1 | 3′-H | 0.69 | 2.24% | (2.02%–2.46%) | 1.1×10−72 | 904 | 0.71 |
| 4C | NNNNNDC-3′ | 0 | 3′-H | 0.68 | 8.46% | (8.21%–8.71%) | 0 | 904 | 0.78 |
| 4C | NNNNNDC-3′ | 0 | 3′-H | 0.66 | 8.53% | (8.3%–8.77%) | 0 | 904 | 0.78 |
| 4C | NNNNNAC-3′ | 0 | 3′-H | 0.56 | 7.87% | (7.37%–8.36%) | 2×10−141 | 904 | 0.77 |
| 4C | NNNNNGC-3′ | 0 | 3′-H | 0.61 | 8.06% | (7.62%–8.49%) | 1×10−175 | 903 | 0.77 |
| 4C | NNNNNRC-3′ | 0 | 3′-H | 0.62 | 10.05% | (9.64%–10.46%) | 2×10−249 | 904 | 0.79 |
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Figure panel and descriptions of the 3′-adapter template (3′ sequence, number of RNA nucleotides, and whether the 3′ end was unblocked, i.e., 3′-OH, or blocked, i.e., 3′-hydrogen [3′-H]) are described in the first four columns. See Table 5 for complete adapter template sequences. The mean difference in 5′ precision and a 95% confidence interval were computed from a paired, two-sided Student's t-test, with Bonferroni adjusted P-values. For Figure 4B, all libraries were compared to a library prepared with no UMI in the 3′rC adapter template, and for Figure 4C, all libraries were compared to a library prepared with 3′rC adapter template with 6-RNA ribonucleotides and a 3′-OH with the following 3′ sequence: NNNNNYC-3′.
