Table 2.
Evaluation of protocol variations
| Variation | Category | Trait | Modified group | Control group | Median difference (Modified: Control) | Test | ||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Sample size | Median | Sample size | Median | Statistic | 95% C.I. | Paired/Unpaired | Statistic | p-value | ||||
| No initial cDNA purification | Sensitivity | # genes detected | 6 | 11072 | 6 | 10249 | 782.5 | 293.0 | 1678.0 | Unpaired | 36 | 0.002 |
| Precision | Pairwise correlation across samples | 15 | 0.344 | 15 | 0.306 | 0.039 | 0.028 | 0.051 | Unpaired | 215 | 1.79E-06 | |
| Accuracy | Pairwise correlation with reference | 6 | 0.400 | 6 | 0.383 | 0.019 | 0.008 | 0.049 | Unpaired | 36 | 0.002 | |
| Reduce rounds of cDNA amp. | Sensitivity | # genes detected | 5 | 11936 | 3 | 11062 | 1051.0 | 33.0 | 4122.0 | Unpaired | 15 | 0.036 |
| Precision | Pairwise correlation across samples | 10 | 0.329 | 3 | 0.333 | −0.006 | −0.034 | 0.018 | Unpaired | 12 | 0.692 | |
| Accuracy | Pairwise correlation with reference | 5 | 0.430 | 3 | 0.401 | 0.029 | 0.009 | 0.066 | Unpaired | 15 | 0.036 | |
| Optimized aRNA | Sensitivity | # genes detected | 5 | 11936 | 18 | 10286 | 1810.0 | 942.0 | 3354.0 | Unpaired | 84 | 0.002 |
| Precision | Pairwise correlation across samples | 10 | 0.329 | 153 | 0.306 | 0.019 | 0.002 | 0.035 | Unpaired | 1084 | 0.028 | |
| Accuracy | Pairwise correlation with reference | 5 | 0.430 | 18 | 0.377 | 0.055 | 0.028 | 0.083 | Unpaired | 79 | 0.009 | |
| Add ERCCs | Sensitivity | # genes detected | 5 | 10154 | 4 | 10101 | 138.5 | −397.0 | 798.0 | Unpaired | 13 | 0.556 |
| Precision | Pairwise correlation across samples | 10 | 0.287 | 6 | 0.282 | 0.002 | −0.012 | 0.013 | Unpaired | 34 | 0.713 | |
| Accuracy | Pairwise correlation with reference | 5 | 0.364 | 4 | 0.369 | −0.003 | −0.016 | 0.019 | Unpaired | 9 | 0.905 | |
| Perform strand-specific sequencing | Sensitivity | # genes detected | 17 | 10006 | 17 | 10325 | −267.0 | −331.5 | −218.5 | Paired | 0 | 1.53E-05 |
| Sensitivity | Depth of unique genes | 17 | 6.397 | 17 | 0.848 | 5.46 | 3.68 | 6.75 | Paired | 153 | 1.53E-05 | |
| Sensitivity | # genes not in bulk | 17 | 534 | 17 | 610 | −62.0 | −76.0 | −49.5 | Paired | 0 | 1.53E-05 | |
| Accuracy | Pairwise correlation with reference | 17 | 0.362 | 17 | 0.376 | −0.013 | −0.017 | −0.009 | Paired | 2 | 4.58E-05 | |
Comparison of dilution replicates generated using modified protocols with control dilution replicates. Sample information can be found in Additional file 1 and protocol information in Methods. # genes detected only considers genes observed in bulk HBR or UHR. Kendall correlation was calculated excluding zeros in either sample. Unpaired comparisons were made using Wilcoxon two-way rank sum test for difference in medians. Paired comparisons were made using Wilcoxon two-way rank sign test for difference in medians. The null hypothesis of no difference was rejected at p < 0.05. Median difference between groups, with 95% CI, was calculated using the Hodges-Lehman statistic