Assessing characteristics of RNA amplification methods for single cell RNA sequencing

Dueck, Hannah R.; Ai, Rizi; Camarena, Adrian; Ding, Bo; Dominguez, Reymundo; Evgrafov, Oleg V.; Fan, Jian-Bing; Fisher, Stephen A.; Herstein, Jennifer S.; Kim, Tae Kyung; Kim, Jae Mun (Hugo); Lin, Ming-Yi; Liu, Rui; Mack, William J.; McGroty, Sean; Nguyen, Joseph D.; Salathia, Neeraj; Shallcross, Jamie; Souaiaia, Tade; Spaethling, Jennifer M.; Walker, Christopher P.; Wang, Jinhui; Wang, Kai; Wang, Wei; Wildberg, Andre; Zheng, Lina; Chow, Robert H.; Eberwine, James; Knowles, James A.; Zhang, Kun; Kim, Junhyong

doi:10.1186/s12864-016-3300-3

BMC Genomics

Table 2 Evaluation of protocol variations

From: Assessing characteristics of RNA amplification methods for single cell RNA sequencing

Variation	Category	Trait	Modified group		Control group		Median difference (Modified: Control)			Test
Variation	Category	Trait	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

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ISSN: 1471-2164

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