Experimental validation of methods for differential gene expression analysis and sample pooling in RNA-seq

AP Rajkumar, P Qvist, R Lazarus, F Lescai, J Ju… - BMC genomics, 2015 - Springer
BMC genomics, 2015Springer
Background Massively parallel cDNA sequencing (RNA-seq) experiments are gradually
superseding microarrays in quantitative gene expression profiling. However, many
biologists are uncertain about the choice of differentially expressed gene (DEG) analysis
methods and the validity of cost-saving sample pooling strategies for their RNA-seq
experiments. Hence, we performed experimental validation of DEGs identified by Cuffdiff2,
edgeR, DESeq2 and Two-stage Poisson Model (TSPM) in a RNA-seq experiment involving …
Background
Massively parallel cDNA sequencing (RNA-seq) experiments are gradually superseding microarrays in quantitative gene expression profiling. However, many biologists are uncertain about the choice of differentially expressed gene (DEG) analysis methods and the validity of cost-saving sample pooling strategies for their RNA-seq experiments. Hence, we performed experimental validation of DEGs identified by Cuffdiff2, edgeR, DESeq2 and Two-stage Poisson Model (TSPM) in a RNA-seq experiment involving mice amygdalae micro-punches, using high-throughput qPCR on independent biological replicate samples. Moreover, we sequenced RNA-pools and compared their results with sequencing corresponding individual RNA samples.
Results
False-positivity rate of Cuffdiff2 and false-negativity rates of DESeq2 and TSPM were high. Among the four investigated DEG analysis methods, sensitivity and specificity of edgeR was relatively high. We documented the pooling bias and that the DEGs identified in pooled samples suffered low positive predictive values.
Conclusions
Our results highlighted the need for combined use of more sensitive DEG analysis methods and high-throughput validation of identified DEGs in future RNA-seq experiments. They indicated limited utility of sample pooling strategies for RNA-seq in similar setups and supported increasing the number of biological replicate samples.
Springer
以上显示的是最相近的搜索结果。 查看全部搜索结果