title: Experimental validation of methods for differential gene expression analysis and sample pooling in RNA-seq.
abstract: 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.
authors.affiliation: Department of Biomedicine, Aarhus University, 6, Bartholins Allé, Aarhus C, Aarhus, 8000, Denmark. apr@biomed.au.dk.|Mental Health of Older Adults and Dementia Clinical Academic Group, South London and Maudsley NHS foundation trust, London, UK. apr@biomed.au.dk.|Wolfson Centre for Age Related Diseases, Institute of Psychiatry, Psychology, & Neuroscience, King's College, London, UK. apr@biomed.au.dk.|The Initiative for Integrative Psychiatric Research, iPSYCH, Aarhus, 8000, Denmark. apr@biomed.au.dk.|Center for Integrative Sequencing, iSEQ, Aarhus University, Aarhus, 8000, Denmark. apr@biomed.au.dk.
authors.forename: Anto P
authors.initials: AP
authors.lastname: Rajkumar
authors.affiliation: Department of Biomedicine, Aarhus University, 6, Bartholins Allé, Aarhus C, Aarhus, 8000, Denmark. per.q@biomed.au.dk.|The Initiative for Integrative Psychiatric Research, iPSYCH, Aarhus, 8000, Denmark. per.q@biomed.au.dk.|Center for Integrative Sequencing, iSEQ, Aarhus University, Aarhus, 8000, Denmark. per.q@biomed.au.dk.
authors.forename: Per
authors.initials: P
authors.lastname: Qvist
authors.affiliation: Computational Biology, Baker IDI heart and diabetes institute, Victoria, 8008, Australia. Ross.Lazarus@bakeridi.edu.au.
authors.forename: Ross
authors.initials: R
authors.lastname: Lazarus
authors.affiliation: Department of Biomedicine, Aarhus University, 6, Bartholins Allé, Aarhus C, Aarhus, 8000, Denmark. lescai@biomed.au.dk.|The Initiative for Integrative Psychiatric Research, iPSYCH, Aarhus, 8000, Denmark. lescai@biomed.au.dk.|Center for Integrative Sequencing, iSEQ, Aarhus University, Aarhus, 8000, Denmark. lescai@biomed.au.dk.
authors.forename: Francesco
authors.initials: F
authors.lastname: Lescai
authors.affiliation: Beijing Genomics Institute, Shenzhen, 518083, China. jvjia@genomics.org.cn.
authors.forename: Jia
authors.initials: J
authors.lastname: Ju
authors.affiliation: Department of Biomedicine, Aarhus University, 6, Bartholins Allé, Aarhus C, Aarhus, 8000, Denmark. nyegaard@biomed.au.dk.|The Initiative for Integrative Psychiatric Research, iPSYCH, Aarhus, 8000, Denmark. nyegaard@biomed.au.dk.|Center for Integrative Sequencing, iSEQ, Aarhus University, Aarhus, 8000, Denmark. nyegaard@biomed.au.dk.
authors.forename: Mette
authors.initials: M
authors.lastname: Nyegaard
authors.affiliation: The Initiative for Integrative Psychiatric Research, iPSYCH, Aarhus, 8000, Denmark. nielmors@rm.dk.|Center for Integrative Sequencing, iSEQ, Aarhus University, Aarhus, 8000, Denmark. nielmors@rm.dk.|Research Department P, Aarhus University Hospital, Risskov, Denmark. nielmors@rm.dk.
authors.forename: Ole
authors.initials: O
authors.lastname: Mors
authors.affiliation: Department of Biomedicine, Aarhus University, 6, Bartholins Allé, Aarhus C, Aarhus, 8000, Denmark. anders@biomed.au.dk.|The Initiative for Integrative Psychiatric Research, iPSYCH, Aarhus, 8000, Denmark. anders@biomed.au.dk.|Center for Integrative Sequencing, iSEQ, Aarhus University, Aarhus, 8000, Denmark. anders@biomed.au.dk.|Research Department P, Aarhus University Hospital, Risskov, Denmark. anders@biomed.au.dk.|Translational Neuropsychiatry Unit, Aarhus University, Aarhus, 8240, Denmark. anders@biomed.au.dk.
authors.forename: Anders D
authors.initials: AD
authors.lastname: Børglum
authors.affiliation: Beijing Genomics Institute, Shenzhen, 518083, China. liqb@bgitechsolutions.com.
authors.forename: Qibin
authors.initials: Q
authors.lastname: Li
authors.affiliation: Department of Biomedicine, Aarhus University, 6, Bartholins Allé, Aarhus C, Aarhus, 8000, Denmark. jhc@biomed.au.dk.|The Initiative for Integrative Psychiatric Research, iPSYCH, Aarhus, 8000, Denmark. jhc@biomed.au.dk.|Center for Integrative Sequencing, iSEQ, Aarhus University, Aarhus, 8000, Denmark. jhc@biomed.au.dk.
authors.forename: Jane H
authors.initials: JH
authors.lastname: Christensen
mesh_terms: D000818:Animals; D018076:DNA, Complementary / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D051379:Mice; D017423:Sequence Analysis, RNA; D012984:Software
chemical_list: D018076:DNA, Complementary