Rigor and reproducibility of RNA sequencing analyses

High-throughput sequencing (HTS) of nucleic acids has revolutionized the biological sciences and ushered in new prospects for molecular medicine. Also referred to as next-generation sequencing (NGS) or massive(ly) parallel sequencing (MPS), HTS has in part superseded techniques such as Sanger sequencing and hybridization or amplification microarrays. As inputs into the HTS workflow, both DNA and RNA can be used, but the latter must be converted first into complementary DNA (cDNA). HTS for RNA (RNA sequencing or RNA-Seq) allows the analysis of the transcriptome at single-nucleotide resolution, identifying RNA sequences without a priori knowledge of genomic DNA, splicing, or posttranscriptional modifications. The technique can thus complement genomic DNA sequencing by capturing a “snapshot” of the transcriptomic output within an organism, tissue, cell, or even extracellular environment.