RNA-Seq describes the abundance and sequence of RNA transcripts. The method, first published by several groups in 20081-5 has effectively displaced older methods such as serial analysis of gene expression (SAGE) and massively parallel signature sequencing (MPSS). With over 8000 references in PubMed, RNA-Seq is by far the most abundantly cited NGS method.6 All RNA-Seq methods are based on the use of a reverse transcriptase to convert the RNA to cDNA. Two basic variations use either random primers or oligo(dT) primers for this reaction. Oligo(dT) primers are highly 3' biased and mostly suitable for mRNA abundance (expression) analysis. Random primers also result in some bias, which can be reduced by fragmentation of the input RNA.7
Additional refinements, such as the use of Moloney murine leukemia virus reverse transcriptase (MMLV-RT) and template-switching oligonucleotides produce a higher yield of full-length transcripts for gene annotation and splice-variant detection. These methods include those based on switch mechanism at the 5' end of RNA templates (Smart), such as Smart-Seq and Smart-seq2 ; cap-analysis gene expression (CAGE), such as NanoCAGE and CAGEscan ; RNA-Seq from single nuclei (snRNA-Seq) ; and sequencing of fixed and recovered intact single-cell RNA (FRISCR).