Technical considerations for functional sequencing assays


The measurement of RNA expression is a foundation of many experiments done in biomedical research. It is therefore natural that the sequencing of long and short RNA both for quantification and discovery is the most popular functional sequencing assay (Fig. 1a). Quantification of mRNA transcripts in RNA-seq is performed by calculating values reported in units of reads per kilobase per million mapped reads (RPKM) with a paired-end fragment equivalent, fragments per kilobase per million reads (FPKM), also commonly used for each gene (Fig. 1b). RPKM normalizes for differences in gene size and makes the comparison of genes within the same sample meaningful in terms of molar equivalents (Fig. 1b). As RNA-seq is not based on predetermined DNA probes to known genes, it is a powerful tool for the discovery of new exons, splice junctions, transcripts and genes as well as new small RNAs (Fig. 1c). The reads can be used to assemble transcripts that result from gene rearrangements and can also help to identify disease-associated genomic abnormalities. Properly filtered RNA-seq reads can be mined for sequence variants and RNA-editing events with tuned analysis and filtering pipelines (Fig. 1d).

  • Zeng W, Mortazavi A. (2012) Technical considerations for functional sequencing assays. Nat Immunol 13(9), 802-7. [abstract]