RNA Sequencing with Next-Generation Sequencing

Sequencing of RNA has been an important application of DNA sequencing technology since its invention. RNA is usually sequenced by first converting it to complementary DNA (cDNA) with the reverse transcriptase enzyme (RNA-dependent DNA polymerase). Reverse transcriptase was originally isolated from Rous sarcoma retrovirus and Rauscher mouse leukemia retrovirus (R-MLV) by Baltimore (1970) and independently by Temin (1970). In 1972, Verma et al. and Bank et al. developed efficient systems to copymessenger RNA (mRNA) to cDNAbyaddingDNAnucleotide triphosphates and and short pieces of oligo(dT), which hybridize to the poly(A) tail of the mRNAs and act as a primer. cDNA is frequently the subject of sequencing studies, because this an efficient method to discover the coding sequence of expressed genes or for finding gene coding regions in genomic DNA sequence. Craig Venter expanded this method by collecting large numbers of short single reads from the 30 ends of mRNA, which were called expressed sequence tags (ESTs). Early EST sequencing of human cells was extraordinarily productive, resulting in the discovery of many thousands of new genes (Adams et al. 1991, 1992). The EST method allowed for a rough form of gene expressionmeasurements in a variety of cell types and some differential expression studies were conducted in this manner. EST sequencing also became a valuable component of de novo sequencing projects, providing a layer of gene expression information and seeding annotation and gene finding efforts. Microarray technology, developed in the 1990s, measures the hybridization of labeled cDNA to an array of DNAprobes that correspond to the sequences of known genes (or ESTs). The microarray method allows for the discovery, in a genome-wide fashion, of gene expression changes (as reflected in changes of mRNA levels) resulting from any biological treatment or condition.