While identification of open reading frames within a DNA sequence is straightforward, identifying coding sequences is not, because the cell translates only a subset of all open reading frames to proteins. Currently CDS prediction uses sampling and sequencing of mRNA from cells, although there is still the problem of determining which parts of a given mRNA are actually translated to protein. CDS prediction is a subset of gene prediction, the latter also including prediction of DNA sequences that code not only for protein but also for other functional elements such as RNA genes and regulatory sequences.
In both prokaryotes and eukaryotes, gene overlapping occurs relatively often in both DNA and RNA viruses as an evolutionary advantage to reduce genome size while retaining the ability to produce various proteins from the available coding regions. For both DNA and RNA, pairwise alignments can detect overlapping coding regions, including short open reading frames in viruses, but would require a known coding strand to compare the potential overlapping coding strand with. An alternative method using single genome sequences would not require multiple genome sequences to execute comparisons but would require at least 50 nucleotides overlapping in order to be sensitive.