Computational approaches followed by experimental validation were implemented to examine how many GENCODE pseudogenes appeared to be transcribed (Pei et al. 2012). Briefly, transcribed pseudogenes were identified manually and tagged by the HAVANA team examining locus-specific transcription evidence (by aligning of mRNAs or ESTs). This identified 171 transcribed processed and 309 unprocessed pseudogenes. The locus-specific transcriptional evidence must indicate a best-in-genome alignment and clear differences compared with the parent locus. Interestingly, there was over one-third more unprocessed pseudogenes annotated as transcribed compared with processed pseudogenes, even though there are approximately four times as many processed pseudogenes present in the genome than unprocessed pseudogenes (see Supplemental Table 4). In addition, automated pipeline analysis of RNA-seq data from the total RNA of ENCODE cell line GM12878 and K562 plus HBM RNA-seq resource (Pei et al. 2012) generated an additional 110 and 344 transcribed processed and unprocessed pseudogenes, respectively. Specific primers could be designed for 162 potentially transcribed pseudogenes and have been subjected to experimental validation of transcription by the RT-PCR-seq pipeline within the GENCODE Consortium (Howald et al. 2012). After the validation experiments, 63 pseudogenes were found to be transcribed within at least one of eight tissues.
