1. Breakdown of 244 total (Yale+Vega) Pseudogenes Amongst Various ENCODE Regions 211 Yale, 178 Vega, Union is 244 More pseudogenes in the manually picked regions. Numbers of genes and pseudogenes are weakly correlated in ENCODE regions. Half of total are processed pseudogenes – Note: many pseudogenes in ENm007 and ENm009 are fragment-like. No. of Genes No. of Pseudogenes r 2 =0.35 ManualRandom

2. Intersection of Pseudogenes with Transcriptional Data Pseudogenes Transcription factors binding sites from ChIP-Chip Genes Sequence conservation in rat, mouse and chimp EST ENm004 Affymetrix TARs using Oligo-microarray GIS-PET Yale TARs using Oligo- microarray CAGE

3. Example of a Pseudogene with Various Transcriptional Evidence Yale_Pgene_58

4. Intersection of Pseudogenes with Transcriptional Data: Comparison of Duplicated and Processed Pseudogenes (just Yale set) Duplicated (confident assignment) Processed (confident assignment) RestTotal # 19103 89211 Yale TAR S (in all 5 expts) 12 (-15) 47 (-60) 87 (-98) Affy Transfrags (in all 6 expts) 9 (-13) 43 (-59) 54 (-84) Some pseudogenes can be confidently annotated as "duplicated" (having intron and exon structure) or "processed" (retrotransposed and disabled) These can be intersected with transcriptional data in 11 experiments at Yale and Affy (Total intersection over all is shown in big type. Just occurring in one experiment is shown in small type.) By random chance, 20-30 of 211 Yale pseudogenes will intersect with TARs Expectation (confirmed) is that duplicated pseudogenes will have higher transcription

5. Intersection of Pseudogenes with Transcriptional Data: Manual vs Random Picks, Vega v Yale Yale PseudogenesVega Pseudogenes ENm*ENr*TotalENm*ENr*Total Pseudogenes1367521111266178 Yale-TARS54-6133-3987-9847-5736-4783-103 Affy-TARs28-4826-3654-8427-4335-4362-85 GIS-PET123235 CAGE510155712 EST1051596 By random chance, 20-30 Yale pseudogenes will intersect with TARs. ~40% ENCODE pseudogenes intersect with TARs.

6. EXTRA

7. Intersection of TARs with Pseudogenes Not-”unique” TAR: one with a sequence of 60 bp (~3 probes) mapping to > 1 genomic locations (≥ 95% identity). 7 No. of TARs Overlapping a Pseudogene 7 Yale-Unique-TAR Yale-not-Unique-TAR No. of TARs Affy-not-Unique-TAR Affy-Unique-TAR

8. Summary 211 Pseudogenes (253, Yale + Vega) in ENCODE regions. Some pseudogenes (< 7%) might be transcribed based on combination of GIS- PET, CAGE or EST data. About one half of pseudogenes overlap with TARs. Non-unique TARs intersect with pseudogenes 5 times more often than unique TARs, probably due to cross-hybridization. Comparison with previous analysis: A more detailed survey found that 12-16% of chr22 pseudogenes intersected with TARs from tiling microarray (Zheng et al., 2005). Both a chr22 and a whole genome analysis showed that ~5% human pseudogenes are likely transcribed (Zheng et al., 2005; Harrison et al., 2005). Cheng et al. (2005) also reported that pseudogene-overlapping TARs are usually not unique. We repeat their analysis using ENCODE pseudogenes and find the same. Refs: Cheng et al., 2005, Transcriptional maps of 10 human chromosomes at 5-nucleotide resolution. Science. 308(5725): 1149-54. Harrison et al., 2005, Transcribed processed pseudogenes in the human genome: an intermediate form of expressed retrosequence lacking protein-coding ability. Nucleic Acids Res. 33(8): 2374-83. Zheng et al., 2005, Integrated pseudogene annotation for human chromosome 22: evidence for transcription. J Mol Biol. 349(1):27-45. 8

9.  genes with Transcription Evidence, Merging Vega and Yale  genes ENm*ENr*Total 15787244 Yale-TARS a 6646112 Affy-TARs b 454186 Union-TARs9463157 GIS-PET224 CAGE61016 EST11718 By random chance, ~25 pseudogenes will intersect with TARs. ~40% ENCODE pseudogenes intersect with TARs. So high percentage? a,b – average of TARs from 5, individual experiments. 2

10. 113 VEGA 40 Yale 75 23 ENSEMBL 2 9 Pseudogenes in ENCODE Regions 211 pseudogenes were identified using an updated computational pipeline (Zhang et al. 2003) and manual curation. Compare Yale pseudogenes with pseudogenes from VEGA group and the ENSEMBL group. 2

11. Break Down of 211 Yale Pseudogenes Among Encode Regions More pseudogenes in the manually picked regions. 211 Pseudogenes can be separated into 104 processed, 19 duplicated and 88 others. Others – those can’t be clearly binned to processed or duplicated, e.g., fragments. Numbers of genes and pseudogenes are weakly correlated in ENCODE regions. 3 Manually Picked (ENm*) Randomly Picked (ENr*) No. of Genes No. of Pseudogenes r2=0.31