1. International Conference on Bioinformatics HKUST, Hong Kong 2007
The complexity of the transcriptional landscape of the human genome Roderic Guigó Center for Genomic Regulation, Barcelona
November 9, 2018
International Conference on Bioinformatics HKUST, Hong Kong 2007

2. International Conference on Bioinformatics HKUST, Hong Kong 2007
genes and proteins
One gene, one enzyme Beadle and Tatum
The Central Dogma Francis Crick
November 9, 2018
International Conference on Bioinformatics HKUST, Hong Kong 2007

3. The standard model of the eukaryotic gene
most of the transcriptional output of the human genome is localized in well defined genomic loci, which encode mRNAs that, when exported into the cytosol, are translated into proteins
November 9, 2018
International Conference on Bioinformatics HKUST, Hong Kong 2007

4. International Conference on Bioinformatics HKUST, Hong Kong 2007
GENSCAN HMM (Burge & Karlin).
Slide from M. Alexandersson
November 9, 2018
International Conference on Bioinformatics HKUST, Hong Kong 2007

5. International Conference on Bioinformatics HKUST, Hong Kong 2007
November 9, 2018
International Conference on Bioinformatics HKUST, Hong Kong 2007

6. International Conference on Bioinformatics HKUST, Hong Kong 2007
1% of the genome. 44 regions
target selection. commitee to select sequence targets
manual targets – a lot of information
radom targets – stratified by non exonic conservation with mouse gene density
November 9, 2018
International Conference on Bioinformatics HKUST, Hong Kong 2007

7. Cis-regulatory elements
DNase Hypersensitive
Sites
DNA
Replication
Epigenetic 
Genes and Transcripts
Cis-regulatory elements
(promoters, transcription factor binding sites)
Long-range regulatory elements
(enhancers, repressors/silencers, insulators)

8. International Conference on Bioinformatics HKUST, Hong Kong 2007
November 9, 2018
International Conference on Bioinformatics HKUST, Hong Kong 2007

9. gencode: encyclopedia of genes and gene variants
identify all protein coding genes in the ENCODE regions:
identify one complete mRNA sequence for at least one splice isoform of each protein coding gene.
eventually, identify a number of additional alternative splice forms.
Roderic Guigó, IMIM-UPF-CRG
Stylianos Antonarakis, Geneve Alexandre Reymond
Ewan Birney, EBI
Michael Brent, WashU
Lior Pachter, Berkeley
Manolis Dermitzkakis, Sanger
Jennifer Ashurst, Tim Hubbard
November 9, 2018
International Conference on Bioinformatics HKUST, Hong Kong 2007

10. International Conference on Bioinformatics HKUST, Hong Kong 2007
the gencode pipeline
mapping of known transcripts sequences (ESTs, cDNAs, proteins) into the human genome
manual curation to resolve conflicting evidence
additional computational predictions
experimental verification
FINAL ANNOTATION
November 9, 2018
International Conference on Bioinformatics HKUST, Hong Kong 2007

11. International Conference on Bioinformatics HKUST, Hong Kong 2007
the gencode pipeline
mapping of known transcripts sequences (ESTs, cDNAs, proteins) into the human genome
manual curation to resolve conflicting evidence
additional computational predictions
experimental verification
FINAL ANNOTATION
November 9, 2018
International Conference on Bioinformatics HKUST, Hong Kong 2007

12. The gencode pipeline manual curation: havana (sanger) experimental verification: geneva bioinformatics: imim
2608 transcripts in 487 loci
137 transcripts in 53 non-coding loci
1097 coding transcripts and non-coding transcripts in protein coding loci
most of protein coding loci encode
a mixture of protein coding and
non-coding transcripts

13. one gene - many proteins very complex transcription units
November 9, 2018
International Conference on Bioinformatics HKUST, Hong Kong 2007

14. Distribution of DNaseI HSs vs. TSS in Different Gene Annotation Sets
from the ENCODE Chromatin and Replication Group, John Stamatoyannopoulos
November 9, 2018
International Conference on Bioinformatics HKUST, Hong Kong 2007

15. International Conference on Bioinformatics HKUST, Hong Kong 2007
EGASP’05
the complete annotation of 13 regions was released in january 30, 2005.
The annotation of the remaining 31 regions was being obtained, and it was withheld.
gene prediction groups were asked to submit predictions by april 15, 2005 in the remaining 31 regions.
18 groups participated, submiting 30 prediction sets
predictions were compared to the annoations in an NHGRI sponsored workshop at the Wellcome Trust Sanger Institute, on may 6 and 7, 2005.
November 9, 2018
International Conference on Bioinformatics HKUST, Hong Kong 2007

16. International Conference on Bioinformatics HKUST, Hong Kong 2007
EGASP’05
two main goals:
to assess how automatic methods are able to reproduce the (costly) manual/computational/experimental gencode annotation
how complete is the gencode annotation. are there still genes consistenly predicted by computational methods
November 9, 2018
International Conference on Bioinformatics HKUST, Hong Kong 2007

17. International Conference on Bioinformatics HKUST, Hong Kong 2007
accuracy measures
November 9, 2018
International Conference on Bioinformatics HKUST, Hong Kong 2007

18. accuracy at the coding exon level
evidence-based
dual genome
“ab intio”
November 9, 2018
International Conference on Bioinformatics HKUST, Hong Kong 2007

19. International Conference on Bioinformatics HKUST, Hong Kong 2007
EGASP’05
programs are quite good at calling the protein coding exons (accuracy at 80%) Not as good at calling the transcribed exons), but the best of the programs predict correctly only 40% of the complete transcripts (considering only the coding fraction)
November 9, 2018
International Conference on Bioinformatics HKUST, Hong Kong 2007

20. International Conference on Bioinformatics HKUST, Hong Kong 2007
EGASP’05
many novel exons predicted: - 8,634 unique exons predicted in intergenic regions - we ranked the exons according to the accuracy of te predicted programs - tested 238 exon pairs by RT-PCR in 24 tissues - only 7 (less than 3%) were confirmed positive
November 9, 2018
International Conference on Bioinformatics HKUST, Hong Kong 2007

21. International Conference on Bioinformatics HKUST, Hong Kong 2007
November 9, 2018
International Conference on Bioinformatics HKUST, Hong Kong 2007

22. Cis-regulatory elements
DNase Hypersensitive
Sites
DNA
Replication
Epigenetic 
Genes and Transcripts
Cis-regulatory elements
(promoters, transcription factor binding sites)
Long-range regulatory elements
(enhancers, repressors/silencers, insulators)

23. International Conference on Bioinformatics HKUST, Hong Kong 2007
Genome tiling arrays
Slide from
Salk Institute Genomic Analysis Laboratory
November 9, 2018
International Conference on Bioinformatics HKUST, Hong Kong 2007

24. International Conference on Bioinformatics HKUST, Hong Kong 2007
TRANSCRIPTION OF PROCESSED POLY A+ RNA based on a number of high throughput tecnologies
Total # of nucleotides : 29,998,060
non repeat masked : 14,707,189
Nb of nucleotide covered
% nucleotides covered
Annotated exons
1,650,821
9.8%
transfrag/tar
1,278,588
9.3%
CAGE Tags*
151,149
0.5%
Ditags*
24,939
0.1%
TOTAL UNIQUE Transcribed Bases
2,355,238
14.7%
November 9, 2018
International Conference on Bioinformatics HKUST, Hong Kong 2007

25. PROCESSED TRANSCRIPTS (PT)
Table 1: Summary of Transcriptional
Coverage of ENCODE Regions.
PROCESSED TRANSCRIPTS (PT)
PRIMARY TRANSCRIPTS
Total Bases 1
Total Interro-gated Bases 2 %
bp in Exons 3 (%)*
bp in CAGE tags 4 (%)*
bpin PET 5 (%)*
bp in TF 6 (%)*
Total  Bases in PT 7 (%)*
Bases in PT (ESTs included) 8 (%)*
Bases in Exons and Introns 9 (%)*
Bases with 5'RACE 10(%)*
Bases between PETs 11 (%)*
Total  Bases 12 (%)*
TOTAL (interrogated and uninterrogated)
49.
(5.9%)
151149
(0.5%)
24939
(0.1%)
(4.6%)
(8.4%)
(16.1%)
(59.2%)
(77.7%)
(65.5%)
(91.1%)
INTERROGATED
(9.8%)
116013
(0.8%)
19629
(9.3%)
(14.7%)
(24.1%)
(64.6%)
(80.0%)
(66.4%)
(92.6%)

26. PROCESSED TRANSCRIPTS (PT)
Table 1: Summary of Transcriptional
Coverage of ENCODE Regions.
PROCESSED TRANSCRIPTS (PT)
PRIMARY TRANSCRIPTS
Total Bases 1
Total Interro-gated Bases 2 %
bp in Exons 3 (%)*
bp in CAGE tags 4 (%)*
bpin PET 5 (%)*
bp in TF 6 (%)*
Total  Bases in PT 7 (%)*
Bases in PT (ESTs included) 8 (%)*
Bases in Exons and Introns 9 (%)*
Bases with 5'RACE 10(%)*
Bases between PETs 11 (%)*
Total  Bases 12 (%)*
TOTAL (interrogated and uninterrogated)
49.
(5.9%)
151149
(0.5%)
24939
(0.1%)
(4.6%)
(8.4%)
(16.1%)
(59.2%)
(77.7%)
(65.5%)
(91.1%)
INTERROGATED
(9.8%)
116013
(0.8%)
19629
(9.3%)
(14.7%)
(24.1%)
(64.6%)
(80.0%)
(66.4%)
(92.6%)

27. International Conference on Bioinformatics HKUST, Hong Kong 2007
Other recent studies
Many individual studies suggest unanticipated complexity of the transcriptional map of the human genome:
Kapranov et al. (2007) RNA onto tiling arrays, novel RNA classes, hundreds of thousands of novel sites of transcription
Peters et al. (2007) LongSage, evidence for thousands of novel transcripts
Roma et al. (2007) gene trap sequence tags in mouse embryonic stem cells, thousands of novel transcripts
Unneberg and Claverie (2007) interchromosomal transcript chimerism
Denoeud et al. (2007) RACEarrays. Doubling the number of annotated exons in protein coding transcripts, widespread transcript chimerism
November 9, 2018
International Conference on Bioinformatics HKUST, Hong Kong 2007

28. tiling arrays reveal many novel sites of transcription
TRANSCRIPTION MAP of HL-60 DEVELOPMENTAL TIME COURSE (data by Tom Gingeras, affymerix)
check if this is after induction with retinoic acid
November 9, 2018
International Conference on Bioinformatics HKUST, Hong Kong 2007

29. characteristics of unannotated transfrags
short: 78bp on average compared with 121 for exonic transfrags
very gc-rich: 56% vs 42% in the background of unannotated regions
lack splice sites
no matches to protein or domain databases
lack of selective constraints
HOWEVER:
reproducible across cell lines
support by independent evidence of transcription (mostly unspliced ESTs).
enriched for RNA structures.
November 9, 2018
International Conference on Bioinformatics HKUST, Hong Kong 2007

30. International Conference on Bioinformatics HKUST, Hong Kong 2007
Rozowsky et al, 2007
Novel tar/transfrags are associated to known genes by identifying novel tars that are co-expressed with known genes across 11 cell lines and conditions
November 9, 2018
International Conference on Bioinformatics HKUST, Hong Kong 2007

31. International Conference on Bioinformatics HKUST, Hong Kong 2007
Rozowsky et al., 2007
November 9, 2018
International Conference on Bioinformatics HKUST, Hong Kong 2007

32. International Conference on Bioinformatics HKUST, Hong Kong 2007
Denoeud et al, 2007
The ENCODE experiments
5’ RACE on 12 tissues
primers in internal exons of 399 protein coding loci
RACE products hybridized into genome tiling arrays
4573 race exons detected novel
Summary of the racearray experimens
November 9, 2018
International Conference on Bioinformatics HKUST, Hong Kong 2007

33. International Conference on Bioinformatics HKUST, Hong Kong 2007
5’ RACE from TMEM15 Gene (region Enr232) identifies
several tissue specific distal 5’ exons.
Target gene
November 9, 2018
International Conference on Bioinformatics HKUST, Hong Kong 2007

34. International Conference on Bioinformatics HKUST, Hong Kong 2007
11/9/2018
International Conference on Bioinformatics HKUST, Hong Kong 2007

35. more than 30% of RACEfrags more than 3Mb away from the index exon
November 9, 2018
International Conference on Bioinformatics HKUST, Hong Kong 2007

36. distal RACEfrags are associated to independently predictes sites of transcription initiation

37. cloning and sequencing of RACEarray products
November 9, 2018
International Conference on Bioinformatics HKUST, Hong Kong 2007

38. cloning and sequencing of RACEarray products
almost 30% of the sequenced products incorporate exons from upstream genes in chimeric structures
International Conference on Bioinformatics HKUST, Hong Kong 2007

39. International Conference on Bioinformatics HKUST, Hong Kong 2007
RACEarrays: an strategy for normalization of RACE libraries, and exhaustive identification of alternative transcripts
November 9, 2018
International Conference on Bioinformatics HKUST, Hong Kong 2007

40. Array based normalization of RACE libraries
If we select 40 clones at random from the RACE reaction, the probability of selecting a clone from the less abundant form is (assuming a multinomial distribution)
However, if the transcript forms could be segregated by RT-PCR, then by selecting again 40 random clones, 10 from each RT-PCR, the probability of selecting the less abundant form is now, 0.6
November 9, 2018
International Conference on Bioinformatics HKUST, Hong Kong 2007

41. RT-PCR cloning and sequencing pilot (Kourosh Salehi-Ashtiani, DFCI)*
24 novel RACEfrags tested by RT-PCR, including 6 cases previously confirmed in Denoeud et al. (2007)
Positive RT-PCR cloned, and 32 randomly selected clones sequenced.
RESULTS
14 positive RT-PCR, 13 confirmed by sequencing.
42 novel transcript variants. Compared with the 52 previously know for the RT-PCR positive loci.
Nearly all canonical splice boundaries
Genomic extensions from 2.5 to 145Kb *
Difficulties in obtaining sequences for long cDNAs (which correlate with long genomic extensions)--but even with previously verified cases.
Problems with RACEfrag assigntation to loci

42. International Conference on Bioinformatics HKUST, Hong Kong 2007
A very efficient strategy for targeted large scale transcript discovery
RACEarray normalization 448 atempted clone sequences  42 novel transcripts
1 novel transcript per 10 clones sequenced.
Carnici et al. (Genome Research 2003, 13: ) 1,989,385 ESTs  70,214 transcripts (mouse)
1 transcript after 30 sequenced ESTs. (and the majority of transcripts already known)
November 9, 2018
International Conference on Bioinformatics HKUST, Hong Kong 2007

43. International Conference on Bioinformatics HKUST, Hong Kong 2007
CONCLUSIONS
there is substantial amount of transcription which does not appear to be associated to protein coding loci
only a fraction of the transcript diversity of protein coding loci appears to have been surveyed so far.
in particular, protein coding loci appear to have tissue specific distal alternative transcriptional start sites
RACEarrays are an effective normalization strategy for identifciation of rear transcripts
ENCODE transcriptional landscape: network of overlapping coding and non-coding transcripts, resulting in a continuum of transcription (more than 90% of the ENCODE regions are transcribed in at least one strand)
November 9, 2018
International Conference on Bioinformatics HKUST, Hong Kong 2007

44. PROVING THE FUNCTIONALITY OF NOVEL TRANSCRIPTS
November 9, 2018
International Conference on Bioinformatics HKUST, Hong Kong 2007

45. The GENCODE annotation
487 loci transcripts
53 non-coding loci. 137 transcripts
434 protein coding loci.
1097 coding transcripts
1374 non-coding transcripts
5.7 transcripts per protein coding locus
2.5 coding transcripts per locus
1.7 proteins per locus
November 9, 2018
International Conference on Bioinformatics HKUST, Hong Kong 2007

46. International Conference on Bioinformatics HKUST, Hong Kong 2007
the combined analysis of BioSapiens, Kellis and Goldman identified 184 annotated protein coding transcripts which challenged (from the structural, functional and/or evolutionary standpoint) our current view of proteins.
Footnote: removing these 184 proteins from the set of 738 GENCODE proteins, will leave 554 proteins for 434 loci; barely 1,3 proteins per locus
November 9, 2018
International Conference on Bioinformatics HKUST, Hong Kong 2007

47. Structural Effects of Pepsinogen C Alternative
Splice Variant
Locus RP11-298J23.1 codes for pepsinogen C. The structure of pepsinogen C is 1htrA.
Isoform -003 is missing 80 residues with respect to pepsinogen C. Here the missing section of -003 is in light green.
The missing section in this isoform would remove the core from both subdomains of the structure. Both the N-terminal sub-domain (on the left) and the C-terminal sub-domain would have to refold.
This is the view from above looking down into the active cleft of the proteinase. Active site aspartates are shown in ball and chain. One of the two active site residues is in the missing section.
The symmetry apparent in this isoform suggests that although it will have to refold it may very well be able to reform into a single subdomain.
Michael Tress & Alfonso Valencia
CNB, Madrid

48. Expression levels alternative vs constitutive
Q-PCR in three cell lines:
SKNAS
GM06990
HelaS3
November 9, 2018
International Conference on Bioinformatics HKUST, Hong Kong 2007

49. Polysomal association alternative vs constitutive
November 9, 2018
International Conference on Bioinformatics HKUST, Hong Kong 2007

50. ACKNOWLEDGEMNTS ENCODE GT GROUP Jan Korbel (Yale)
Julien Lagarde (IMIM)
Jeff Long (Affx)
Todd Lowe (UCSC)
G. Madhavan (Affx)
Anton Nekrutenko (Penn State)
David Nix (Affx)
Jakob Pedersen (UCSC)
Alex Reymond Geneva)
Joel Rozowsky (Yale)
Yijun Runan (GIS)
Albin Sandelin (RIKEN)
Mike Snyder (Yale)
Peter F. Stadler (U. Vienna)
Kevin Struhl (Harvard)
Hari Tammana (Affx)
Scott Tennenbaun (SUNY, Albany)
Chia Lin Wei (GIS)
Matt Weirauch (UCSC)
Deyou Zheng (Yale)
Addam Frankish(Sanger)
Tom Gingeras (Affymetrix)
Roderic Guigó (CRG)
ENCODE GT GROUP
Stilyanos Antonarakis (Geneva)
Robert Baertsch (UCSC)
Ian Bell (Affx)
Ewan Birney (EBI)
Robert Castelo (IMIM)
Jill Cheng (Affx)
Evelyn Cheung (Affx)
Hiram Clawson (UCSC)
France Denoeud (IMIM)
Sam Deustch(Geneva)
Sujit Dike (Affymetrix)
Jorg Drenkow (Affymetrix)
Olof.Emanuelsson (Yale)
Paul Flicek (Sanger)
Mark Gerstein (Yale)
Srinka Ghosh (Affx)
Jenn Harrow (Sanger)
Greg Helt (Afffx)
Ivo Hofacker (U. Vienna)
Tim Hubbard (Sanger)
Phil Kapranov (Affx)
Damian Keefe (EBI)
Before we get into the data it is important to acknowledge the efforts of those whose efforts will be represented in this data.
-AFFX group
-Kevin’ group
-Scott
-Brad

51. International Conference on Bioinformatics HKUST, Hong Kong 2007
November 9, 2018
International Conference on Bioinformatics HKUST, Hong Kong 2007

52. International Conference on Bioinformatics HKUST, Hong Kong 2007
CENTER FOR GENOMIC REGULATION, PRBB,
BARCELONA
November 9, 2018
International Conference on Bioinformatics HKUST, Hong Kong 2007