1 Q1-Q3 results. 2 RF lengths 3 Filtered RF length distribution

1

Q1-Q3 results

2

RF lengths

3

Filtered RF length distribution

Min Max AverageQ1 28 1462 78.2Q2 20 372 42.3Q3 28 826 77.2

4

Q1 filtered RF length distribution

5


6


7

RF position when compared to genes and exons

8

Q1-Q2-Q3: Projected filtered RF distribution(internal=overlap target gene ; projection done by pool)

39% internal

46% exonic

54% intronic

61% external

71% genic

79% exonic22% overlap most 5' ex. of tr.

21% intronic29% intergenic

86% internal

88% exonic

12% intronic

14% external

78% genic



21% internal

47% exonic

53% intronic

79% external

78% genic



Q1 Q3Q2

® chimeric transcripts?

9

Why are Q3 RF mostly external (79%) ?

Existence of a systematic swap between certain pairs of pools?

For each RF we have computed the overlap with all genes of Q3 and then compared:

RF pool withRF overlapping gene pool

10

RF overlapping gene pool

Pool 1

1

8

2

12

6

5

Pool 2

2

13

15

1

5

11

Pool 3

3

16

11

12

2

8

Pool 8

8

11

1

13

16

4

6

7

Pool 9

9

6

11

1

10

13

11

Q3 RF compared to Q3 genes

Pool1 73 31 104 70.192 136 31 167 81.443 30 26 56 53.574 25 0 25 1005 26 12 38 68.426 41 19 60 68.337 49 29 78 62.828 10 17 27 37.049 16 42 58 27.5910 42 35 77 54.5511 47 16 63 74.612 58 7 65 89.2313 32 38 70 45.7114 40 36 76 52.6315 26 25 51 50.9816 56 20 76 73.68

Nb RF with at least one overlapping gene

in its pool

Nb RF with no overlapping gene

in its pool

Nb RF with at least one overlapping

gene

Proportion of RF with at least one overlapping gene

in its pool

® Q3 RF are more overlapping genes of their pool than genes of other pools (no clear pool swap)

12

6 genes of Q3 are in two different pools

® generates pool unspecific RF

55 CHODL,NM_024944, 6,10, Same gene

47 NM_021090,MTMR3, 15,16, Same gene

27 12,3, Same gene

16 14,15, Same gene

12 ZNF74,NM_003426, 10,13, Same gene

2 NM_005446,P2RXL1, 8,9, Same gene

1 14,16, Not the same gene

Nb RF with two Q3

overlapping genes

Q3 genes overlapping the RF

Pools of these genes

Are these genes the same one?

NM_015367,BCL2L13,

RP5-1042K10.2,NM_0157

05,

NM_001013676,MTMR3,

Problematic pools are:● 6-10-13● 8-9● 12-3● 14-15-16

13

Q3 RF overlapping Q3 genes

Pool Nb assigned RF1 261 104 39.85 73 27.972 260 167 64.23 136 52.313 164 56 34.15 30 18.294 172 25 14.53 25 14.535 354 38 10.73 26 7.346 229 60 26.2 41 17.97 222 78 35.14 49 22.078 183 27 14.75 10 5.469 212 58 27.36 16 7.5510 184 77 41.85 42 22.8311 256 63 24.61 47 18.3612 173 65 37.57 58 33.5313 169 70 41.42 32 18.9314 209 76 36.36 40 19.1415 104 51 49.04 26 2516 259 76 29.34 56 21.62

Total 3411 1091 32.95 707 20.8

RF with at least one overlapping gene

RF overlapping its assigned gene

14

Position of Q3 filtered projected RF when filtering RF shorter than a threshold

All >30 bp > 60 bp > 90 bp

Internal 21 21.04 29.1 34.86

Exonic 47 52.08 55.29 54.87

Intronic 53 47.92 44.71 42.75

External 79 78.96 70.9 65.14

Genic 78 79.09 83.12 85.3

Exonic 69 70.44 73.03 73.82

Intronic 31 29.56 26.97 26.18

Intergenic 22 20.91 16.88 14.7

15

Q2 vs Encode 2005

86% internal

88% exonic

12% intronic

14% external

78% genic



68% internal

49% exonic

51% intronic

32% external

80% genic



Q2 Encode 2005

433 out of 1577 (27.5%)are novel projected RF

2859 out of 4951 (57.8%)are novel projected RF

16

Distance of RF to closest gene within pool (target gene)

17

Q1, Q3: proportion of RF > 3Mb away from target gene

Q1:

983/10387= 9.4% filtered RF > 3Mb away from target gene

Q3:

1789/3411 = 52.4% RF > 3Mb away from target gene

839/1249 = 67.2% external non exonic RF > 3Mb away from target gene

18

Min Max Average0 10.7Mb 556kb

19

Min Max Average0 32Mb 5.1Mb

20

Min Max Average0 32Mb 6.8Mb

21

Proportion of Q3 filtered RF >3 Mb away from target gene

All

Projected RF 3411> 3Mb away 1789Proportion 52.4

>30bp


>60bp


>90bp


22

23

24

25

Do external exonic projected RF overlap most 5' exons of transcripts more than

other exons of transcripts ?

26

Proportion of external exonic projected RF overlapping most 5' exons of transcripts

Real: 22.3% (63)

Same strand: 68.3% (43)

Opposite strand: 31.7% (20)

Random: 19.8% (56)



Real: 23.0% (335)



Random: 15.8% (230)



Real: 46.5% (206)



Random: 30.7% (136)



Q1 Q3Q2

27

Does the most 5' RF of a particular gene and a particular tissue overlap most 5'

exons of transcripts more than other RF?

All filtered assignable RF

Total RF External RF External exonic RF

Q1 10387 821 504 61.39 282 55.95 63 22.34 43 68.25

Q2 17477 4561 645 14.14 443 68.68 206 46.5 94 45.63

Q3 3796 3411 2704 79.27 1455 53.81 335 23.02 208 62.09

Encode 2005 24361 4951 1605 32.42 901 56.14 207 22.97 101 48.79

Most 5' filtered assignable RF for each gene and each tissue

Total RF External RF External exonic RF

Q1 575 134 92 68.66 57 61.96 16 28.07 7 43.75

Q2 416 174 111 63.79 102 91.89 49 48.04 47 95.92

Q3 87 78 74 94.87 31 41.89 7 22.58 3 42.86

Encode 2005

Projected RF

Overlapping a most 5' exon of a transcript

Overlapping a most 5' exon of a

transcript on the same strand

Projected RF

Overlapping a most 5' exon of a transcript

Overlapping a most 5' exon of a

transcript on the same strand

28

Correlation of most 5' RF with CAGE tags

29

Correlation of most 5' racefrags with cage tags

5 prime most rf Random1 Random2 Random3 Random4 Random5 Random6 Random7 Random8 Random9 Random1050 7 1 1 2.5 0 0 3.5 0 1 1 1

250 22 3.5 7 7 7 3.5 7 1 7 1 8500 29 5.5 11.5 10.5 10.5 4.5 10.5 4.5 9 8 12.5

1000 45 10.5 18.5 17 14 9 15 10.5 12.5 12.5 18.5

Most 5'RF

5'

30

Pool unspecific RF

31

Pool unspecific unique RF(USPP-filtered)

Most pool unspecific unique RF are:

Q1: internal exonic (72%)

Q2: internal exonic (87%)

Q3: external (91%) (of which 63% are exonic)

Q1 Q2 Q3

1736 94.40% 3032 62.70% 3217 93.00%

102 5.60% 1804 37.30% 243 7.00%

Unique RF in 1 pool

Unique RF in more than 2

pools

20 unique RF are in more than 4 pools

32

Pool unspecific unique Q3 RF(filtered)

- Hits found by blat.- Need to be done again using our highlighted probe simulator.

Unique RF Total

20 14

In 1 pool 20 5

With multiple >80% Id hits in chr21 and 22

In more than 4 pools

33

1 2 3 4 5 6 80

500

1000

1500

2000

2500

3000

3500

4000 Q31 2

0

200

400

600

800

1000

1200

1400

1600

1800

2000 Q1

1 2 3 4 5 6 7 8 9 100

500

1000

1500

2000

2500

3000

3500 Q2

Q1-Q3: Number of pools a unique RF appears in (unfiltered/filtered)

34

Pool-unspecific RFs in Q3Possibly due to...

1 - cross-hybridization?is there a correlation between number of pools a RF is found in and the number of non-unique probes it overlaps?

noby the way 135,380 / 2,191,331 (6%) of probes from chr21/22 chip have multiple perfect matches in genome

35


2 - high GC content?

Number of pools the RACEfrag appears in Number of RACEfrags Average GC content

1 3413 52%2 191 55%3 41 55%4 12 57%5 4 56%6 3 57%7 0 N/A8 1 42%

-> Answer: NO!

36


3 - mis-priming on unknown transcripts of chr21 or chr22 (missed by the simulator)?

4 - genuine chimeric transcripts?

5 - Pooling errorsthe same gene is present in >1 pool because it has 2 different identifiers (UCSC known genes / RefSeq nomenclature discrepancy

we found a few cases like this, not sure yet how widespread it is (systematic survey to come)

37

Genes present in several pools

5 genes present in 2 pools:

RP5-1042K10.2,NM_015705 (pools 14,15)

CHODL,NM_024944 (pools 6,10)

NM_005446,P2RXL1 (pools 8,9)

ZNF74,NM_003426 (pools 10,13)

NM_015367,BCL2L13 (pools 12,3)

1 gene present in 3 pools:

NM_021090,NM_001013676,MTMR3 (pools 15,14,16)

Eliminate RF present in these pairs/triplets of pools (problematic pool RF)

38

Effect of filtering problematic pool RF on Q3 pool unspecificity

2 3 4 5 6 80

20

40

60

80

100

120

140

160

180

200

Initial RFFiltering problematic pool RF

# pools

# R

F

-48

-1Genes present in several pools do not explain all pool unspecific RF of Q3

39

Distribution of pool specific and pool unspecific unique Q3 RF

Total Q3 genic Q3 exonic Genic Exonic

3217 977 30.37 504 15.67 2668 82.93 1664 51.73

243 52 21.4 37 15.23 206 84.77 154 63.37

194 34 17.53 21 10.82 158 81.44 116 59.79

Pool specific unique Q3 RF

Pool unspecific

unique Q3 RFSame without problematic pool Q3 RF

Pool unspecific Q3 RF are more:● external to Q3 genes,● exonic,

compared to pool specific Q3 RF

40

Pool unspecific RF

Unassigned

Overlap target gene

Overlap a Q3 gene

Overlap a gene

Do not overlap a gene

Pool specific and unspecific RFregarding gene overlap

Total Unassigned Overlap target gene Overlap a Q3 gene Overlap a gene

Pool specific RF 3217 324 10.07 661 20.55 234 7.27 1498 46.57 500 15.54

480 55 11.46 29 6.04 39 8.13 269 56.04 88 18.33


Pool unspecific RF

Pool specific RF

Unassigned

Overlap target gene

Overlap a Q3 gene

Overlap a gene


Pool specific RF overlap their target gene more than pool unspecific RF

41

Two other criteria for comparing Q3 pool specific and unspecific RF

Total

Pool specific RF 3217 1732 53.84 956 55.2

480 308 64.17 172 55.84

Overlap a gene other than target

Overlap a gene other than target

but in same orientation

Pool unspecific RF

Overlap with gene in same orientation as target gene

Min Max Avg

0 32Mb 4.9Mb

191 28Mb 7Mb

Pool specific RF

Pool unspecific RF

Distance to target gene

Pool unspecific RF are more distant to their target gene

Pool unspecific RF behave similarly as pool specific RF

42

6 genes of Q3 are in two different pools

® generates pool unspecific RF

55 CHODL,NM_024944, 6,10, Same gene

47 NM_021090,MTMR3, 15,16, Same gene

27 12,3, Same gene

16 14,15, Same gene

12 ZNF74,NM_003426, 10,13, Same gene

2 NM_005446,P2RXL1, 8,9, Same gene

1 14,16, Not the same gene

Nb RF with two Q3

overlapping genes

Q3 genes overlapping the RF

Pools of these genes

Are these genes the same one?

NM_015367,BCL2L13,

RP5-1042K10.2,NM_0157

05,

NM_001013676,MTMR3,

Problematic pools are:● 6-10-13● 8-9● 12-3● 14-15-16

43

Impact of index exon position on RF coverage

44

45

USPP filter results

46

Initial set of RF

Exonic Intronic Intergenic Total

Pool1 3567 1192 1043 5802

Pool1 61.48 20.54 17.98 100.00%

Pool2 4100 1196 336 5632

Pool2 72.8 21.24 5.97 100.00%

Total 7667 2388 1379 11434Total 67.05 20.89 12.06 100.00%

Filtered RF

Exonic Intronic Intergenic Total

Pool1 3023 1038 747 4808

Pool1 62.87 21.59 15.54 100

Pool2 4100 1143 336 5579

Pool2 73.49 20.49 6.02 100

Total 7123 2181 1083 10387

Total 68.58 21 10.43 100Removed 7.1 8.67 21.46 9.16

The USPP filter removes more intergenicthan genic RF

Q1: proportion of exonic, intronic and intergenic RF before and after USPP-based filtering

47

Initial set of RF

Genic <50kb <100kb <200kb <500kb <1Mb >1Mb >5Mb Non genic TotalPool1 3618 762 777 1126 1148 1176 1008 295 2184 5802Pool1 34.89 35.58 51.56 52.56 53.85 46.15 13.51 100Pool2 4026 764 773 787 900 935 671 196 1606 5632Pool2 47.57 48.13 49 56.04 58.22 41.78 12.2 100Total 7644 1526 1550 1913 2048 2111 1679 491 3790 11434Total 40.26 40.9 50.47 54.04 55.7 44.3 12.96 100

Filtered RF

Genic <50kb <100kb <200kb <500kb <1Mb >1Mb >5Mb Non genic TotalPool1 3300 752 767 820 842 870 638 272 1508 4808Pool1 49.87 50.86 54.38 55.84 57.69 42.31 18.04 100Pool2 3973 764 773 787 900 935 671 196 1606 5579Pool2 47.57 48.13 49 56.04 58.22 41.78 12.2 100Total 7273 1516 1540 1607 1742 1805 1309 468 3114 10387Total 48.68 49.45 51.61 55.94 57.96 42.04 15.03 100Removed 4.85 0.66 0.65 16 14.94 14.5 22.04 4.68 17.84 9.16

The USPP filter removes more RF located:- from 100 to 200 kb - from 1 to 5 Mb to closest gene within pool

Q1: Distance of RF to closest gene within poolbefore and after the USPP-based filter

48

0 1 2

21629 21917 22316

Number of genes generating USPPs

Number of Experimental RFs overlapping USPPs 259 263 283

Number of genes with RFs overlapping USPPs 8 8 8

class 0 RFs removed by USPP filter 0: 0% 0: 0% 0: 0%

class 1 RFs removed by USPP filter 2: 0% 2: 0% 2: 0%

class 3 RFs removed by USPP filter 185: 18.4% 185: 18.4% 188: 18.7%

class 5 RFs removed by USPP filter 72: 0.5% 76: 0.5% 93: 0.6%

Number of RACE product vs. probe mismatches allowed for the probe to be considered positive in simulation

Number of Simulated Positive Probes (SPPs)Number of positive probes outside of target locus (Unspecific Simulated Positive Probes, USPPs)

6969 / 21629 7063 / 21917 7279 / 22316

19 / 41 23 / 41 34 / 41

Q2: Class 0, 1, 3, 5 RF removed by USPP-based filter (using 0, 1 and 2 Race/probe mismatches)

The USPP filter:- removes 37 times more 3' RF than 5' RF- is ~ independent of the number of RACE/probe mismatches

49

Proportion of RF and projected RF eliminated by the USPP-based filter

(projections made by pool)

Initial number of RF

Q1 11434 10387 9.16 785 734 6.5

Q2 17740 17477 1.48 1620 1577 2.65

Q3 4012 3796 5.38 3102 2933 5.45

ENCODE 2005 25605 24361 4.86 5104 4951 3

Number of RF after filter

Percentage of RF filtered

Initial number of projected RF

Number of projected RF after filter

Percentage of projected RF filtered

50

Proportion of RF and projected RF eliminated by the USPP-based filter

(projections made by pool)

Initial number of RF

ENCODE 2005 initial 35129 30688 12.64 9178 8569 6.64

38001 33426 12.04 9178 8671 5.52

25605 24361 4.86 5104 4951 3

Number of RF after filter

Percentage of RF filtered

Initial number of projected RF

Number of projected RF after filter

Percentage of projected RF filtered

ENCODE 2005 fragmented initial

ENCODE 2005 fragmented filtered by

France

51

Tissue specificity results

521 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47

0

100

200

300

400

500

600

700

800

900

1000

Q1: number of tissues a unique RF appears in (unfiltered/filtered)

531 2 3 4 5 6 7 8 9 10 11 12

0

500

1000

1500

2000

2500

3000

Q2: Number of tissues a unique RF appears in (unfiltered/filtered)

54

Generating RF from probes

55

Generating RF from probes

Initial Filtered Genic ExonicExp Q3 4012 3796 94.62 3322 82.8 2174 65.44

Sim Q3 (2 probes) 7448 7178 96.37 5786 77.69 3562 61.56Sim Q3 (3 probes) 4024 3806 94.58 3333 82.83 2191 65.74

56

Comparison between Encode 2005 and Q2

57

Intersection between Encode 2005 and Q2 RF sets

Total RF All Globally common All All All

25605 3020 1407 46.59 1096 615 56.11 1096 339 30.93 280 46 16.43

#RF in Q2 17740 17740 3579 20.17 807 561 69.52 807 460 57 64 33 51.56

RF corresponding to genes common to Encode 2005 and

Q2

RF corresponding to experiments common to

Encode 2005 and Q2

RF corresponding to 14 experiments common to

Encode 2005 and Q2

Novel RF corresponding to experiments common to

Encode 2005 and Q2

Common in corresponding

experiment


experiment and tissue


experiment

#RF in Encode 2005

58

Comparison between Q1 and Q3

59

Overlap between Q1 and Q3 RF assigned to genes common to Q1-Q3

TCP10L 1213 397 32.73 25 18 72

TPTE 588 302 51.36 53 30 56.6

C21orf13 998 632 63.33 47 30 63.83

C21orf59 1316 665 50.53 53 18 33.96

YWHAH 995 392 39.4 73 13 17.81

APP 1446 981 67.84 43 18 41.86

RFPL3S 1008 2 0.2 22 2 9.09

TBX1 232 121 52.16 15 8 53.33

DSCR2 948 357 37.66 45 18 40

TSPO 645 205 31.78 30 8 26.67

All 9389 4054 43.18 406 163 40.15

Q1 RF of this gene

Overlapped by a Q3 RF assigned to this

geneQ3 RF of this gene

Overlapped by a Q1 RF assigned to this

gene

40% overlap between Q1 and Q3 RF assigned to genes common to both experiments ® problem in gene assignment?

Documents

1 Q1-Q3 results. 2 RF lengths 3 Filtered RF length distribution