1. Homology Based Analysis of the Human/Mouse lncRNome
Cédric Notredame
Giovanni Bussotti
Comparative Bioinformics lab
CRG

2. Part 1: GENCODE v10 lncRNA screening vs human and mouse genomes
Strategy: PipeR one2many homolog assignment
Template:
PipeR Parameters:
Blast
- Freyhult parametrization
- Lower case masking
- Low complexity masking
Exonerate
- est2genome model
- 70% coverage required
- seed extension 2X
(the span of the genomic size of the query on both sides)
genes
10840
transcripts
17547
exons
58857
sum of mature transcript length (nt)
16·927·027
real coverage (nt)
13·083·478
non overlapping loci
7428

3. PipeR: a pipeline for mapping lncRNAs
blast-exonerate based framework to map lncRNAs
against target genomes
algorithm used:
chromosome
2 Blast hits
mapping
extension
Exonerate
spliced transcript
lncRNA

4. PipeR: lncRNA Homology Mapping
GENECODE lncRNAs Vs
Complete Genomes
PipeR: lncRNA Homology Mapping
Anchor points: ENCODE vs Mouse with tuned Blast
Extension: Exonerate
Filtering: Id and Coverage
Validation of the GFF annotation
Overlap with Annotation
Overlap with Cufflink Models
RPKM on target genome
Further Mapping Parameter Space Exploration using Experimental Evidences
GFF File
Notredame, Bussotti

5. Mapping overview Query species Multiple Homologues Target species
Gene A
Gene B
Query species
Transcript 1
Transcript 3
Transcript 2
Multiple Homologues
Blast/Exonerate
failed
Homolog 1
Homolog 2
Homolog 3
Homolog 4
Best reciprocal
Conserved exon number
High repeat coverage
Overlap with protein
Target species

6. GENCODEv10 vs human genome
mapped transcripts out of 17547
many lncRNAs found in multiple copies
(lncRNA families)
- found homologs
corresponding to exons
Annotations of discovered homologs
are readily available

7. Homolog repeat coverage
About the 10% of all our homolog
predictions are fully covered by repeats

8. Homolog repeat coverage
We could sub-group the homologs
in 3 set according with the repeat
coverage:
<= 20
< = 80
< = 100

9. Mapping statistics HUMAN <= 20% <= 80% <= 100%
genV10 mapped genes
6088
10425
10698
genV10 mapped transcripts
9318
16856
17327
Total homologs
35399
102250
144566
Homologs whose exons overlap protein coding exons (same strand)
3621
5076
8988
HUMAN

10. GENCODEv10 vs mouse genome
mapped 3190 transcripts out of 17547
representing 2249 human genes
many lncRNAs found in multiple copies
(lncRNA families)
- found homologs
corresponding to exons
Annotations of discovered homologs
are readily available

11. Exon Number Conservation
Human/Mouse
Exon Number Conservation
Difference between the number of exons in the human transcripts and in the mouse homologs
“0” means that the exon number is the same
Negative bins indicate mouse homologs having more exons than the human query
1160 GENCODE v10 transcripts find at least 1 homolog in mouse with the same exon number
human < mouse
human > mouse

12. Homolog repeat coverage
We could sub-group the homologs
in 3 set according with the repeat
coverage:
<= 20
< = 80
< = 100

13. Mapping statistics MOUSE <= 20% <= 80% <= 100%
Reciprocal homologs
genV10 mapped genes
1867
2172
2249
1445
genV10 mapped transcripts
2586
3076
3190
1966
Total homologs
6108
11141
14936
Homologs whose exons overlap protein coding exons (same strand)
1611
2290
3177
497
Homologs with conserved number of exons
1534
2407
2958
689
MOUSE
Best Candidates: There are 148 transcripts that have < 20% repeat coverage,
conserved exon structure, do not overlap protein coding exons and are best reciprocal homologs
with the human queries

14. PipeR: lncRNA Homology Mapping
GENECODE lncRNAs Vs
Complete Genomes
PipeR: lncRNA Homology Mapping
Anchor points: ENCODE vs Mouse with tuned Blast
Extension: Exonerate
Filtering: Id and Coverage
Validation of the GFF annotation
Overlap with Annotation
Overlap with Cufflink Models
RPKM on target genome
Further Mapping Parameter Space Exploration using Experimental Evidences
GFF File
Notredame, Bussotti

15. BlastR vs The World

16. BlastR vs The World

17. blastnOpt (12487) a) blastn (8749) all (7492) blastr (12093) b) c)
Figure 2: Exon read support.
Venn-diagram indicating the number of exon detected
by different methods (numbers in parentesis) and their intersection (transcripts annotated identically by the three methods).
Average amount of reads per exons
Percent of reads covered by at least one exon
all
(7492)
blastr
(12093) b) c)

18. Part 2: Ensembl.v65 lncRNAs screening vs human and mouse genomes
Strategy: PipeR one2many homolog assignment
Template:
PipeR Parameters:
Blast
- Freyhult parametrization
- Lower case masking
- Low complexity masking
Exonerate
- est2genome model
- 70% coverage required
- seed extension 2X
(the span of the genomic size of the query on both sides)
genes
3845
transcripts
5669
exons
18353
sum of mature transcript length (nt)
real coverage (nt)
non overlapping loci
2790

19. Ensembl.v65 vs human genome
mapped 1187 transcripts out of 5669
many lncRNAs found in multiple copies
(lncRNA families)
- found homologs
corresponding to exons
Annotations of discovered homologs
are readily available

20. Ensembl.v65 vs mouse genome
mapped 5622 transcripts out of 5669
many lncRNAs found in multiple copies
(lncRNA families)
- found homologs
corresponding to exons
Annotations of discovered homologs
are readily available

21. Exon Number Conservation
Mouse/Human
Exon Number Conservation
Difference between the number of exons in the mouse transcripts and in the human homologs
“0” means that the exon number is the same
Negative bins indicate human homologs having more exons than the mouse query
481 Ensemblv65 transcripts find at least 1 homolog in human with the same exon number
mouse < human
mouse > human

22. Homolog repeat coverage
Not observed a peak of homolog
predictions fully covered by repeats

23. Ensemble.65 and GENCODEv10 repeat coverage
Input lncRNA datasets have similar repeat distributions

24. Mapping statistics HUMAN MOUSE 879 1187 3815 13193 5622 3642 41005
ensV65 mapped genes
879
ensV65 mapped transcripts
1187
Total homologs
13193
Homologs whose exons overlap protein coding exons (same strand)
3642
Homologs whose exons do not overlap any gencode v10 element (same strand)
6085
Homologs with conserved number of exons
4925
ensV65 mapped genes
3815
ensV65 mapped transcripts
5622
Total homologs
41005
Homologs whose exons overlap protein coding exons (same strand)
10086
MOUSE
HUMAN

25. Part 3: GENCODE v10 lncRNA coding potential check
Strategies: 1) GeneId ORF score comparison between mRNAs and lncRNAs
2) BlastX against human proteins (ensembl 65)
3) Overlap with protein coding gene exon annotations (gencodeV10)
4) PipeR filtering routines

26. 1) ORF scores as returned by GeneID
2) blastX against human proteins indicates that GENCODE v10 lncRNAs match proteins
Parameters:
seg low complexity filtering, repeat filtering , evalue 10e-10, search just the plus strand.
Human Ensembl 65 protein set

27. 3)
Checked the overlap between GENCODE v10 lncRNA exons
and GENCODE v10 protein coding exons.
- Found 846 lncRNA having at least one exon overlapping with a protein coding gene exon
Example 1
Example 2

28. 4) Extensive filtering
7813 GENCODE v10 transcripts passed *ALL* PipeR filtering routines
Filtering rules:
- overlap with protein coding exons
- geneID ORF score similar to the ones of mRNA
- blastX to uniprot database (50% redundancy)
- blastX to nr database
- rpsBlast to pfam domain families
- blast against Rfam