Presentation by J Lichtenberg at BOSC2012 - Discovery of motif-based regulatory signatures in NextGen Sequencing Experiments

1. Discovery of motif-based
regulatory signatures in NextGen
Sequencing Experiments
http://code.google.com/p/nextgen-signatures
GNU General Public License, version 3.0 (GPLv3)
Jens Lichtenberg
Hematopoiesis Section, Genetics and Molecular Biology Branch
National Human Genome Research Institute, National Institutes of Health

2. Motivation
● Large variety of omics approaches that produce
sequencing data
● Common threads in the
Methylation
evaluation process Seq
● Few approaches exist RNA Seq ChIP Seq
that attempt the large
Comprehensive
scale analysis of omics Analysis
data Protein Seq Histone Seq
● Direct correlation of Systems
Biology Insights
multiple omics data into
actual biological insights

3. Requirements
● General
○ Quantification of sequencing data requires dynamic
pipeline allowing for frequent adjustments
○ Close interaction between bench and analysis
personnel
● Specific
○ Quantitative analysis
○ Functional analysis
○ Regulatory analysis
○ Visualizations

4. General Analysis Approach

5. Hematopoietic Stem Cell
Differentiation in Mouse
Microarray Data curated in BloodExpress
RNA Seq Data
Methylation Seq Data
ChIP Seq Data (EKLF)
Histone Seq Data

6. Methylation Seq
Peak Calling Expression Correlation
Motif Discovery Occupancy Validation
Transcription Occupied Sites Number Exp. Z-Score P-Value
Factors Overlapping Overlapping
ERG 36166 966 1983 -20.80 2.16e-96
FLI1 19601 348 1075 -21.32 3.70e-101
GATA2 9234 278 507 -9.87 2.81e-23
GFI1B 8853 235 486 -11.04 1.23e-28
...
RUNX1 5269 97 290 -11.11 5.61e-29
SCL 7096 146 389 -12.26 7.42e-35

7. ChIP Seq
Peak Calling Methylation Correlation
ERY (Meth.) MEP (Meth.)
Total 1187 587
Dist. Prom. 210 102
Prox. Prom. 29 21
Downstream 345 207
RefSeq 983 513
Functional Analysis Motif Discovery
● EKLF control in MEP can be found in the first intron (Siatecka and Bieker, Blood, 2011)
● During erythropoiesis EKLF is restricted to hematopoietic organs (Siatecka and Bieker,
Blood, 2011)
● Down-regulation of EKLF expression in MEP cells leads megakaryopoiesis (Siatecka
and Bieker, Blood, 2011)

8. Histone Seq
Peak Calling EKLF/Methylation Correlation
Functional Analysis Motif Discovery
MEME (OOPS) MEME (ZOOPS)
TomTom Lookup: TomTom Lookup:
● THI2, ZincFinger ● THI2, ZincFinger
● NKx2-5, Homeobox ● NKx2-3, Homeobox
● NKx2-6, Homeobox ● NKx2-5, Homeobox
● NKx2-6, Homeobox
● NKx3-1, Homeobox

9. RNA Seq
Peak Calling Functional Analysis
MEG
Pathway Name ERY, MEP, MEG MEG, MEP ERY, MEG ERY, MEP
241
ERK/MAPK Sig. 1.83E-09 4.47E-16 5.01E-10
IGF-1 Sig. 1.04E-15 1.25E-10
47 1308
3338 MolMech. 3.72E-10 1.59E-22 1.13E-10 3.72E-10
Cancer
216 966 2408 ...
PI3K/AKT Sig. 3.22E-20 2.84E-24 6.24E-18 1.33E-15
ERY MEP
mRNA Differentiation Motif Discovery
Increase Decrease
MEP -> MEG 1238 7323
MEP -> ERY 1198 9307

10. Comprehensive Approach
Current Status
● Perl Framework
○ Commonly used applications and repositories
● Next-Generation Sequencing
○ Read Mapping
■ UCSC Genomic Data
○ Peak Calling/Partitioning
■ UCSC Genomic Data
○ Transcript Quantification
■ UCSC/Ensembl Genomic Data
● Functional Genomics ● Regulatory Genomics
○ Expression Correlation ○ Enumerative motif discovery
■ BloodExpress Database ■ Transfac/Jaspar
○ Pathway Analysis Database
■ KEGG/IPA ○ Occupancy validation
○ Ontology Analysis ■ Literature specific data
■ GO/IPA sets

11. Future Issues
Data
● Complete case study for Protein Seq
Implementation
● Complete implementation of all analysis facets
● Transition Perl framework to C++ architecture
● Parallelize software architecture for higher
performance/throughput
Support
● Update web-interface and documentation to allow
unassisted data analysis

12. Conclusions and Availability
● A comprehensive approach is possible
● Meaningful results can be extracted using the approach
● Regulatory genomics can be used as a suitable post-
processing analysis
● Comprehensive hematopoiesis study is feasible
● http://code.google.com/p/nextgen-signatures (GNU
General Public License, version 3.0)

13. Acknowledgements
NHGRI - GMBB - Hematopoiesis Section
David Bodine and Amber Hogart
NHGRI Intramural Training Program