Presented at the NGS Tech and Applications Congress: USA. To find out more, visit:
www.global-engage.com
Masayoshi Itoh is a Senior Scientist at the RIKEN Center for Life Science Technologies (CLST) and Coordinator of the RIKEN Preventive Medicine and Diagnostics Innovation Program (PMI). In this presentation Masayoshi introduces CAP trapper technologies and presents the findings of the FANTOM5 project.
Plasmapheresis - Dr. E. Muralinath - Kalyan . C.pptx
CAP Trapper Technologies and Applications, CAP Analysis of Gene Expression (CAGE) and FANTOM5 Project
1. Cap trapper technologies and applications,
Cap Analysis of Gene Expression (CAGE)
and FANTOM5 project
Masayoshi Itoh
Division of Genomic Technologies (DGT), RIKEN Center for Life
Science Technologies (CLST)
RIKEN Preventive Medicine and Diagnostics Innovation Program (PMI)
2. Transcriptome
• Transcriptome Analysis Technologies by NGS
• Digital Gene Expression
• 3’ terminal sequencing and quantification
• RNA-seq
• Whole transcribed region sequencing by fragmentation
• CAGE and TSS-seq
• 5’ terminal sequencing and quantification
• CAGE (Cap Analysis of Gene Expression) is based on Cap Trapper Technology
3. Cap Trapper Technology
• Cap structure-specific enrichment technology
• Ribose diol-specific biotinylation after reverse
transcription
• Single strand RNA digestion to remove 3’ end
biotinylation
• Recovery of cDNA reached 5’ end of RNA
• Originally developped by Piero Carninci in 1994
O
O OH
P-O O
O
O
O OH
P-O O
O
O
O OH
P-O O
O
A/G/U/C
A/G/U/C
A/G/U/C
OPOPOPO
O
OH OH
O- O- O-
O O O
7mG/m3G/mP
O
O OH
P-O O
O
O
OH OH
A/G/U/C
A/G/U/C
matured mRNA
cap structure
target
target
4. Cap Trapper Technology
• Cap Trapper Technology
• Cap structure specific enrichment
• Enable to prepare cDNA reached 5’ end of RNA by RT reaction
• Only from capped RNA transcribed by RNA polymerase II
• Different from another full length cDNA preparation
• Template-switching can prepare cDNA reached 5’ end of RNA from any RNA
templates
• Precise 5’ end of matured RNA = Precise Transcription Start Sites (TSSs)
• Independent on base of cap structure
• Enable to prepare cDNA from unknown cap base carrying RNA
5. Cap Trapper Technology
• Applied Technologies
• Full length cDNA cloning
• Only from matured polyA+ mRNA by oligo dT primer
• Achieved the FANTOM full length cDNA clone sets in FANTOM1•2
• Cap Analysis of Gene Expression (CAGE)
• Comprehensive analysis of 5’ end of RNA by random primer
• Precise TSS mapping on genome
• Revealed large number of ncRNA existance and sense-antisense transcriptions in FANTOM3
• Revealed gene regulation network in the time course of monocytic differentiation in FANTOM4
FANTOM1 FANTOM2
FANTOM3 FANTOM4
6. FANTOM5
• Expansion of FANTOM3•4 activity on
various cell lines, primary cell types,
cellular differentiation time courses, and
mouse developmental time courses
• CAGE Technology
• FANTOM3•4: cDNA was amplified, tagged,
concatenated, cloned and sequenced by
Sanger sequencers
• –> less quantitativeness for the regulation
network analysis
• FANTOM5: Adapted on HeliScope single
molecule sequencer to eliminate any
amplification, ligation, or other troublesome
steps
• –> enabled highly quantitative analysis
9. 9
Cell specific network models
Key transcription factors, Key motifs
Integrated transcript sequencing
• CAGE promoter map
• RNA-seq transcript map
• Short RNA processing map
Transcript discovery
• Better gene models
• New lncRNAs
• New insights on processing
• Promoter-centered expression map
1000 human samples types (500 mice) + time courses perturbations
Primary
cell
compendium
39635 TPM
13210 TPM
0
0
Expression
level
Expression
level
Median
0
Median
5067
947 samples
ACTB
GFAP
Alistair R. R. Forrest et al., Nature 507, 462 (2014)
10. 10
Astrocyte donor1
Astrocyte donor2
Astrocyte donor3
CD14+ donor1
CD14+ donor2
CD14+ donor3
CD4+ donor1
CD4+ donor2
CD4+ donor3
B4GALT1
~270bp, unprecedented high resolution
SW-13 cell line
Higher tissue and tag coverage:
understanding composite promoter architectures and its mixed modes of regulation
223,428 in human and 162,264 in mouse of
reference TSS
Cell4
CpGCpG CpG CpG CpG CpGTATA
Cell1 Cell3Cell2
• Blue, yellow and green:
Broadly used
• Red:
Cell2 specific and highly
expressed
TSS preferences:
• B4GALT1 core promoter
• Primary Astrocytes
• CD14+ monocytes
• CD4+ T-cells
11. 11
CAGE locates known enhancers in vivo
Based on this, we make a rule
to locate novel transcribed
enhancers over the whole
FANTOM collection.
Enhancers have bidirectional
CAGE transcription.
Bidirectional transcription
identifies the nucleosome
boundary.
Robin Andersson et al., Nature 507, 455 (2014)
12. 12
CAGE enhancer expression identify
cell-specific, active, enhancers..
CAGE-
defined
enhancer
expression
DHSS
H3K27ac
H3K4me1
Robin Andersson et al., Nature 507, 455 (2014)
13. 13
CRE2
CRE3
0.0
0.5
1.0
1.5
Neuron
Astrocyte
Brain
nt
nt
ysl
ysl
mu
100μm 100μm
fp
fp
ne
100μm
100μm
100μm
100μm
Blood vessel
of epithlial cell
Endothelial cell of
hepatic sinusoid
Kidney
epithelial cell
Urothelial cell
Respiratory
epithelial cell
Spleen
Heart
Brain
Cardiac fibroblast
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Neuronal
stem cell
Neuron
Astrocyte
Spinal cord
Brain
Systematic in vivo characterization of
active enhancers across the human body
We identified 65,423 and 44,459 enhancers
in human and mouse.
60% are over-represented in one cell/tissue
group
15. 15
http://fantom.gsc.riken.jp/
Databases by Hideya Kawaji, Takeya Kasukawa et al.
ZENBU: Jessica Severin et al., Nature Biotechnology 32, 217 (2014)
SSTAR: Imad Abugessaisa et al., Database pii: baw105 (2015)
Number of page view a year
Total: 9,217,440
ZENBU: 2,927,780 (FY2015)
16. FANTOM5: How many lncRNA genes are there?
Number of Human lncRNA Genes
63,132
permissive
9,318
stringent
27,919robust
Versus GENCODEv25 lncRNA Catalogue
(number of genes)
with
CAGE
support
without
CAGE
support
19,668 7,228 7,790
FANTOM CAT GENECODE
v25
Chung-Chau Hon et al., Nature 543, 199 (2017)
17. An atlas of human lncRNAs with high-confidence 5’ ends
Identified 19,175 potentially
functional lncRNAs in human
Co-expression of lncRNA-mRNA
pairs linked by eQTL
Chung-Chau Hon et al., Nature 543, 199 (2017)
18. Current CAGE Technology
• HeliScope CAGE
• SeqLL (MA, USA) can provide sequencing
service, and early access of their HeliScope-
type benchtop sequencer
• nAnT-iCAGE (no-Amplified non-Tagging
illumina CAGE)
• After Helicos company collapsed, we have
developed as an alternative CAGE
technology
• After releasing cap trapped cDNA, adapters
are ligated directionally, 2nd strand is
synthesized, and then ready to be
sequenced
• No amplification steps to reduce any biases
• nAnT-iCAGE library preparation kit is
available from Dnaform K.K. (Japan)
• nAnT-iCAGE analysis service is provided by
RIKEN GeNAS and Dnaform K.K.
19. Clinical Application
• Biomarkers for lymph node metastasis of endometrial cancer
• Purpose
• To avoid unnecessary lymphadenectomy for patients with low risk of recurrence
despite the risk of complications suck as lymphedema
• Method
• Comparison of CAGE profiles of the primary lesions of endometrial cancers
between lymph node metastasis + and –
• Identification of differentially expressed promoters between LN+ and LN–
• Validation
• qRT-PCR of TSS markers with control TSS of stably expressed genes
E. Yoshida et al. in submission
20. New Technology
• Cap Trap RNA-seq
• Full length cDNA from Matured PolyA+
mRNA by Cap Trapper Technology
• Unique Molecular Index (UMI) at both
ends of cDNA
• –> Generate Quantitative Directional
TSS same as CAGE
• –> Generate Quantitative Directional
Transcription Termination Site (TTS)
• Tn5 Transposase Tagmentation for
RNA-seq
• –> Generate RNA-seq
• A library involves RNA-seq, CAGE
and DGE for transcribed regions with
countable TSS and TTS
23. Conclusion
• Cap Trapper-based technologies enabled to prepare full length
cDNA from only capped polyA+ RNA
• CAGE enabled to determine and measure quantitative TSSs
comprehensively
• CTR-seq enabled to determine and measure quantitative TSSs
and TTSs with transcribed regions by RNA-seq within a library
• FANTOM5 project revealed comprehensive lncRNAs,
promoters, and enhancers of various cell lines, primary cultures
and some time courses of differentiation
25. FANTOM Collaborators
Australia Western Australian Institute for Medical Research
Peter KLINKEN, Louise WINTERINGHAM
Canada McGill University
Hisashi MIURA, Josee DOSTIE
The University of British Columbia, Center for Molecular Medecine and Therapeutics
Thomas Jonghyun HA
Denmark University of Copenhagen, Department of Biology
Robin ANDERSSON, Albin SANDELIN, Eivind VALEN
Finland University of Helsinki, Department of Medical Genetics
Alessandro BONETTI
France University Pierre & Marie Curie, Laboratoire Microorganisms Genomics
Hugues RICHARD
Germany Charité - Universitätsmedizin Berlin, Allergy Center
Magda BABINA
University Hospital Regensburg
Christian SCHMIDL, Michael REHLI
Italy Dulbecco Telethon Institute
Valerio ORLANDO, Beatrice BODEGA
Fondazione Bruno Kessler (FBK)
Marco CHIERICI, Cesare FURLANELLO, Marco RONCADOR
International School for Advanced Studies (SISSA)
Stefano GUSTINICH, Silvia ZUCCHELLI
National Lab of Italian Consortium for Biotechnology (L.N.C.I.B.)
Silvano PIAZZA, Claudio SCHNEIDER, Roberto VERARDO
Japan Database Center for Life Science
Hidemasa BONO
Keio University, School of Medicine
Shigeo KOYASU, Kazuyo MORO, Jun-ichi FURUSAWA
Thanks!!
