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Francois Vigneault Church Lab CEGS meeting Oct 16th 2008. Technology developement for high – throughput sequencing of microRNAs. How to clone miRNAs?. 5’. 3’. X. PO 4. 5’. 3’-OH. PO 4. T4 RNA ligase. +. 5’. +. PO 4. 3’. X. ATP. How to capture miRNAs?. 5’. 3’. X. PO 4. - PowerPoint PPT Presentation
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Francois Vigneault
Church Lab CEGS meeting
Oct 16th 2008
How to clone miRNAs?
X3’
3’-OH5’PO4
5’PO4
5’PO4
+
X3’
+T4 RNA ligase
ATP
How to capture miRNAs?
X3’
3’-OH5’PO4
5’App
5’PO4
+
X3’
+T4 RNA ligase
ATPX
3’5’PO4
How does one make app-Oligo
Lau et al. Science 2001 (Bartel lab)
Yield 10-20%
IDT (5 nmole $ 595)
Enzymatic adenylation
X5’PO4
3’
5’3’
AppT4 DNA Ligase
.
ATP
Pre-adenylation
3’adapter ligation
5’ adapter ligation
High-throughput sequencing
Illumina ~5 000 000 reads/track for 1200$ Less than ~1000 miRNAs in human cells Multiplexing up to 50 samples per track
(100 fold dynamic range) bring cost per samples to 24$
Bar-coded adenylation
3’GCGC X
5’PO4
CCAA X5’PO4
3’
TAGC X5’PO4
3’
ATAT X5’PO4
3’
NNNN5’3’
NNNN5’3’
NNNN5’3’
NNNN5’3’
App
App
App
App
T4 DNA Ligase
T4 DNA Ligase
T4 DNA Ligase
T4 DNA Ligase
.
Bar-coded adenylation
3’GCGC X
5’
CCAA X5’ 3’
TAGC X5’ 3’
ATAT X5’ 3’
App
App
App
App
miRNAs ligation
3’GCGC X
5’
CCAA X5’ 3’
TAGC X5’ 3’
ATAT X5’ 3’
App
App
App
App
3’-OH5’PO4
3’-OH5’PO4
3’-OH5’PO4
3’-OH5’PO4
T4 DNA Ligase
T4 DNA Ligase
T4 DNA Ligase
T4 DNA Ligase
miRNAs ligation
3’GCGC X
CCAA X3’
TAGC X3’
XATAT3’5’PO4
5’PO4
5’PO4
5’PO4
T4 DNA Ligase
T4 DNA Ligase
T4 DNA Ligase
T4 DNA Ligase
5’adapter ligation
3’NNNN X5’PO4
5’ 3’-OHT4 DNA Ligase
5’adapter ligation
3’NNNN X5’ T4 DNA
Ligase
.
RT-PCR
3’NNNN X5’
RT pol
TAQ pol
Sequencing
miRNA barcode
Sequencing
ATC
TGA ATCACG
GCC
TCA
AAT
GTG
GGG
ATAT
GCGC
TAGC
CCAA
TTG…
GTC
TTC
CCA
ATC
TCA
AAT
GGG
TTG…
TTG…
TTG…
ATC
TGA
GCC
GTG
ATC
TCA
AAT
GGGATC
TGA
GCC
GTGATCTGA
GCC
GTG
Sequencing
.
TGA ATCACG GCGCAAT TTG…ATC ATCGGGATC
GCC GGG TAGCTTCATC TTG…TGAGTGATCTGA
ATC AAT ATATTTG…CCATCA GCC AATTGA GCC
TCAGTG CCAAGTCGGG TTG…GTG TCAGCC GTG
Analysis
Exp
ress
ion
leve
ls
miRNA sequences
miRNA profiling
TGA
ATCACG
GCGC
AAT
TTG…
ATC
ATCGGG
ATC
GCC
GGG
TAGC
TTC
ATC
TTG…
TGAGTGATCTGA
TCA
GTG
CCAA
GTC
GGG
TTG…
GTG
TCA
GCC
GTG
Analysis
ATC
AAT
ATATTTG…
CCA
TCA
GCC
AAT
TGA
GCC
ATC
AAT
ATATTTG…
CCA
TCA
GCC
AAT
TGA
GCC
ATC
AAT
ATATTTG…
CCA
TCA
GCC
AAT
TGA
GCC
ATC
AAT
ATATTTG…
CCA
TCA
GCC
AAT
TGA
GCC
TGA
ATCACG
GCGC
AAT
TTG…
ATC
ATCGGG
ATC
TGA
ATCACG
GCGC
AAT
TTG…
ATC
ATCGGG
ATC
TGA
ATCACG
GCGC
AAT
TTG…
ATC
ATCGGG
ATC
GCC
GGG
TAGC
TTC
ATC
TTG…
TGAGTGATCTGA
GCC
GGG
TAGC
TTC
ATC
TTG…
TGAGTGATCTGA
TCA
GTG
CCAA
GTC
GGG
TTG…
GTG
TCA
GCC
GTG
TCA
GTG
CCAA
GTC
GGG
TTG…
GTG
TCA
GCC
GTG
TCA
GTG
CCAA
GTC
GGG
TTG…
GTG
TCA
GCC
GTG
TCA
GTG
CCAA
GTC
GGG
TTG…
GTG
TCA
GCC
GTG
Sample1 Sample2 Sample3 Sample4
.
Projections
Personal Genome Project (PGP) Induced pluripotent stem cells (iPS) VDJ-ome
VDJ-ome
Acknowledgment
George M Church
Michael Sismour Greg Porreca Andrew Z Fire and lab All Church lab members…
