Targeted Sequencing of Human Genomes, Transcriptomes, and Methylomes

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Targeted Sequencing of Human Genomes,
Transcriptomes, and Methylomes

• Jin Billy Li
• George Church Lab
• Harvard Medical School
• jli_at_genetics.med.harvard.edu

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Genetic Loci X Sample Size Information
PCR seq Mass-spec
SNP array
samples
Shotgun seq RNA-seq ChIP-seq
genetic loci
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Target Capturing with Padlock Probes (aka MIPs)
pol
lig

feature 1
feature n
PCR (or RCA)

Porreca et al., Nat Methods 2007
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Mass Production of Padlock Oligos
150 nt
100 nt
50 nt
55k features of up to 200nt
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10,000-fold Improvement Since Nov 20071. longer
hybridization time 2. more probes 3. right
dNTP
1
2
3

Li et al., in prepration
20-fold improvement already by better probe
design and synthesis
6
10,000-fold Improvement Since Nov 20071. longer
hybridization time 2. more probes 3. right
dNTP
1
2
3

Li et al., in prepration
20-fold improvement already by better probe
design and synthesis
7
10,000-fold Improvement Since Nov 20071. longer
hybridization time 2. more probes 3. right
dNTP
1
2
3

Li et al., in prepration
20-fold improvement already by better probe
design and synthesis
8
Improved Technology -gt Better Performance
Sensitivity Uniformity
Correlation
Current
Current
Nov 2007
Nov 2007
95 captured 85 within 100-fold range 55 within
10-fold range
Li et al., in prepration
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Summary of Improvements
Nov 2007 Current
Specificity 100 100
Sensitivity/Multiplexity (of 55k) 18 95
Uniformity (in 100-fold range) 16 85
Correlation of replicates (r) 0.35 0.98
Accuracy (heterozygous calls) 31 99
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Targeted Capturing of

• Genomes
• Exome PGP etc.
• Contiguous regions or gene panels
• SNPs
• Hypermutable CpG dinucleotides
• Transcriptomes
• Alleotyping
• RNA editing sites
• Methylomes
• CpG methylation

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Targeted Capturing of

• Genomes
• Exome PGP etc.
• Contiguous regions or gene panels
• SNPs
• Hypermutable CpG dinucleotides
• Transcriptomes
• Alleotyping
• RNA editing sites
• Methylomes
• CpG methylation

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Predicting Putative Editing Sites
A -gt I (G) RNA Editing

• Post-transcriptional A -gt I
• I is read as G during translation
• Only 10 targets are known in human coding regions

A in the genome

G in some mRNAs or ESTs
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Discovery of 100s of Novel Editing Sites
36,000 predicted editing sites gDNA 7 tissue
cDNAs from an individual
Padlock Solexa 239 sites found to be edited
Validation (PCR Sanger) 18 of 20 random sites
are obviously edited
with Erez Levanon, in preparation
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Genomic DNA
Example VEZF1
RNA - cerebellum
RNA - corpus callosum
RNA - frontal lobe
RNA - diencephalon
RNA - intestine
RNA - kidney
RNA - adrenal
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Bisulfite Padlock Probes (BSP) CpG Methylation
Bisulfite-treated genome
3-base genome
High specificity of padlock
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Methylation Level Accurately Measured
BSP-BSP correlation
BSP-Sanger correlation
Methylation level estimated by Sanger sequencing
Methylation level, replicate 2
r 0.979
r 0.966
Methylation level measured by BSP sequencing
Methylation level, replicate 1
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Methylation Pattern around GenesGene-Body
Methylation
with Madeleine Price Ball, in preparation (poster)
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Acknowledgements
Padlock technology Kun Zhang John Aach Abraham
Rosenbaum Jay Shendure Greg Porreca Annika
Ahlford RNA editing Erez Levanon Jung-Ki
Yoon CpG methylation Madeleine Price
Ball Church Lab
Sequencing Yuan Gao Bin Xie Bob Steen
Agilent Emily Leproust Wilson Woo
George Church
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Superior Quality of Padlock Oligos
55k features of up to 200nt
PCR (2x)
Solexa sequencing
150 nt
Fraction of probes
100 nt
50 nt
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From Agilent Oligos to Padlock Probesamplificatio
n and selection
DpnII
T
18bp
Agilent oligo, 136 bp 18bp
PCR
UA

p
? exonuclease
U

Annealed with DpnII guide oligo
U

NN
USER DpnII
Padlock probe
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Heterozygous Genotypes Correctly Called
before
after
Homozygous wild type Heterozygous
variation Homozygous variation
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Methods in Comparison
Padlock Array-based hyb
Upfront probe cost (10-20 of exome) 12,000 per 55k 100mers 600 per 385k 70mers
Probes amplifiable? Yes No
Reaction phase Solution, 10-20 µl Surface, 200 µl
Enzymatic hyb? Yes No
gDNA required 0.5-1 µg 20 µg (WGA)
Efficiency (-gtaccuracy) 1 N/A (lt0.1?)
Uniformity 100-fold range 10-fold range
Specificity 100 on target 30-80 on or near target
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Differential Clamping at Ligation Junction
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GC VS Capturing Efficiency
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99 Concordance Between Padlock and HapMap
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The Editing Calls Are Well Correlated
r 0.964
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Bisulfite Padlock Probes (BSP) CpG Methylation
Bisulfite-treated genome

• 10k CpG sites tiling the ENCODE regions
• 1 CpG site every 3kb region on average
• High specificity
• 79 of 80 Sanger reads match correct locations

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collected in a tube
B
P
shearing, end polishing
PCR
B
P
adapter ligation
? exonuclease
B
hybridization in closed-tube solution
strep
B
denaturing, PCR
Li et al., unpublished
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Methods in Comparison
Padlock Array-based hyb Biotin-coupled hyb
Upfront probe cost (10-20 of exome) 12,000 per 55k 100mers 600 per 385k 70mers 500 per 244k 60mers
Probes amplifiable? Yes No Yes
Reaction phase Solution, 10-20 µl Surface, 200 µl Solution, 10-20 µl
Enzymes in hyb? Yes No No
gDNA required 0.5-1 µg 20 µg (WGA) 0.5-1 µg
Efficiency (-gtaccuracy) 1 N/A (lt0.1?) 10?
Uniformity 100-fold range 10-fold range 10-fold range?
Specificity 100 on target 30-80 on or near target 55 on or near target
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Two Tech Replicates Are Well Correlated
Uniformity
Correlation of counts
Number of reads per site
Counts, replicate 2
Counts, replicate 1
Ranked target sites