Targeted Sequencing of Human Genomes, Transcriptomes, and MethylomesJin Billy LiGeorge Church LabHarvard Medical [email protected]
Genetic Loci X Sample Size = Information# samples# genetic lociPCR seqMass-specShotgun seqRNA-seqChIP-seqSNP array
Target Capturing with Padlock Probes (aka MIPs)feature 1feature npolligPCR (or RCA)Porreca et al., Nat Methods 2007
Mass Production of Padlock Oligos100 nt150 nt50 nt55k features of up to 200nt
~10,000-fold Improvement Since Nov 20071. longer hybridization time; 2. more probes; 3. right [dNTP]123** 20-fold improvement already by better probe design and synthesisLi et al., in prepration
Chart3
0.002411
0.002791.1576763485
0.014055.8298755187
0.028711.9087136929
0.0316513.132780083
0.036615.1867219917
0.0312.4481327801
0.127552.9045643154
0.4445184.4398340249
0.5985248.3402489627
0.00943.9004149378
1.05435.6846473029
0.4445184.4398340249
0.282.9875518672
0.076531.7427385892
Capturing efficiency (%)
Fold improvement
Sheet1
10:1 ratio, different hyb time
10.00%10.52
1.00%14.06
0.10%17.78
0.01%20.94
0.00%24.57
0.00%27.85
5 days18.2315 mins4.00E-05
2 days18.31 hour4.00E-03
1 day + cycling18.571 day4.00E-02
1 day19.091 day + cycling5.78E-02
1 hour22.782 days6.97E-02
15 mins29.035 days7.32E-02
CG55k; 6/13 MIPs10:1, dNTP 100x18.72
50:1, dNTP 100x17.12
250:1, dNTP 100x14.77
100:1, dNTP 10,000x17.86
100:1, dNTP 1,000x16.47
100:1, dNTP 100x15.2
100:1, dNTP 10x13.96
100:1, dNTP 1x20.450.00
6/10 MIPs15 mins23.56probe:gDNA = 10:115 mins0.000.001.00
1 hour23.351 hour0.000.011.16
1 day20.31 day0.010.035.83
1 day + cycling19.271 day + cycling0.030.0611.91
2 days19.132 days0.030.0613.13
5 days18.925 days0.040.0715.19
standards; 10%11.541 day + cycling10:10.030.0612.45
1%15.0350:10.130.2652.90
0.10%18.47100:10.440.89184.44
0.01%21.36250:10.601.20248.34
0.00%25.83dNTP1x0.00940.023.90
10x1.052.10435.68
100x0.44450.89184.44
1,000x0.20.4082.99
10,000x0.07650.1531.74
dNTPratiotime0.00
100x dNTPprobe:gDNA = 10:1variable hyb time15 mins0.000.001.00
1 hour0.000.011.16
1 day0.010.035.83
1 day + cycling0.030.0611.91
2 days0.030.0613.13
5 days0.040.0715.19
100x dNTP2 day hyb timevariable probe:gDNA10:10.030.0612.45
50:10.130.2652.90
100:10.440.89184.44
250:10.601.20248.34
probe:gDNA = 100:11 day hyb timevariable dNTP amount1x0.00940.023.90
10x1.052.10435.68
100x0.44450.89184.44
1,000x0.20.4082.99
10,000x0.07650.1531.74
Sheet1
Capturing efficiency (%)
Sheet2
[dNTP]
Capturing efficiency (%)
Sheet3
Capturing efficiency (%)
Capturing efficiency (%)
Fold of improvement
Capturing efficiency (%)
Fold improvement
~10,000-fold Improvement Since Nov 20071. longer hybridization time; 2. more probes; 3. right [dNTP]123** 20-fold improvement already by better probe design and synthesisLi et al., in prepration
Chart3
0.002411
0.002791.1576763485
0.014055.8298755187
0.028711.9087136929
0.0316513.132780083
0.036615.1867219917
0.0312.4481327801
0.127552.9045643154
0.4445184.4398340249
0.5985248.3402489627
0.00943.9004149378
1.05435.6846473029
0.4445184.4398340249
0.282.9875518672
0.076531.7427385892
Capturing efficiency (%)
Fold improvement
Sheet1
10:1 ratio, different hyb time
10.00%10.52
1.00%14.06
0.10%17.78
0.01%20.94
0.00%24.57
0.00%27.85
5 days18.2315 mins4.00E-05
2 days18.31 hour4.00E-03
1 day + cycling18.571 day4.00E-02
1 day19.091 day + cycling5.78E-02
1 hour22.782 days6.97E-02
15 mins29.035 days7.32E-02
CG55k; 6/13 MIPs10:1, dNTP 100x18.72
50:1, dNTP 100x17.12
250:1, dNTP 100x14.77
100:1, dNTP 10,000x17.86
100:1, dNTP 1,000x16.47
100:1, dNTP 100x15.2
100:1, dNTP 10x13.96
100:1, dNTP 1x20.450.00
6/10 MIPs15 mins23.56probe:gDNA = 10:115 mins0.000.001.00
1 hour23.351 hour0.000.011.16
1 day20.31 day0.010.035.83
1 day + cycling19.271 day + cycling0.030.0611.91
2 days19.132 days0.030.0613.13
5 days18.925 days0.040.0715.19
standards; 10%11.541 day + cycling10:10.030.0612.45
1%15.0350:10.130.2652.90
0.10%18.47100:10.440.89184.44
0.01%21.36250:10.601.20248.34
0.00%25.83dNTP1x0.00940.023.90
10x1.052.10435.68
100x0.44450.89184.44
1,000x0.20.4082.99
10,000x0.07650.1531.74
dNTPratiotime0.00
100x dNTPprobe:gDNA = 10:1variable hyb time15 mins0.000.001.00
1 hour0.000.011.16
1 day0.010.035.83
1 day + cycling0.030.0611.91
2 days0.030.0613.13
5 days0.040.0715.19
100x dNTP2 day hyb timevariable probe:gDNA10:10.030.0612.45
50:10.130.2652.90
100:10.440.89184.44
250:10.601.20248.34
probe:gDNA = 100:11 day hyb timevariable dNTP amount1x0.00940.023.90
10x1.052.10435.68
100x0.44450.89184.44
1,000x0.20.4082.99
10,000x0.07650.1531.74
Sheet1
Capturing efficiency (%)
Sheet2
[dNTP]
Capturing efficiency (%)
Sheet3
Capturing efficiency (%)
Capturing efficiency (%)
Fold of improvement
Capturing efficiency (%)
Fold improvement
~10,000-fold Improvement Since Nov 20071. longer hybridization time; 2. more probes; 3. right [dNTP]123** 20-fold improvement already by better probe design and synthesisLi et al., in prepration
Chart3
0.002411
0.002791.1576763485
0.014055.8298755187
0.028711.9087136929
0.0316513.132780083
0.036615.1867219917
0.0312.4481327801
0.127552.9045643154
0.4445184.4398340249
0.5985248.3402489627
0.00943.9004149378
1.05435.6846473029
0.4445184.4398340249
0.282.9875518672
0.076531.7427385892
Capturing efficiency (%)
Fold improvement
Sheet1
10:1 ratio, different hyb time
10.00%10.52
1.00%14.06
0.10%17.78
0.01%20.94
0.00%24.57
0.00%27.85
5 days18.2315 mins4.00E-05
2 days18.31 hour4.00E-03
1 day + cycling18.571 day4.00E-02
1 day19.091 day + cycling5.78E-02
1 hour22.782 days6.97E-02
15 mins29.035 days7.32E-02
CG55k; 6/13 MIPs10:1, dNTP 100x18.72
50:1, dNTP 100x17.12
250:1, dNTP 100x14.77
100:1, dNTP 10,000x17.86
100:1, dNTP 1,000x16.47
100:1, dNTP 100x15.2
100:1, dNTP 10x13.96
100:1, dNTP 1x20.450.00
6/10 MIPs15 mins23.56probe:gDNA = 10:115 mins0.000.001.00
1 hour23.351 hour0.000.011.16
1 day20.31 day0.010.035.83
1 day + cycling19.271 day + cycling0.030.0611.91
2 days19.132 days0.030.0613.13
5 days18.925 days0.040.0715.19
standards; 10%11.541 day + cycling10:10.030.0612.45
1%15.0350:10.130.2652.90
0.10%18.47100:10.440.89184.44
0.01%21.36250:10.601.20248.34
0.00%25.83dNTP1x0.00940.023.90
10x1.052.10435.68
100x0.44450.89184.44
1,000x0.20.4082.99
10,000x0.07650.1531.74
dNTPratiotime0.00
100x dNTPprobe:gDNA = 10:1variable hyb time15 mins0.000.001.00
1 hour0.000.011.16
1 day0.010.035.83
1 day + cycling0.030.0611.91
2 days0.030.0613.13
5 days0.040.0715.19
100x dNTP2 day hyb timevariable probe:gDNA10:10.030.0612.45
50:10.130.2652.90
100:10.440.89184.44
250:10.601.20248.34
probe:gDNA = 100:11 day hyb timevariable dNTP amount1x0.00940.023.90
10x1.052.10435.68
100x0.44450.89184.44
1,000x0.20.4082.99
10,000x0.07650.1531.74
Sheet1
Capturing efficiency (%)
Sheet2
[dNTP]
Capturing efficiency (%)
Sheet3
Capturing efficiency (%)
Capturing efficiency (%)
Fold of improvement
Capturing efficiency (%)
Fold improvement
Improved Technology -> Better Performance95% captured85% within 100-fold range55% within 10-fold rangeSensitivity + UniformityCorrelationNov 2007Nov 2007CurrentCurrentLi et al., in prepration
Summary of Improvements
Nov 2007CurrentSpecificity~100%~100%Sensitivity/Multiplexity (of 55k)18%95%Uniformity (in 100-fold range)16%85%Correlation of replicates (r)0.350.98Accuracy (heterozygous calls)31%99%
Targeted Capturing ofGenomesExome: PGP etc.Contiguous regions or gene panelsSNPsHypermutable CpG dinucleotides
TranscriptomesAlleotypingRNA editing sites
MethylomesCpG methylation
Targeted Capturing ofGenomesExome: PGP etc.Contiguous regions or gene panelsSNPsHypermutable CpG dinucleotides
TranscriptomesAlleotypingRNA editing sites
MethylomesCpG methylation
Predicting Putative Editing SitesA in the genomeG in some mRNAs or ESTs A -> I (G) RNA EditingPost-transcriptional A -> I I is read as G during translationOnly 10 targets are known in human coding regions
36,000 predicted editing sitesgDNA + 7 tissue cDNAs from an individualPadlock + Solexa: 239 sites found to be editedValidation (PCR + Sanger):18 of 20 random sites are obviously editedDiscovery of 100s of Novel Editing Siteswith Erez Levanon, in preparation
Genomic DNARNA - intestineRNA - kidneyRNA - diencephalonRNA - frontal lobeRNA - corpus callosumRNA - cerebellumRNA - adrenal Example:VEZF1
Bisulfite Padlock Probes (BSP): CpG MethylationBisulfite-treated genome3-b
