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Peter L. Nagy MD, PhD;  [email_address] ;  PS12-420F Director, Personalized Genomic Medicine, Department of Pathology Capt...
Division of Personalized Genomic Medicine <ul><li>Mission Statement:  To provide state of the art comprehensive genetic di...
Our agenda <ul><li>Screen specific patient populations for mutations in known disease causing genes using NextGen sequenci...
Nextgene sequencing lab <ul><li>Laboratory personnel: </li></ul><ul><ul><li>Library preparation and sequencing </li></ul><...
Roche <ul><li>454 JR  FLX </li></ul>
Illumina <ul><li>GAIIx  MySeq </li></ul>
Navigare necesse est <ul><li>Options </li></ul><ul><ul><li>PCR based methods </li></ul></ul><ul><ul><ul><li>Traditional PC...
General considerations for choosing the capture method <ul><li>Size of region of interest  </li></ul><ul><ul><li>< 50 kb  ...
Mitochondrial genome sequencing
<ul><li>Long Range PCR from genomic DNA and use of long read technlogy, Roche 454, protects from errors derived from pseud...
Long range PCR alerts to heteroplasmic deletions
Raindance <ul><li>Covaris S series  Raindance RDT1000 </li></ul><ul><li>Fragmentation of Genomic DNA to about 5000 bp frag...
Raindance projects <ul><li>Cardiomyopathy panel /Howard Wormann  </li></ul><ul><li>ABCA4 locus/ Rando Alikmets </li></ul><...
Raindance cardiomyopathy data  Mean coverage: 71 Std Dev: 58
Illumina exome capture cardiomyopathy data  Mean coverage: 50 Std Dev: 27
Fluidigm <ul><li>IFC Controller AX  Fluidigm FC1 Cycler </li></ul>
Fluidigm project <ul><li>ABCA4 exons/ Rando Alikmets </li></ul><ul><ul><li>Findings </li></ul></ul><ul><ul><ul><li>Design ...
Comparison of capture methods on the ABCA4 exons Illumina Exome capture  Mean:110; STD:39 Fluidigm Mean:313; STD:207 Raind...
Agilent Sureselect custom capture Alzheimer panel data  Mean coverage: 487 Std Dev: 185
<ul><li>Rapid & Economical </li></ul><ul><ul><li>Up to 384 amplicons per sample, 96 samples per plate ( 36,864 reactions) ...
Conclusion <ul><li>For clinical applications quality of the sequence data is paramount </li></ul><ul><ul><li>Hybridization...
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Peter Nagy, Columbia Agilent Symposium, Jan, 27 2012

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Peter Nagy, Columbia Agilent Symposium, Jan, 27 2012

  1. 1. Peter L. Nagy MD, PhD; [email_address] ; PS12-420F Director, Personalized Genomic Medicine, Department of Pathology Capture Strategies for Nextgen Sequencing in Clinical Practice
  2. 2. Division of Personalized Genomic Medicine <ul><li>Mission Statement: To provide state of the art comprehensive genetic diagnosis for familial disorders and cancer </li></ul><ul><ul><li>Location: 17. floor of Physicians & Surgeons Bldg </li></ul></ul><ul><ul><li>Directors: </li></ul></ul><ul><ul><ul><li>Dr. Mahesh Matsukhani : Cancer Genetics </li></ul></ul></ul><ul><ul><ul><li>Ali Naini : Neurogenetics </li></ul></ul></ul><ul><ul><ul><li>Brynn Levy and Vajdehi Jobanputra : Cytogenetics </li></ul></ul></ul><ul><ul><ul><li>Lorraine Clark: Array genotyping </li></ul></ul></ul><ul><ul><ul><li>Peter L. Nagy: Nextgen sequencing; constitutional genetics </li></ul></ul></ul>
  3. 3. Our agenda <ul><li>Screen specific patient populations for mutations in known disease causing genes using NextGen sequencing panels </li></ul><ul><ul><ul><ul><li>Mitochondrial disorders </li></ul></ul></ul></ul><ul><ul><ul><ul><li>ALS and ALS-related disorders </li></ul></ul></ul></ul><ul><ul><ul><ul><li>Parkinson/ Alzheimer disease </li></ul></ul></ul></ul><ul><ul><ul><ul><li>Peripheral neuropathies </li></ul></ul></ul></ul><ul><ul><ul><ul><li>Muscular dystrophies </li></ul></ul></ul></ul><ul><ul><ul><ul><li>Cardiomyopathies </li></ul></ul></ul></ul><ul><ul><ul><ul><li>Cancer </li></ul></ul></ul></ul><ul><li>Sequence full exome in patients with unknown cause </li></ul><ul><li>Organize and mine the accumulating genomic data to improve identification of novel pathogenic mutations and modifier variants </li></ul>
  4. 4. Nextgene sequencing lab <ul><li>Laboratory personnel: </li></ul><ul><ul><li>Library preparation and sequencing </li></ul></ul><ul><ul><ul><li>Zhenming Yu, Ph.D </li></ul></ul></ul><ul><ul><ul><li>Endre Hegedus, Ph.D </li></ul></ul></ul><ul><ul><ul><li>Amanda Kahn </li></ul></ul></ul><ul><ul><ul><li>Stephanie Sheikh </li></ul></ul></ul><ul><ul><li>IT support </li></ul></ul><ul><ul><ul><li>Marissa Chang </li></ul></ul></ul><ul><ul><ul><li>Nick Rouse </li></ul></ul></ul>
  5. 5. Roche <ul><li>454 JR FLX </li></ul>
  6. 6. Illumina <ul><li>GAIIx MySeq </li></ul>
  7. 7. Navigare necesse est <ul><li>Options </li></ul><ul><ul><li>PCR based methods </li></ul></ul><ul><ul><ul><li>Traditional PCR </li></ul></ul></ul><ul><ul><ul><li>Raindance </li></ul></ul></ul><ul><ul><ul><li>Fluidigm </li></ul></ul></ul><ul><ul><ul><li>Illumina TruSeq Custom Amplicon </li></ul></ul></ul><ul><ul><li>Hybridization based methods </li></ul></ul><ul><ul><ul><li>Agilent Sureselect; Exome/ Custom Enrichment </li></ul></ul></ul><ul><ul><ul><li>Roche Nimblegen; Exome/ Custom Enrichment </li></ul></ul></ul><ul><ul><ul><li>Illumina; TruSeq Exome/ Custom Enrichment </li></ul></ul></ul>Captura necesse est
  8. 8. General considerations for choosing the capture method <ul><li>Size of region of interest </li></ul><ul><ul><li>< 50 kb -long range PCR </li></ul></ul><ul><ul><li>50kb – 1Mb -PCR based capture </li></ul></ul><ul><ul><li>100kb - 60Mb -Hybe capture </li></ul></ul><ul><ul><li>>60Mb -Genome sequencing </li></ul></ul><ul><li>Number of samples to analyze </li></ul><ul><ul><li>The larger the sample size, the more the PCR based methods make sense </li></ul></ul><ul><ul><li>Illumina allows capture of 24 combined libraries </li></ul></ul>
  9. 9. Mitochondrial genome sequencing
  10. 10. <ul><li>Long Range PCR from genomic DNA and use of long read technlogy, Roche 454, protects from errors derived from pseudogenes </li></ul>Mitochondrial genome amplification using traditional PCR 16566 16331 3729 3460 8753 9158 10 16494
  11. 11. Long range PCR alerts to heteroplasmic deletions
  12. 12. Raindance <ul><li>Covaris S series Raindance RDT1000 </li></ul><ul><li>Fragmentation of Genomic DNA to about 5000 bp fragments </li></ul><ul><li>Fusion of fragmented genome with about 5 primer pairs in a single droplet </li></ul><ul><li>Generate millions of such emulsion droplets and perform PCR in a single tube </li></ul><ul><li>Supposed to work up to 20,000 primer pairs </li></ul><ul><li>Primers are limiting in PCR reactions to normalize PCR product amounts </li></ul>
  13. 13. Raindance projects <ul><li>Cardiomyopathy panel /Howard Wormann </li></ul><ul><li>ABCA4 locus/ Rando Alikmets </li></ul><ul><li>Findings </li></ul><ul><ul><li>Primer design pipeline is inadequate </li></ul></ul><ul><ul><li>Upfront cost and running cost is high </li></ul></ul><ul><ul><ul><li>$10 per primer pair plus $350/sample </li></ul></ul></ul><ul><ul><li>Instrument is tedious, temperamental </li></ul></ul><ul><ul><ul><li>About 10 percent of the runs have to be redone for unexplained instrument failure </li></ul></ul></ul><ul><ul><li>Reagent packaging is awkward </li></ul></ul><ul><ul><ul><li>Each vial contains 8 sample worth of reagent </li></ul></ul></ul><ul><ul><ul><li>Has to be used in one setting </li></ul></ul></ul><ul><ul><ul><li>Each run takes about an hour </li></ul></ul></ul><ul><ul><ul><li>Sample prep is time consuming individual fragmentation + individual library preps </li></ul></ul></ul>
  14. 14. Raindance cardiomyopathy data Mean coverage: 71 Std Dev: 58
  15. 15. Illumina exome capture cardiomyopathy data Mean coverage: 50 Std Dev: 27
  16. 16. Fluidigm <ul><li>IFC Controller AX Fluidigm FC1 Cycler </li></ul>
  17. 17. Fluidigm project <ul><li>ABCA4 exons/ Rando Alikmets </li></ul><ul><ul><li>Findings </li></ul></ul><ul><ul><ul><li>Design of primers is relatively straightforward </li></ul></ul></ul><ul><ul><ul><li>Setup is not very time consuming </li></ul></ul></ul><ul><ul><ul><li>Machine is relatively easy to use and reliable </li></ul></ul></ul><ul><ul><ul><li>Setup of experiments is rigid; 48 amplicons in 48 samples </li></ul></ul></ul><ul><ul><ul><li>Coverage is uneven, allele representation is significantly biased </li></ul></ul></ul>
  18. 18. Comparison of capture methods on the ABCA4 exons Illumina Exome capture Mean:110; STD:39 Fluidigm Mean:313; STD:207 Raindance Mean:159;STD:102
  19. 19. Agilent Sureselect custom capture Alzheimer panel data Mean coverage: 487 Std Dev: 185
  20. 20. <ul><li>Rapid & Economical </li></ul><ul><ul><li>Up to 384 amplicons per sample, 96 samples per plate ( 36,864 reactions) </li></ul></ul><ul><ul><li>Plate based processing </li></ul></ul><ul><ul><li><8 hrs from DNA to sequencing-ready library </li></ul></ul><ul><ul><li>No gels, no fragmentation – uses standard lab equipment </li></ul></ul><ul><li>Fully customized target probes and capture </li></ul><ul><ul><li>Extension and ligation based assay </li></ul></ul><ul><li>Interactive probe design and ordering </li></ul><ul><ul><li>Personalized and easy to use design tool </li></ul></ul><ul><ul><li>Rapid design turnaround – as little as 10 days from design to assay shipment </li></ul></ul>TruSeq Custom Amplicon Sequencing for high throughput tests
  21. 21. Conclusion <ul><li>For clinical applications quality of the sequence data is paramount </li></ul><ul><ul><li>Hybridization based capture provides more even coverage and much more reliable allele ratios (closer to the expected 50-50) </li></ul></ul><ul><ul><li>With the development of technology allowing capturing multiple libraries simultaneously sample preparation time is significantly shortened and cost is reduced </li></ul></ul><ul><ul><li>For very high throughput tests Illumina’s new Trueseq amplicon system is recommended </li></ul></ul>

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