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Peter Nagy, Columbia Agilent Symposium, Jan, 27 2012
 

Peter Nagy, Columbia Agilent Symposium, Jan, 27 2012

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    Peter Nagy, Columbia Agilent Symposium, Jan, 27 2012 Peter Nagy, Columbia Agilent Symposium, Jan, 27 2012 Presentation Transcript

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