peter nagy, columbia agilent symposium, jan, 27 2012
TRANSCRIPT
Peter L. Nagy MD, PhD; [email protected]; 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
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Mitochondrial genome amplification using traditional PCR
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: 71Std Dev: 58
Illumina exome capture cardiomyopathy data
Mean coverage: 50Std 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 captureAlzheimer panel data
Mean coverage: 487Std 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