Peter L. Nagy MD, PhD; pln2104@columbia.edu; 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