oligonucleotides for next generation sequencing research and clinical diagnostics
TRANSCRIPT
John Havens, PhD
VP, Business Development
Integrated DNA Technologies
AMP Workshop 2014
Oligonucleotides for Next Generation Sequencing
Research and Clinical Diagnostics
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INTEGRATED DNA TECHNOLOGIES
IDT Hybridization Enrichment Panels
• Stocked panels• Acute Myeloid Leukemia
• 264 genes, recurrently mutated in 200 AML patients, 1.4 Mb target space• Developed with TCGA, Wash U Genome Institute, Tim Ley• Published in NEJM 368, 2059 (2013)
• Pan-Cancer• 127 genes, from 12 cancers analyzed in 3281 tumors, 800 kb target space• Developed with TCGA, Wash U Genome Institute, Li Ding• Published in Nature 502, 333 (2013)
• Inherited Diseases
• 4503 genes, from Human Genetic Mutation Database
• Developed with Emory Genetics Laboratory, Madhuri Hegde
• Custom panels
• Supplements to stocked panels—fast, inexpensive
• Completely custom sets—fast, inexpensive if more than a few samples
• Wide range in target space from 700 bases to 2 Mb
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INTEGRATED DNA TECHNOLOGIES
xGen® Predesigned Gene Capture Pools
• Pools of xGen® Lockdown®
probes targeting all exons of designated genes
• Available as individual pools for each gene or in one tube combining pools for all genes
• Shipped in solution and ready to use
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INTEGRATED DNA TECHNOLOGIES
Acute Myeloid Leukemia Targeted Enrichment
• IDT AML set covering 264 genes spiked into NimbleGen exome
• ~100% coverage of the AML target space
• Coverage depths for AML set ~500X, allowing clone detection down to ~5%
The Genome Institute, Washington University
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INTEGRATED DNA TECHNOLOGIES
High-Fidelity Individual Oligo Synthesis
• Per-step coupling efficiency has a big influence on full-length yield
• 99.6% coupling efficiency enables oligos of 200 bases
• Full-length desalted yields:• 60mers: 79%
• 120mers: 62%
• 200mers: 45%
• 5′ chemical biotinylation improves effective purity
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INTEGRATED DNA TECHNOLOGIES
ESI Mass Spectroscopy for Quality Control
BioGCGGCGAGCGGAGATCCGGGGCCTGCGCTGCGCACTCGAGCCTGGCGGGCCGGCACGGTGCGGGCCATGAGCGGGGCGGTGCCCCAGGACCTAGCGGTGAGTGGCGGCCGAGTCGGGCAC
ESI-MS trace of an
xGen® Lockdown® probe
with 78% GC content
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INTEGRATED DNA TECHNOLOGIES
Synthesis Failures
• 120mer DNA probes with 5′ biotin
• Each probe requires ~5000 steps to manufacture
• ~116,000 probes for inherited diseases panel of ~4500 genes
• 62% full-length product
• Most truncation products capped and not biotinylated
• QC failures remade and included in pool if they pass
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ass
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Probe GC Content
Number of Probes First Pass Fail Rate
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INTEGRATED DNA TECHNOLOGIES
Enhanced Hybridization Probes for NGS Target Enrichment
• Individually synthesized
• Individually QCed by ESI MS
• Individually normalized
• Chemically biotinylated
• Failures resynthesized
• Normally 120mers, but range can be60–200mers
• Processed robotically
• Available as 21 CFR Part 820 (GMP)
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INTEGRATED DNA TECHNOLOGIES
High-Efficiency Universal Blockers
• Blockers remove adapter participation in hybridization enrichment
• Universal blockers contain inosines that hybridize to barcodes and proprietary modifications for improved affinity
• On-target percentages can show 60% improvement
• This translates to lower sequencing costs, particularly for multiplexed samples
Foundation Medicine
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INTEGRATED DNA TECHNOLOGIES
Barcoded Adapters for Library Preparation
Custom adapters available:• More barcodes
• Dual indexing to minimize cross contamination
• Degenerate barcodes for molecular indexing
• TruGrade™ processing
The Genome Institute, Washington University
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INTEGRATED DNA TECHNOLOGIES
TruGrade™ Analysis from the Sanger Institute
• “With HPLC (Company A) or PAGE (Company B) purification, approximately 0.56% and 0.34% mapped to missing barcodes.”
• “With TruGrade this was dramatically reduced to just 0.03%.”
Mike Quail
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INTEGRATED DNA TECHNOLOGIES
gBlocks® Gene Fragments as NGS Standards
• dsDNA of 125–2000 bp
• Copy number variants
• Troubleshooting library preparation
• Rare-allele detection
• Sequencing accuracy as a function of sequence composition
Carlson, et al., Nature Comm. 2013.
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INTEGRATED DNA TECHNOLOGIES
Summary of IDT NGS Products
• Individually synthesized, QCed, normalized hybridization probes offer:• Complete, uniform capture of the target space
• 4-hour hybridization time
• Insensitivity to targets with minor mismatches
• Capability for SNV, indel, CNV, LOH, and translocation detection
• 21 CFR Part 820 manufacture for clinical research and diagnostics
• Stocked or custom panels
• Universal blockers improve on-target performance for multiplexed samples
• Custom adapters available to improve sequencing performance
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INTEGRATED DNA TECHNOLOGIES
Additional Resources• xGen® Lockdown® Panels
www.idtdna.com/xGen
• Videoswww.youtube.com/idtdnabio
• Improving NGS Target Capture
• Expanding Your Research Capabilities Using Targeted NGS
• IDT DECODED newsletter articleswww.idtdna.com/DECODED
• Core Concepts: Target Enrichment Facilitates Focused Next Generation Sequencing
• Your Research: Insertion Site Detection and Targeted RNA Capture Using Next Generation
Sequencing (Cofactor Genomics, St. Louis, MO)
• Your Research: Next Generation Sequencing in the Clinic: A Perspective from Dr Elaine Mardis
(Washington University, St. Louis, MO)
• Your Research: Target Enrichment Identifies Mutations that Confer Fitness Effects (University of
Texas, Austin, TX)