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
0
5,000
10,000
15,000
20,000
25,000
30,000
35,000
40,000
0%
2%
4%
6%
8%
10%
12%
Nu
mb
er
of
Cap
ture
Pro
be
s
Firs
t-P
ass
Fail
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e
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)