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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25,000

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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)