IDT provides oligonucleotides and panels for targeted sequencing including stocked and custom gene panels. Their panels include 264 genes for acute myeloid leukemia, 127 genes for pan-cancer analysis, and 4503 genes for inherited diseases. IDT probes are individually synthesized and quality controlled before being pooled. Universal blockers improve on-target rates by blocking adapter participation. Additional services include custom barcoded adapters and gBlocks fragments for quality control.

1. Oligonucleotides for Next Generation Sequencing
Research and Clinical Diagnostics
John Havens, PhD
VP, Business Development
Integrated DNA Technologies
AMP Workshop 2014

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IDT Hybridization Enrichment Panels
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• 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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xGen® Predesigned Gene Capture Pools
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• 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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Acute Myeloid Leukemia Targeted Enrichment
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• 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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High-Fidelity Individual Oligo Synthesis
• Per-step coupling efficiency has a big
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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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ESI Mass Spectroscopy for Quality Control
BioGCGGCGAGCGGAGATCCGGGGCCTGCGCTGCGCACTCGAGCCTGGCGGGCCGGCACGGTGCGGGCC
ATGAGCGGGGCGGTGCCCCAGGACCTAGCGGTGAGTGGCGGCCGAGTCGGGCAC
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ESI-MS trace of an
xGen® Lockdown® probe
with 78% GC content

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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
Number of Probes First Pass Fail Rate
40,000
35,000
30,000
25,000
20,000
15,000
10,000
5,000
0
12%
10%
8%
6%
4%
2%
0%
Number of Capture Probes
First-Pass Fail Rate
Probe GC Content

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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 be
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60–200mers
• Processed robotically
• Available as 21 CFR Part 820 (GMP)

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High-Efficiency Universal Blockers
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• 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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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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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
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reduced to just 0.03%.”
Mike Quail

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gBlocks® Gene Fragments as NGS Standards
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• 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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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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Additional Resources
• xGen® Lockdown® Panels
www.idtdna.com/xGen
• Videos
www.youtube.com/idtdnabio
• Improving NGS Target Capture
• Expanding Your Research Capabilities Using Targeted NGS
• IDT DECODED newsletter articles
www.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)