Custom, Affordable Gene Panels with Superior Coverage and Uniformity

Custom, Affordable Gene Panels with

Superior Coverage and Uniformity

xGen® Predesigned Gene Capture Pools

Rami Zahr, NGS Product Manager

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INTEGRATED DNA TECHNOLOGIES

Overview

• xGen® Lockdown® Probes—performance advantages

• xGen Predesigned Gene Capture Pools—new custom enrichment options

• Advantages of xGen Predesigned Gene Capture Pools Better customizing ability

Decreased optimization time

Lower cost

Easy supplementing and augmenting of existing panels

• Summary

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Enrichment OverviewGenomic DNA

Fragment

Attach Adapters

Sequence

Generate Amplicons

Perform Hybrid Capture

Whole Genome Sequencing

Target Enrichment

Samples in Experiment ~1 10s-100s

Target Analysis Size 3 Gb Variable: 5 kb–60 Mb

Primary Applications• Discovery• Building a reference (de

novo)

• Rare variant discovery• Variant detection

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What Are xGen® Lockdown® Probes?

• In-solution hybridization probes

• 120-nucleotide DNA oligos with a 5′ biotin modification

• Synthesized using IDT’s proprietary Ultramer® synthesis process to ensure more full-length product

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Easy Ordering With Online Tool

1. Visit idtdna.com/xgen

2. Select “Learn more about xGen® Lockdown® Probes”

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Technical Overview—Target Enrichment

• Filter out unwanted sequences

• Focus only on the regions of interest

Begin with prepared library (using e.g., Illumina TruSeq® HT kit or Ion Torrent library prep kit)

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1. Incubate library with Cot-1 DNA and xGen® Blocking Oligos.

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2. Hybridize xGen Lockdown® Probes to target library fragments.

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3. Capture target fragments using streptavidin-bound magnetic beads.

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3. Capture target fragments using streptavidin-bound magnetic beads.

4. Apply magnet to isolate target fragments from non-target sequences.

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Various Formats of xGen® Lockdown® Probes

• xGen® Lockdown® Probes Mini (3 pmol/probe or 1 rxn/probe)

Standard (24 pmol/probe or 8 rxns/probe)

XL (192 pmol/probe or 64 rxns/probe)

• xGen Lockdown Panels Acute Myeloid Leukemia (264 genes)

Pan-Cancer (127 genes)

Inherited Diseases (4,503 genes)

16 and 96 rxns

• xGen Predesigned Gene Capture Pools—newest addition

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What Advantages Does IDT Offer?

IDT1. Oligos are individually synthesized at

very high coupling rate (99.5%)

2. Each oligo undergoes QC and is normalized

3. 7–10 day turnaround time

4. End-to-end tiling possible, reducing the number of probes

Competitors1. Oligos are synthesized in parallel on a

microarray (~98% coupling)

2. Molecules are cleaved off the array, so individual QC is not possible

3. 6–8 week turnaround time

4. Attempt to compensate coverage issues by “boosting”

5. Oligos for RNA baits may undergo transcription

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A higher coupling rate means more full-length probes

IDT Offers a Higher Base Coupling Rate

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What Advantages Does IDT Offer?

IDT1. Oligos are individually synthesized at

very high coupling rate (99.5%)

2. Each oligo undergoes QC and is normalized

3. 7–10 days turnaround time

4. End-to-end tiling possible, reducing the number of probes

Competitors1. Oligos are synthesized in parallel on a

microarray (~98% coupling)

2. Molecules are cleaved off the array, so individual QC is not possible

3. 6–8 weeks turnaround time

4. Attempt to compensate coverage issues by “boosting”

5. Oligos for RNA baits may undergo transcription

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High Performance of xGen® Lockdown® Probes

• One of the most important aspects of capture is uniformity

• Key targets with low depth of coverage limit the sample size

• High uniformity allows you to multiplex more samples and have more efficient target enrichment and sequencing

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Excellent Coverage of xGen® Lockdown® Probes

• 4 samples multiplexed on a MiSeq® run

• Average depth of coverage is 300X

• >99% covered by ≥30X

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Advantages of the xGen® Rapid Capture Protocol

• xGen® Lockdown® Probes have been optimized with the xGen Rapid Capture Protocol Hybridization time is reduced to 4 hr but can be extended to 18 hr

Total hands-on time is 6–8 hr

Complete library in 1 (long) day or 2 days

• xGen Rapid Capture Protocol provides the flexibility to expand or augment your panel without replacing it Purchase a supplementary panel of xGen Lockdown Probes and add the probes into

your current panel

Added probes do not affect the capture properties of existing probes in your pool

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xGen® Predesigned Gene Capture Pools

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What Are xGen® Predesigned Gene Capture Pools?

• Pools of xGen® Lockdown® Probes designed to target specific genes

• Delivered combined in a single tube or as separate gene pools in a plate

• Shipped wet and ready to use

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Features of xGen® Predesigned Capture Pools

• Target coding regions (CDS) of human RefSeq genes

• Designed with repeat masking and 1X tiling

• Flat price of $100/gene for 16 reactions or $150/gene for 96 reactions

• Ships in 7–10 days

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Supplement Existing Panels with xGen® Predesigned Gene Capture Pools

• Probes pools can be added into panels you have already purchased

• xGen® Predesigned Gene Capture Pools can be used with: IDT stocked panels

IDT custom panels

Panels from other vendors

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Advantages of xGen® Predesigned Gene Capture Pools

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Data from xGen® Predesigned Gene Capture Pools

• Data were generated from samples using: 20-gene panel with 395 exons

71 kb target space

804 total probes

• Samples were sequenced on a MiSeq® sequencer: Multiplex capture of 6 samples

300 bp paired end reads

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High On-Target Performance With xGen® Predesigned Gene

Capture Pools

804 probes

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Consistently Deep Coverage With xGen® Predesigned Gene

Capture Pools

804 probes

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Create Customized Panels for Each Project Using xGen®

Predesigned Gene Capture Pools

• Cancer types have different target genes Kandoth, et al. (2013) Nature

502:333–339

• Labs or cores studying multiple diseases (e.g., different types of cancer) may use a combination of overlapping and different gene groups

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Common: TP53 KRAS PK3CA KMT2C KMT2D

BLCA: EP300 AARD1A KDM6A RB1

AML: NPM1 DNMT3A FLT3

Share xGen® Predesigned Gene Capture Pools Between Multiple Researchers

• Researchers in your lab study different diseases(e.g., bladder urothelial carcinoma and acute myeloid leukemia)

• Some genes are common to both diseases, but others occur in only one of these diseases

• xGen® Predesigned Gene Capture plates allow the researchers to share the common genes and add disease-specific genes for each study

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Competitor Optimization Time is 16–24 Weeks

6–8

wee

ks Order custom capture panel 4

–8 w

eeks Test the

custom panel

6–8

wee

ks Order optimized capture panel (version 2)

Total optimization time: 16–24 weeks (4–6 months)Most of the optimization time spent waiting for panel

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IDT Optimization Time is Only 6–11 Weeks

Order xGen® Predesigned Gene Capture panel

Test the panel

Order supplementary probes for the panel

1–1.5 weeks

4–8 weeks

1–1.5 weeks

Total time: 6–11 weeks (1.5–3 months) Most of the optimization time spent testing the panel

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Decrease the Cost of Target Capture

• xGen® Lockdown® Probes are your target capture solution

• A custom panel is too expensive and may contain too much material

• Example: 125 protein-coding genes of xGen Pan-Cancer Panel

4962 probes

List price on the mini scale $74,430

125 xGen Predesigned Gene Capture Pools cost $12,500 for 16 reactions and $18,750 for 96 reactions

Save $61,970 (83% less than panel) on 16 reactions and save $55,680 (75% less than panel) on 96 reactions

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Expand Existing Panels with xGen® Predesigned Gene Capture Pools

• Expand an existing IDT stocked panel or custom panel No need to purchase a new panel that targets all the regions

Keep your existing panel—spike-in performance shows similar results to original panel

• xGen® Predesigned Gene Capture Pools have a turnaround time of 7–10 days, allowing you to quickly obtain and validate your new panel

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Supplement Existing Panels with xGen® Predesigned Gene

Capture Pools

• Supplemented xGen® Pan-Cancer Panel with xGen Predesigned Gene Capture Pools for 3 genes

• Supplemented probes were added in equal molar quantity with no optimization

• Targets from 8 multiplexed samples captured and sequenced on a MiSeq® sequencer

• Excellent coverage by supplemented probes with no adverse effect on the rest of the panel

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Supplementing Existing Panels

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Supplemented Probes Have Comparable Depth of Coverage to Existing Panel Probes

• xGen® Pan-Cancer Panel average depth of coverage: 308X

• Supplemented Gene Probe Pools:

Target Genes Probes Average Depth of Coverage

LDLR 47 271X

CKM 19 247X

PYGM 37 263X

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Use xGen® Predesigned Gene Capture Pools toAugment Panels From Other Vendors

• Example: You purchased a custom panel from another vendor1. You need to increase the target space of your panel by adding more genes, or

2. You are unhappy with the performance of portions of your panel

• Solution: Add xGen® Predesigned Gene Capture Pools into your panel, even if that gene is already present in the panel

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Summary

• xGen® Lockdown® probes provide highly uniform target capture

• xGen Gene Predesigned Capture Pools: Pools of xGen Lockdown Probes designed to target the CDS of human

genes

An affordable solution for creating your own panels or supplementing existing panels, including panels from other vendors

Decrease optimization time and increase efficiency of sequencing

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Questions?

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Build Panels to Suit Your Budget

• Build your own panel with xGen® Predesigned Gene Capture Pools

• Example: Average cost for 20 coding genes with a custom panel using xGen® Lockdown®

Probes—$6,500

20 xGen Predesigned Gene Capture Pools:

• $2,000 for 16 reactions

• $3,000 for 96 reactions

20-gene panel can be used in multiplex capture so that 10 samples are run in a single capture:

• 160 samples for 16-reaction size

• 960 samples for 96-reaction size

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Decrease the Cost of Target Capture

• Coding regions are not the only targets of interest

• With xGen® Predesigned Gene Capture Pools you can reduce the overall cost of your panels and still target non-coding regions

• Example: You need 30 genes—5 with UTRs, 2 with introns, and 5 intergenicregions Input the 30 genes as xGen Predesigned Gene Capture Pools

Input the remaining regions as custom xGen Lockdown® Probes

Reduce the cost of the 30 genes by $7,000 for 16 reactions and $5,500 for 96 reactions

Custom, Affordable Gene Panels with Superior Coverage and Uniformity

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