IDT provides a range of solutions for targeted next generation sequencing. Labs processing hundreds to thousands of samples can create highly uniform, custom panels using xGen® Lockdown Probes. The new xGen Acute Myeloid Leukemia (AML) panel is a predesigned set of Lockdown Probes that captures 260 genes identified by whole genome and exome sequencing of 200 patient samples. The AML panel can be used as stand-alone or customized with additional probes to detect other targets of interest.
Custom Enrichment Panels for Targeted Next Generation Sequencing
1. Custom Enrichment Panels for Targeted Next
Generation Sequencing
Rami Zahr, NGS Field Application Specialist
Integrated DNA Technologies
2. What is Target Enrichment?
Whole Genome
Sequencing
Target
Enrichment
Genomic DNA
Perform Fragmentation
Attach Adapters
Perform
Hybrid Capture
Generate
Amplicons
Sequence
Samples in Experiment
Target Analysis Size
Primary Applications
1–10 Samples
100s–1000s
3 Gb
Variable: 5 kb–60 Mb
Discovery
Building a reference (de novo)
Rare variant discovery
Variant detection
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3. IDT xGen® Target Enrichment Products
xGen® Lockdown® Probes
Individually synthesized and quality control (QC) tested
Lengths of 60–120 nt
7–10 business day TAT
xGen® Acute Myeloid Leukemia Cancer Panel v1.0
260 genes, 11.7K probes, 1.2 Mb
Based on findings published by The Cancer Genome Atlas Research Network (2013)
[N Engl J Med, 368:2059–2074]
xGen® Universal Blocking Oligos
Single oligo sequence blocks many barcoded adapters simultaneously
Consistent on-target performance even with high multiplex captures
xGen® 4-Hour Capture Protocol
4-hour hybridization, 2–4 hours hands-on time
High uniformity of enrichment
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4. Probe Performance and Validation—Design of Tm Experiment
1, 3, or 7 bp (All T)
7 bp (All T or All C)
Top strand = 121, 123, or 127 bp respectively
120 bp
7 bp (All T or All C)
Top strand = 134 bp
120 bp
1 bp mismatch (G-T or T-T)
120 bp
120 bp
Ultramer® Oligonucleotides with 1, 3, or 7 G-T or T-T mismatches
120 bp
120 bp
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5. Probe Performance and Validation—Conclusion
1–7 base mismatches had <5°C ΔTm
One or two 1–7 base insertions had <4°C ΔTm
These small changes in Tm will not affect capture
Thus use of a 120mer capture probe is sufficient
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8. AML Panel Performance—Uniformity
>0.2 x Mean Coverage
>0.5 x Mean Coverage
>1.0 x Mean Coverage
1
0.9
0.8
% of Targets
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
1
2
Replicate Number
3
4
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10. Summary
xGen® Lockdown® Probes are high quality, individually quality
controlled oligos
The xGen® AML Panel v1.0 provides a large list of genes that
researchers can use as a starting point to create a customized panel
at low cost, for high performance
xGen® Standard Blocking Oligos used with xGen® Lockdown® Probes
increase on-target capture, and xGen® Universal Blocking Oligos can
block many indices
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