The document discusses custom enrichment panels for targeted next-generation sequencing, focusing on the xgen® lockdown® probes and various target enrichment products offered by Integrated DNA Technologies. It highlights the performance and applications of the xgen® acute myeloid leukemia cancer panel, including its design, efficiency, and the use of universal blocking oligos to improve capture rates. Overall, it emphasizes the high quality and customizable nature of these products for genomic research.

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
2

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
3

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
4

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
5

6. AML Panel Performance—Fold Enrichment
300
Average Coverage
Depth
Fold Enrichment
750
700
250
650
600
550
150
500
100
Fold Enrichment
Average Coverage Depth
200
450
400
50
350
0
300
#10
#11
#17
#23
#10
#11
#17
#23
6

7. AML Panel Performance—Alignment Breakdown
Off-target
Duplicate On-Target
500 bp Flank
On-Target
100%
90%
80%
70%
% of Reads
60%
50%
40%
30%
20%
10%
0%
#10
#11
#17
#23
#10
#11
#17
#23
7

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
8

9. xGen® Universal Blocking Oligos
60.00
On-Target Reads (%)
50.00
40.00
30.00
20.00
10.00
xGen® Universal Blocking
Oligos
xGen® Standard Blocking
Oligos
w/ no inosines
Standard Blocking Oligos
w/ inosine barcodes
9

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
10