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Custom Enrichment Panels for Targeted Next Generation Sequencing

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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.

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Custom Enrichment Panels for Targeted Next Generation Sequencing

  1. 1. Custom Enrichment Panels for Targeted Next Generation Sequencing Rami Zahr, NGS Field Application Specialist Integrated DNA Technologies
  2. 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. 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. 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. 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. 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. 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. 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. 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. 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

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