xGen® Lockdown® Probes

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Focus your sequencing on a subset of the genome that is interesting to you with IDT xGen® Lockdown® Probes.

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xGen® Lockdown® Probes

  1. 1. Integrated DNA Technologies xGen® Lockdown® Probes Rami Zahr, Scientific Application Representative
  2. 2. What is target enrichment? Genomic DNA Fragmentation Attach Adapters Sequence Amplicon Generation Hybrid Capture Whole Genome Sequencing Target Enrichment Samples in Experiment 1 – 10 samples 100s-1000s Target Analysis Size 3 Gb Variable: 5 kb – 60 Mb Primary Applications Discovery Building a reference (De Novo) Rare Variant Discovery Variant Detection
  3. 3. The value of enrichment – increase multiplex, save $$$ Run Parameters Human Genome Human Exome Custom 500 kb # of Samples @ 30X coverage / cost per sample 10 / $2k 200 / $500 40,000 / <$1 # of Samples @ 500X / cost per sample 0.5 / $20k 12 / $1.8k 2,400 / ~$8 Example: A HiSeq 2000 run costs approximately $20k Application Human Genome Human Exome Custom 500 kb Reference Building +++ ++ + General Discovery ++ ++ + Diagnostics + ++ +++ Rare Variants + ++ +++ Ease of Analysis + ++ +++
  4. 4. Detecting rare variants requires enrichment 0 500 1000 1500 2000 2500 3000 3500 100% 80% 60% 40% 20% 10% 5% 3% 2% 1% DepthofCoverageRequired Expected Mutation Rate
  5. 5. Comparing enrichment methods Method Hybrid Capture Amplicon Enrichment Workflow Complex (-) Slow [1-2 days] (-) Straightforward (+) Fast [<1 day] (+) Cost* Higher upfront cost (-) Lower cost per sample (+) Lower upfront cost (+) Higher cost per sample (-) Performance High sensitivity (+) Lower specificity (-) Sequence or Tm biases (-) Good sensitivity (+) High specificity (+) Amplification biases (-) Capture Size Large: from 5kb to the entire human exome Small: from 5kb to ~500kb Applications Multiple: Variant Analysis, Indel Analysis, CNV, Splice Variants, Translocations Limited: Variant Analysis *Depends on scale
  6. 6. xGen® Lockdown® Probes – Oligo Specifications  Probes are 60-120 base Ultramers with a 5’ biotin modification  Each probe additionally undergoes mass spectrometry for quality control  The probes are available at three different yields and two formats:  2pmol yield (single pooled tube)  20pmol yield (single pooled tube or 96 well plates)  200pmol yield (single pooled tube or 96 well plates)
  7. 7. Protocol  Simple protocol that that contains all the components of the buffers in the experiment  Compatible with Illumina MiSeq library preparation and can be adjusted for use with other platforms  No specialized equipment needed – all of the reagents and machines are commonly found in molecular biology labs
  8. 8. Protocol – Overview Prepped Library from Illumina kit (or other library prep kit) Hybridize library to probes for 48 hours Use magnetic beads with streptavidin to sequester targets from the remainder of the library Wash the beads and then elute the targets
  9. 9. Do mutations in target hurt capture efficiency?  Long 120mers are very tolerant to mismatch  How tolerant?  Studied Tm of hybridization of a single 120mer bait oligo to different targets having 0-7 bases mismatch (permissive G:T pairing or more disruptive T:T pairings)  Also studied targets with 1, 3, or 7 base insertions (indels)
  10. 10. Design of 120mer Tm experiment 120 bp 120 bp 1, 3, or 7 bp (All T) 7 bp (All T or All C) 7 bp (All T or All C) Top strand = 121, 123, or 127 bp respectively Top strand = 134 bp 1 bp mismatch (G-T or T-T) 120 bp 120 bp 120 bp 120 bp Ultramers had either 1, 3, or 7 G-T or T-T mismatches
  11. 11. DTm with 1-7 base mismatches (SNPs) Mismatches Tm oC Measured DTm oC Mismatch Tm oC Predicted 0 85.7 -- 87.6 1 T-T 85.6 - 0.1 87.1 1 T-T 85.0 - 0.7 86.9 3 T-T 84.2 - 1.5 85.7 7 T-T 80.9 - 4.8 82.9 7 T-G 81.6 - 4.1 85.8 120 bp
  12. 12. DTm with 1, 3, or 7 base insertions (indels) 1, 3, or 7 bp (All T) Top strand = 121, 123, or 127 bp respectively 7 bp (All T or All C) 7 bp (All T or All C) Top strand = 134 bp Bulge Tm oC Measured DTm oC Mismatch None 85.7 -- 1 T 85.3 - 0.4 3 T 84.8 - 0.9 7 T 83.9 - 1.8 7 T + 7 T 82.3 - 3.4 7 C + 7 C 82.4 - 3.3
  13. 13. Conclusions from Tm studies  1-7 base mismatches had < 5°C ΔTm  1 or 2 1-7 base insertions had < 4°C ΔTm  These small changes in Tm will not affect capture  Thus use of 120mer capture probes is sufficient
  14. 14. xGen® Lockdown® Probe design  Design approach: Tile probes on the target region  Design considerations:  Probe length  Tiling depth  Flanking region size  Repeat-masking
  15. 15. Probe Design
  16. 16. Deeper Coverage 0.00% 10.00% 20.00% 30.00% 40.00% 50.00% 60.00% 70.00% 80.00% 90.00% 100.00% 0 500 1000 1500 2000 2500 3000 %ofSequences>XCoverage Coverage Depth 133 kb Capture 29 kb Capture 99.9% of sequences covered >500X 99.9% of sequences covered >750X
  17. 17. Using NextGen seq methods to study Merkel Cell Carcinoma  Merkel Cell Carcinoma (MCC) – rare and highly aggressive neuroendocrine carcinoma of the skin  Usually caused by integration of Merkel cell polyomavirus genome (5.3Kb) into host DNA  Difficult to study - insertion appears to be fairly random with mutation and deletion rates
  18. 18. Duncavage 2011 manuscript
  19. 19. Merkel Cell Polyomavirus capture probes – improved method  Merkel Cell Polyomavirus Capture – 2012 study from the Washington University Genome Center  Employed IDT xGen® Lockdown®TM probes (45 x 120bp, 5’ biotinylated)  26 patient samples: 25 captured with IDT probe and one replicate capture with biotinylated PCR amplicons  2 MiSeq runs each produced ~900Mb per run (13 sample pool)  2 GAIIX lanes each produced ~5Gb per lane (13 sample pool)  Achieved 77x better depth of coverage than the Duncavage study using PCR- generated probes with the same number of reads Washington University Genome Center St. Louis, Missouri
  20. 20. Improve coverage and uniformity Results from Foundation Medicine comparing results of a large set of IDT xGen® Lockdown®TM probes with a focused Agilent SureSelectTM set. IDT xGen®: 100% >150x coverage Agilent: 80.7% >150x coverage # Reads Foundation Medicine Boston, Massachusetts
  21. 21. xGen®TM xGen® Lockdown®TM Probes show less GC bias Foundation Medicine Boston, Massachusetts
  22. 22. Augment Performance of Existing Exome Captures Before supplementation with xGen™ Lockdown™ Probes After supplementation with xGen™ Lockdown™ Probes
  23. 23. Improve Coverage and Uniformity 0% 2% 4% 6% 8% 10% 12% 14% 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 %ofTotalCoverage Normalized Coverage xGen Lockdown Probes RNA Capture RNA Capture More sequences with reduced coverage xGen™ Lockdown™ Probes: 53.4% target covered within X ± 0.2 RNA Capture: 44.4% target covered within X ± 0.2
  24. 24. Blocking Oligos  Blocking Oligos are used to inhibit binding to the adapter sequences  Complimentary to the adapter sequences with modification to inhibit extension  Can be used on indexed adapters  Available for Illumina, Ion Torrent, and Roche platforms
  25. 25. Blocking oligos Two classes of blocking oligos are needed: 1) Cot1 DNA = Alu, LINE repeat elements 2) linkers/adaptors
  26. 26. Blocking Oligos Efficacy
  27. 27. Summary  xGen® Lockdown® probes are high quality, individualy QC’ed oligos  Provide capture of regions with high uniformity even with significant mutations  Blocking oligos used with xGen® Lockdown® probes increase on- target captures and are available for a variety of platforms

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