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

1. Integrated DNA Technologies
xGen® Lockdown® Probes
Rami Zahr, Scientific Application Representative

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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. xGen® Lockdown® Probe design
 Design approach: Tile probes on the target region
 Design considerations:
 Probe length
 Tiling depth
 Flanking region size
 Repeat-masking

15. Probe Design

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. 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. Duncavage 2011 manuscript

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. 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. xGen®TM xGen® Lockdown®TM Probes show less GC bias
Foundation Medicine
Boston, Massachusetts

22. Augment Performance of Existing Exome Captures
Before supplementation with
xGen™ Lockdown™ Probes
After supplementation with
xGen™ Lockdown™ Probes

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. 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. Blocking oligos
Two classes of blocking
oligos are needed:
1) Cot1 DNA = Alu, LINE
repeat elements
2) linkers/adaptors

26. Blocking Oligos Efficacy

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