Integrated DNA Technologies
xGen® Lockdown® Probes
Rami Zahr, Scientific Application Representative
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
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 + ++ +++
Detecting rare variants requires enrichment
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100% 80% 60% 40% 20% 10% 5% 3% 2% 1%
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Expected Mutation Rate
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
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)
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
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
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)
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
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
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
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
xGen® Lockdown® Probe design
Design approach: Tile probes on the target region
Design considerations: Probe length
Tiling depth
Flanking region size
Repeat-masking
Probe Design
Deeper Coverage
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0 500 1000 1500 2000 2500 3000
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Coverage Depth
133 kb Capture 29 kb Capture
99.9% of sequences covered >500X
99.9% of sequences covered >750X
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
Duncavage 2011 manuscript
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
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
xGen®TM xGen® Lockdown®TM Probes show less GC bias
Foundation Medicine Boston, Massachusetts
Augment Performance of Existing Exome Captures
Before supplementation with xGen™ Lockdown™ Probes
After supplementation with xGen™ Lockdown™ Probes
Improve Coverage and Uniformity
0%
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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
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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
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
Blocking oligos
Two classes of blocking oligos are needed: 1) Cot1 DNA = Alu, LINE repeat elements 2) linkers/adaptors
Blocking Oligos Efficacy
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