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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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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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

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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