Target enrichment enables researchers to focus their next generation sequencing (NGS) efforts on regions of interest, allowing them to obtain more sequencing data relevant to their study. In-solution target capture is a method of enrichment using oligonucleotide probes directed to specific regions within a genome. Target capture can be used to enrich multiple samples simultaneously, reducing the cost per sample, while using individually synthesized probes allows researchers to construct gene panels that can be optimized over time.

1. Expanding Your Research Capabilities Using
Targeted Next Generation Sequencing
Rami Zahr, NGS Field Application Specialist
Ibrahim Jivanjee, NGS Product Manager
Integrated DNA Technologies

2. A Brief History of DNA Sequencing
1990 – Human Genome Project
2000 – Draft of human genome
2002 – Capillary sequencers introduced
2003 – Completed human genome
2004 – Pyrosequencing introduced
2005 – “Sequencing by Synthesis”
2007 – “Sequencing by Ligation”
2008 – Heated competition
2011 – Bench-top platforms introduced
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3. Impact of Next Generation Sequencing
Academia
Industry
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4. IDT and Next Generation Sequencing
Work with thought leaders to understand and address distinct
challenges
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The Genome Institute, Washington University (DECODED 3.1)
Foundation Medicine, Inc. (DECODED 2.3)
Cuppen Lab, Hubrecht Institute (DECODED 2.1)
Tsai Lab, North Carolina State University (DECODED 1.3)
Barrick Lab, University of Texas at Austin (DECODED 3.3)
The GenePool, University of Edinburgh (DECODED 2.4)
Find these articles by searching on NGS Your Research at www.idtdna.com
The DECODED newsletter is available at www.idtdna.com/decoded
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5. Enabling Research Through Custom Biology Consumables
Integrated DNA Technologies (IDT) is a leader in the development and
manufacture of custom biology products for the research and diagnostic life
science markets.
• Founded in 1987
• Largest custom oligonucleotide
manufacturer worldwide
Goal for NGS: Leverage knowledge of DNA
synthesis to provide…
• The highest quality, least biased
scientific results
• The greatest level of flexibility and
customization
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6. xGen® Target Capture Products
xGen® Lockdown® Probes
 Individually synthesized and QC’d
 Lengths of 60–120 nt
 7–10 business day TAT
xGen® Standard Blocking Oligos
 Predesigned for easy ordering
 Select for only needed adapters
 2–6 business day TAT
xGen® 48-Hour Capture Protocol
 48-hour hybridization, 2–4 hours hands-on time
 DIY buffers and reagents, recipes provided
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7. xGen® Target Capture Products *New*
xGen® Acute Myeloid Leukemia Cancer Panel v1.0
 260 genes, 11.7 k probes, 1.2 Mb
 Based on findings published by The Cancer Genome Research Network (2013) [N Engl J
Med, 368:2059–2074]
 Next day TAT
xGen® Universal Blocking Oligos
 Single oligo sequence blocks many barcoded adapters simultaneously
 Consistent on-target performance even with high multiplex captures
 Next day TAT
xGen® 4-Hour Capture Protocol
 4-hour hybridization, 2–4 hours hands-on time
 High uniformity of enrichment
 Requires Nimblegen Buffers & Reagents
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8. Applications of Targeted Next Generation Sequencing
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9. What is Target Enrichment?
Whole Genome
Sequencing
Target
Enrichment
Genomic DNA
Fragmentation
Attach Adapters
Hybrid Capture
Amplicon
Generation
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
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10. Protocol – Overview
Begin with prepped library
from Illumina (or other library
prep) kit
Hybridize library to probes for 4
hours
Use magnetic beads with
streptavidin to sequester
targets from the remainder of
the library
Wash the beads and elute the
targets
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11. Probe Performance And Validation
Goal: Validate the performance of the individual probe
 Studied Tm of hybridization of a single 120mer oligo to different
targets having 0–7 bases mismatched (permissive G:T pairing or
more disruptive T:T pairing)
 Also studied targets with 1, 3, or 7 base insertions (indels)
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12. 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 had either 1, 3, or 7 G-T or T-T
mismatches
120 bp
120 bp
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13. Probe Performance And Validation – Conclusion
 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 a 120mer capture probe is sufficient
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14. Applications - Overview
Application strengths of in-solution hybridization:
 Identify integration sites of transposons and viral genomes
 Capture novel translocations and recombinations
 Chromosomal translocation
 V(D)J recombination
 Splice variant
 Capture novel SNPs in regions of interest
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15. Applications – Viral and Transposon Integration Sites
 The known sequence is the viral genome or the transposon
sequence
 The researcher is interested in finding the integration location
 Tile probes against the transposon sequences
 Sequence flanking, unknown sites
Unknown
Integration Site
Known Viral/Transposon Sequence
Unknown
Integration Site
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16. Applications – Viral and Transposon Integration Sites
 Target the regions you want to focus on in various ways:
Target
Unknown
Integration Site
Target
Unknown
Integration Site
Target
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17. Applications – Translocation and Recombination
 The known sequence is one or more regions suspected of moving in
the genome
 The researcher is interested in identifying recombination or
translocation events within their region of interest
 Tile probes against regions of interest
 Sequence flanking unknown sites
Unknown Fusion
Site
Region of Interest
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18. Applications – Translocation and Recombination
Target the regions you want to focus on in various ways:
Unknown Fusion
Site
Target
Sequence unknown translocation
Target 1
Target 2
Target fusion events
Target 1
Target 2
Target different sites to see if they recombine
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with one another or with different sites

19. Applications – Genotyping
 Known sequence contains the SNP or indel
 The researcher wants to find the SNP or indel in their region of
interest
 Center probe on the SNP
Reference Sequence
SNP
Reference Sequence
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20. Applications – Genotyping (Pitfalls)
What if you were trying to enrich with PCR?
Primer
“Reference” Sequence
SNP
“Reference” Sequence
Primer
A single mismatch can cause up to 7°C ΔTm which can dramatically
reduce PCR efficiency
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21. 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
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22. 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
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23. AML Panel Performance – Read Statistics
Off-target
Duplicate On-Target
500 bp Flank
On-Target
10
9
8
Reads (Millions)
7
6
5
4
3
2
1
0
#10
#11
#17
#23
#10
#11
#17
#23
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24. 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
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25. Improve Coverage and Uniformity
# Reads
Data from Foundation Medicine comparing results of a large set of
IDT xGen® Lockdown® Probes with a focused Agilent SureSelect® set.
IDT xGen® Lockdown® Probes: 100% >150X coverage
Agilent SureSelect® set: 80.7% >150X coverage
Foundation Medicine
Boston, MA, USA
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26. xGen® Lockdown® Probes Show Less GC Bias
CDS regions
have GC content
between 0.47
and 0.61
5’ UTR regions
that can affect
expression have
GC content
between 0.48
and 0.72
Zhang L, Kasif S, et al. (2004)
PNAS, 101(48):16855–16860.
Foundation Medicine
Boston, Massachusetts
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27. xGen® Standard Blocking Oligos
 Complimentary to the adapter sequences with modification to
inhibit extension
 Bind to the adapter sequences attached to the library to inhibit
hybridization of the adapters to one another
 Available for Illumina, Ion Torrent, and Roche platforms
 Can be used on indexed adapters
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28. Blocking Oligos—Function
Two classes of blocking
oligos are needed:
I) Cot1 DNA = Alu, LINE
repeat elements
II) linkers/adapters
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29. Blocking Oligos – Efficacy
 ~100% more ontarget reads after
blocking
 Increased reads
enable researchers
to get more depth
or multiplex more
samples
Blumenstiel B, Cibulskis
K, et al. (2010) Curr Protoc
Hum Genet, Chapter
18:Unit 18.4.
Hodges E, Rooks M, et al.
(2009) Nat Protoc,
4(6):960–974.
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30. xGen® Universal Blocking Oligos
 A single sequence that can block multiple indices
 Greatly reduce the number of blocking oligos needed in an
experiment, decreasing cost and complexity of the target
enrichment
 Perform better than the individual index blocking oligos or blocking
oligos with inosines
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31. 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
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32. Summary
 xGen® Lockdown® Probes are high quality, individually QC’d oligos
 xGen® Lockdown® Probes enable researchers to identify insertion
sites, splice junctions, indels, and SNPs
 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 the xGen® Universal Blocking Oligos
can block many indices
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33. More Information
For more information, or if you have questions, about IDT xGen®
products, visit our website: www.idtdna.com/xgen
or e-mail us at xGen@idtdna.com.
See how your
colleagues are
successfully using
these IDT NGS
products by
searching on NGS
Your Research at
www.idtdna.com.
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