expanding your research capabilities using targeted ngs
DESCRIPTION
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.TRANSCRIPT
Integrated DNA Technologies
Expanding Your Research Capabilities Using Targeted Next Generation Sequencing
Rami Zahr, NGS Field Application Specialist Ibrahim Jivanjee, NGS Product Manager
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A Brief History of DNA Sequencing
1990 – Human Genome Project2000 – Draft of human genome2002 – Capillary sequencers introduced2003 – Completed human genome2004 – Pyrosequencing introduced2005 – “Sequencing by Synthesis”2007 – “Sequencing by Ligation”2008 – Heated competition2011 – Bench-top platforms introduced
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Impact of Next Generation Sequencing
IndustryAcademia
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IDT and Next Generation Sequencing
Work with thought leaders to understand and address distinct challenges
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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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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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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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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Applications of Targeted Next Generation Sequencing
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What is Target Enrichment?
Genomic DNA
Fragmentation
Attach Adapters
Sequence
Amplicon GenerationHybrid 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 ApplicationsDiscovery
Building a reference (De Novo)Rare Variant Discovery
Variant Detection
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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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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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Probe Performance and Validation – Design of 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
Ultramer® Oligonucleotides had either 1, 3, or 7 G-T or T-T mismatches
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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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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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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
Known Viral/Transposon SequenceUnknown Integration Site
Unknown Integration Site
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Applications – Viral and Transposon Integration Sites
Target the regions you want to focus on in various ways:
Unknown Integration Site
Unknown Integration Site
Target
Target
Target
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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
Region of InterestUnknown Fusion Site
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Applications – Translocation and Recombination
Target the regions you want to focus on in various ways:
Unknown Fusion Site
Target 1
Target
Target 2
Target 1 Target 2
Sequence unknown translocation
Target fusion events
Target different sites to see if they recombine with one another or with different sites
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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
SNPReference Sequence Reference Sequence
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Applications – Genotyping (Pitfalls)
What if you were trying to enrich with PCR?
SNP“Reference” Sequence “Reference” Sequence
Primer
Primer
A single mismatch can cause up to 7°C ΔTm which can dramatically reduce PCR efficiency
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AML Panel Performance – Fold Enrichment
#10 #11 #17 #23 #10 #11 #17 #23Replicate 1 Replicate 2
0
50
100
150
200
250
300
300
350
400
450
500
550
600
650
700
750Average Coverage Depth
Fold Enrichment
Aver
age
Cove
rage
Dep
th
Fold
Enr
ichm
ent
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AML Panel Performance – Alignment Breakdown
#10 #11 #17 #23 #10 #11 #17 #23Replicate 1 Replicate 2
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%Off-target Duplicate On-Target 500 bp Flank On-Target
% o
f Rea
ds
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AML Panel Performance – Read Statistics
#10 #11 #17 #23 #10 #11 #17 #23Replicate 1 Replicate 2
0
1000000
2000000
3000000
4000000
5000000
6000000
7000000
8000000
9000000
10000000Off-target Duplicate On-Target 500 bp Flank On-Target
Read
s (M
illio
ns)
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AML Panel Performance – Uniformity
1 2 3 40
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1>0.2 x Mean Coverage >0.5 x Mean Coverage >1.0 x Mean Coverage
Replicate Number
% o
f Tar
gets
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Improve Coverage and Uniformity
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
# Reads
Foundation MedicineBoston, MA, USA
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xGen® Lockdown® Probes Show Less GC Bias
Foundation MedicineBoston, Massachusetts
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.
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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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Blocking Oligos—Function
Two classes of blocking oligos are needed:
I) Cot1 DNA = Alu, LINE repeat elements
II) linkers/adapters
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Blocking Oligos – Efficacy
~100% more on-target reads after blocking
Increased reads enable researchers to get more depth or multiplex more samples
Hodges E, Rooks M, et al. (2009) Nat Protoc, 4(6):960–974.
Blumenstiel B, Cibulskis K, et al. (2010) Curr Protoc Hum Genet, Chapter 18:Unit 18.4.
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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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xGen® Universal Blocking Oligos
xGen® Universal Blocking Oligos
xGen® Standard Blocking Oligos
w/ no inosines
Standard Blocking Oligos w/ inosine barcodes
-
10.00
20.00
30.00
40.00
50.00
60.00
On-
Targ
et R
eads
(%)
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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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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 [email protected].
See how your colleagues are successfully using these IDT NGS products by searching on NGS Your Research at www.idtdna.com.