Expanding Your Research Capabilities Using Targeted NGS

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 xGen@idtdna.com.

See how your colleagues are successfully using these IDT NGS products by searching on NGS Your Research at www.idtdna.com.