Expanding Your Research Capabilities Using Targeted NGS

3,328 views

Published on

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.

Published in: Health & Medicine, Technology
0 Comments
1 Like
Statistics
Notes
  • Be the first to comment

No Downloads
Views
Total views
3,328
On SlideShare
0
From Embeds
0
Number of Embeds
132
Actions
Shares
0
Downloads
69
Comments
0
Likes
1
Embeds 0
No embeds

No notes for slide
  • NGS usage is becoming more wide-spread as continuous cost-reductions enable a greater number of applications.Academic and corporate customers use NGS in various fields of researchers whether studying marine biology or microorganisms, developing diagnostics, doing drug development, and understanding new crops, NGS has enabled a diverse set of genetically oriented applications.
  • Expanding Your Research Capabilities Using Targeted NGS

    1. 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. 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 2
    3. 3. Impact of Next Generation Sequencing Academia Industry 3
    4. 4. 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 4
    5. 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 5
    6. 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 6
    7. 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 7
    8. 8. Applications of Targeted Next Generation Sequencing 8
    9. 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 9
    10. 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 10
    11. 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) 11
    12. 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 12
    13. 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 13
    14. 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 14
    15. 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 15
    16. 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 16
    17. 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 17
    18. 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 18 with one another or with different sites
    19. 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 19
    20. 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 20
    21. 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 21
    22. 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 22
    23. 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 23
    24. 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 24
    25. 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 25
    26. 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 26
    27. 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 27
    28. 28. Blocking Oligos—Function Two classes of blocking oligos are needed: I) Cot1 DNA = Alu, LINE repeat elements II) linkers/adapters 28
    29. 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. 29
    30. 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 30
    31. 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 31
    32. 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 32
    33. 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. 33

    ×