Digital DNA-seq Technology: Targeted Enrichment for Cancer Research
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Targeted DNA sequencing has become a powerful approach by achieving high coverage of the region of interest while keeping the cost of sequencing and complexity of data interpretation manageable. However, existing PCR-based target enrichment approaches introduce errors due to PCR amplification bias and artifacts, which significantly affects quantification accuracy and limit the ability to confidently detect low-frequency DNA variants. This webinar introduces a new digital sequencing approach that is based on the use of unique molecular indices (UMIs) - QIAseq Targeted DNA Panels. With UMIs, each unique DNA molecule is barcoded before any amplification takes place to correct for PCR errors. Detailed workflow and applications in cancer research will be presented. Join us and learn about this exciting novel digital DNAseq technology
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Digital DNA-seq Technology: Targeted Enrichment for Cancer Research
- 1. Sample to Insight Mutational analysis using QIAGEN’s QIAseq® panels and Sample to Insight® NGS solutions Raed Samara, PhD Global Product Manager, QIAGEN 1QIAseq Targeted NGS for Cancer Research, 10.10.2016
- 2. Sample to Insight 2 Legal disclaimer QIAseq Targeted NGS for Cancer Research, 10.10.2016 • QIAGEN products shown here are intended for molecular biology applications. These products are not intended for the diagnosis, prevention or treatment of a disease. • For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.QIAGEN.com or can be requested from QIAGEN Technical Services or your local distributor.
- 3. Sample to Insight 3 Precision medicine: Right drug, right patient, right time and dose QIAseq Targeted NGS for Cancer Research, 10.10.2016 “One size fits all” does not work
- 4. Sample to Insight Mutations AGCTCGTTGCTCAGCTC Reference genome AGCTCGTTGCTCAGCGTTC Insertion AGCTC---GCTCAGCTC Deletion Indels Copy number variations T G CA T G A C RS 4 DNA variants for precision medicine QIAseq Targeted NGS for Cancer Research, 10.10.2016
- 5. Sample to Insight RS 5 Actionable DNA variants for precision medicine QIAseq Targeted NGS for Cancer Research, 10.10.2016 Mutations AGCTCGTTGCTCAGCTC Reference genome AGCTCGTTGCTCAGCGTTC Insertion AGCTC---GCTCAGCTC Deletion Indels Copy number variations Only a handful of mutations are actionable Actionable DNA Variant BRAF V600E EGFR E746-750 + Kinase domain mutation HER2 Disease Melanoma Lung adenocarcinomas IDC-Breast cancer Therapy Vemurafenib (PLX4032) Erlotinib / Gefitinib Trastuzumab
- 6. Sample to Insight RS 6 Actionable DNA variants for precision medicine QIAseq Targeted NGS for Cancer Research, 10.10.2016 How many?
- 7. Sample to Insight EGFR (L858R) KRAS (G12C) + Response rates of >70% in patients with non-small cell lung cancer treated with either erlotinib or gefitinib Poor response rate in patients with non-small cell lung cancer treated with either erlotinib or gefitinib KRAS 25% EGFR 23% EML4-ALK 6% BRAF 3%PIKC3A 3% MET 2% ERBB2 1% MAP2K1 0.4% NRAS 0.2% Unknown 37% RS 7 Precision medicine for lung cancer QIAseq Targeted NGS for Cancer Research, 10.10.2016 Current lung cancer biomarker landscape • How many mutations to test for? • How to test for these mutations ◦ Sequential testing ◦ Parallel testing Adapted from: Govindan, R. et al. (2012). Genomic landscape of non-small cell lung cancer in smokers and never-smokers. Cell 150, 1121–34.
- 8. Sample to Insight Attribute / Parameter Information level Cost per sample Coverage achieved DNA input No. of samples multiplexed Whole Genome Sequencing 3 x 109 bps $5000 30x 1 µg 1 Whole Exome Sequencing 5 x 107 bps $2000 100x 100–200 ng 2 Targeted DNA Sequencing 6 x 104 bps $200 1000x 10 ng 96 Benefits of Targeted DNA Sequencing: More relevant data More cost effective Detect low-frequency mutations Lower DNA requirements Higher multiplexing capabilities RS 8 Actionable DNA variants for precision medicine QIAseq Targeted NGS for Cancer Research, 10.10.2016 Targeted DNA sequencing delivers accurate information required for precision medicine Clinical utility requires targeted analysis
- 9. Sample to Insight • Well-defined content • Small target size • More reads per sample RS 9 Why choose targeted DNA sequencing? QIAseq Targeted NGS for Cancer Research, 10.10.2016 Targeted DNA sequencing limits the genes or targets to be sequenced Features Benefits • Examine variants that matter • Multiplex many samples to save money • Detect low frequency variants
- 10. Sample to Insight Sample Insight The principle of targeted enrichment is to simultaneously sequence millions of small DNA fragments that represent the region of interest gDNA Variants Report: KRAS G12D EGFR T790M IDH1 R132H KRAS EGFR IDH1 RS 10 Sample isolation Library construction & targeted enrichment NGS run Data analysis Interpretation Targeted DNA Sequencing (TDS) QIAseq Targeted NGS for Cancer Research, 10.10.2016 Shrink the genome
- 11. Sample to Insight RS 11 Why choose PCR-based targeted enrichment? QIAseq Targeted NGS for Cancer Research, 10.10.2016 • Offers specificity that beats capture-based approaches Features Benefits • Lets you use sequencing capacity on regions targeted by the panel, with minimal off-target sequencing • Lets you achieve more uniform enrichment for more sequencing efficiency It delivers unmatched specificity and uniformity (compared to capture-based methods)
- 12. Sample to Insight 12 Sample Insight • Turnaround time, and limited amounts of DNA • Uniformity of enrichment • Coverage of GC-rich regions • Platform- dependent challenges • Data processing & variant calling • Isolation of high-quality DNA samples • Quantification of amplifiable (not total) amounts of DNA • Clinical & biological interpretation of data RS Sample QC Library QC Variant confirmation Sample isolation Library construction & targeted enrichment NGS run Data analysis Interpretation Sample to Insight: Integrated universal targeted NGS workflow QIAseq Targeted NGS for Cancer Research, 10.10.2016 To overcome NGS challenges
- 13. Sample to Insight 13 Inability to detect low- frequency mutations Inefficient enrichment and sequencing of GC-rich regions PCR and sequencing errors • Limits sensitivity and accuracy of calling low-frequency variants o Doesn’t let you confidently call variants down to 1% variant allele frequency (VAF) Suboptimal uniformity of enrichment and sequencing Suboptimal, GC-rich region-incompatible PCR chemistry • Limits comprehensiveness of panel coverage o Doesn’t let you efficiently sequence clinically-relevant genes such as CEBPA or CCND1 – or clinically-relevant regions such as TERT promoter Conventional PCR protocols and two-primer amplicon design • Increases variability in coverage across targeted genomic regions o Causes you to over-sequence to accommodate the under- sequenced o Doesn’t let you call variants in low-depth regions Mainly due to inferior PCR amplification approaches Challenges of current DNA targeted sequencing approaches QIAseq Targeted NGS for Cancer Research, 10.10.2016
- 14. Sample to Insight 14 DNA dsDNA PCR amplification & sequencing PCR and sequencing errors The necessary evil: PCR amplification QIAseq Targeted NGS for Cancer Research, 10.10.2016 PCR amplification is required for target enrichment, but…
- 15. Sample to Insight 15 5 reads OR library fragments that look exactly the same. Cannot tell whether they represent: 1. 5 unique DNA molecules, or 2. Quintuplets of the same DNA molecule (PCR duplicates) Conventional targeted DNA sequencing EGFR exon 21 Quantification based on non-unique reads does not reflect quantities of original DNA molecules Challenges of conventional targeted DNA sequencing QIAseq Targeted NGS for Cancer Research, 10.10.2016 PCR duplicates limit accurate quantification
- 16. Sample to Insight 16 Conventional targeted DNA sequencing EGFR exon 21 * Variant calling based on non-unique reads does not reflect the mutational status of original DNA molecules Challenges of conventional targeted DNA sequencing QIAseq Targeted NGS for Cancer Research, 10.10.2016 A mutation is seen in 1 out of 5 reads that map to EGFR exon 21. Cannot accurately tell whether the mutation is: 1. A PCR or sequencing error (artifact) / false positives, or 2. A true low-frequency mutations PCR and sequencing errors (artifacts) limit variant calling accuracy
- 17. Sample to Insight 17 • Proprietary PCR chemistry to enrich even GC-rich regions • Primers based on single primer extension (SPE) approach for enhanced uniformity Panel box (kit) QIAGEN’s solutions to overcome challenges of targeted NGS QIAseq Targeted NGS for Cancer Research, 10.10.2016 QIAseq targeted DNA panels • Molecularly-barcoded library adapters to incorporate unique molecular indices (UMIs). Index box (kit)
- 18. Sample to Insight 18 With the QIAseq targeted DNA panels, variant detection is done by analyzing unique DNA molecules instead of total reads Overcoming current challenges QIAseq Targeted NGS for Cancer Research, 10.10.2016 Current approach Challenges How QIAseq targeted DNA panels overcome challenges of current approaches • Conventional targeted DNA sequencing for variant detection • PCR and sequencing errors • UMIs that enable digital sequencing to correct for PCR and sequencing errors • Inefficient sequencing of GC- rich regions • Proprietary chemistry to efficiently sequence GC-rich regions • Suboptimal uniformity of enrichment and sequencing • SPE-based primer design to increase uniformity For optimal variant detection
- 19. Sample to Insight 19 TATCGTACAGAT (12 nucleotides long) Incorporate this random barcode (signature) into the original DNA molecules before amplification to preserve their uniqueness What is a UMI (molecular barcode)? QIAseq Targeted NGS for Cancer Research, 10.10.2016 Tag (barcode) to identify unique DNA molecules
- 20. Sample to Insight 20 DNA dsDNA TATCGTACAGAT Molecularly-barcoded adapter Incorporate this random barcode (signature) into the original DNA molecules before amplification to preserve their uniqueness PCR amplification & sequencing Correct for PCR duplicates & errors How are UMIs incorporated? QIAseq Targeted NGS for Cancer Research, 10.10.2016 Ligate molecularly-barcoded adapters to unique DNA molecules before amplification
- 21. Sample to Insight 21 5 unique DNA molecules since 5 molecular barcodes are detected Quintuplets of the same DNA molecule (PCR duplicates) since 1 molecular barcode is detected UMI Digital sequencing with UMIs UMIs before any amplification Achieve accurate quantification with molecular barcodes QIAseq Targeted NGS for Cancer Research, 10.10.2016 Count and analyze single original molecules (not total reads) = digital sequencing 5 reads OR library fragments that look exactly the same. Cannot tell whether they represent: 1. 5 unique DNA molecules, or 2. Quintuplets of the same DNA molecule (PCR duplicates) Conventional targeted DNA sequencing EGFR exon 21
- 22. Sample to Insight 22 False variant is present in some fragments carrying the same UMI True variant is present in all fragments carrying the same UMI UMI UMIs before any amplification * ***** Digital sequencing with UMIs Achieve accurate variant calling with molecular barcodes QIAseq Targeted NGS for Cancer Research, 10.10.2016 Conventional targeted DNA sequencing EGFR exon 21 *A mutation is seen in 1 out of 5 reads that map to EGFR exon 21. Cannot accurately tell whether the mutation is: 1. A PCR or sequencing error (artifact) / false positives, or 2. A true low-frequency mutations Count and analyze single original molecules (not total reads) = digital sequencing
- 23. Sample to Insight 23 Sample isolation Library construction & targeted enrichment NGS run Data analysis InterpretationSample Insight Panels and molecularly- barcoded adapters Barcode-aware variant calling pipeline QIAseq targeted DNA panels: Sample to Insight QIAseq Targeted NGS for Cancer Research, 10.10.2016 Panels, molecularly-barcoded adapters and data analysis algorithms
- 24. Sample to Insight 24 Specifications of QIAseq targeted DNA panels QIAseq Targeted NGS for Cancer Research, 10.10.2016 DNA input As little as 20 ng DNA Primer multiplexing level 11,500 / 9600 primers (Catalog / Custom DNA) Number of primer pools 1 Enrichment technology SPE-based with molecularly-barcoded adapters Amplicon size Average 150 bp Sample multiplexing level 384 (Illumina), 96 (Ion Torrent) Total workflow time 8–9 hours Number of libraries per sample 1 Sequencer compatibility Illumina and Ion Torrent platforms Variant allele frequency called 1%
- 25. Sample to Insight End repair and A tailing Adapter ligation / library construction (incorporation of adapters, molecular barcodes and sample indexes) 5’ 5’ 5’ 5’ MB MB Adapter 5’ IL-F RSP Add GSPs and UP* 5’ IL-U SIP Add indexes and UP* Universal PCR amplification Sample indexing and amplification Sequencing-ready library MB: Molecular barcode RSP: Region-specific primer FP: Forward primer UP: Universal primer SIP: Sample index primer A A 5’MB Target enrichment by SPE 5’ *Preceded by bead cleanup Lib quant* Enzyme-based random DNA fragmentation DNA 5’ 5’ 1.5Days QIAseq Targeted DNA Panel: Workflow (Illumina®) QIAseq Targeted NGS for Cancer Research, 10.10.2016 25
- 26. Sample to Insight QIAseq Targeted DNA Panel: Workflow (Ion Torrent™) QIAseq Targeted NGS for Cancer Research, 10.10.2016 26 End repair and A tailing Adapter ligation / library construction (incorporation of adapters, molecular barcodes and sample indexes) 5’ 5’ 5’ 5’ MB MB Adapter 5’ LT-F RSP Add GSPs and UP* 5’ LT-U P1 Add indexes and UP* Universal PCR amplification Sample indexing and amplification Sequencing-ready library MB: Molecular barcode RSP: Region-specific primer FP: Forward primer UP: Universal primer P1: P1 primer A A 5’MB Target enrichment by SPE 5’ *Preceded by bead cleanup Lib quant* Enzyme-based random DNA fragmentation DNA 5’ 5’ 1.5Days
- 27. Sample to Insight 27 1. Exonic regions of genes plus 10 bases to cover intron / exon junctions 2. Mix of type of coverage 1 (for tumor suppressor genes) and HotSpots for oncogenes 3. SNPs 4. Full chromosome QIAseq targeted DNA panels QIAseq Targeted NGS for Cancer Research, 10.10.2016 Panel Variant (Cat) number Number of genes Number of primers Type of coverage Breast cancer panel DHS-001Z 93 4831 1 Colorectal cancer panel DHS-002Z 71 2929 1 Myeloid Neoplasms panel DHS-003Z 141 5887 1 Lung cancer panel DHS-005Z 72 4149 1 Actionable solid tumor panel DHS-101Z 23 651 2 BRCA1 and BRCA2 panel DHS-102Z 2 223 1 BRCA1 and BRCA2 Plus panel DHS-103Z 6 348 1 Pharmacogenomics panel DHS-104Z 39 146 3 Mitochondria panel DHS-105Z Chromosome M 222 4 Inherited diseases panel DHS-3011Z 298 11,579 1 Comprehensive cancer panel DHS-3501Z 275 11,311 1 List of panels Types of coverage:
- 28. Sample to Insight 28 QIAseq targeted DNA panels QIAseq Targeted NGS for Cancer Research, 10.10.2016 List of panels Panel Variant (Cat) number Panel size (bases) Specificity (reads with primers, %) Uniformity (0.2x mean baseMT, %) Breast cancer panel DHS-001Z 370,942 96.47 99.84 Colorectal cancer panel DHS-002Z 215,328 90.39 99.79 Myeloid Neoplasms panel DHS-003Z 436,672 95.31 99.71 Lung cancer panel DHS-005Z 318,059 97.3 99.91 Actionable solid tumor panel DHS-101Z 15,160 90.48 99.85 BRCA1 and BRCA2 panel DHS-102Z 16,405 99.59 100 BRCA1 and BRCA2 Plus panel DHS-103Z 25,590 99.46 99.92 Pharmacogenomics panel DHS-104Z 3313 93.43 99.34 Mitochondria panel DHS-105Z 16,570 99.72 99.08 Inherited diseases panel DHS-3011Z 838,627 97.29 99.21 Comprehensive cancer panel DHS-3501Z 836,670 97.42 199.76 Uniformity and specificity are defined based on NA12878 tests
- 29. Sample to Insight Extended and Custom What is the list of your targets? RS 29 Extended panels Custom panels • Extend the contents of an existing cataloged panel • Turnaround time = 14 days • Bioinformatically target any gene(s) or genomic region(s) within the human genome • Turnaround time = 14 days Customized panels QIAseq Targeted NGS for Cancer Research, 10.10.2016
- 30. Sample to Insight 30 CEBPA GC content Coverage GC content Coverage CCND1 The proprietary PCR chemistry used in the QIAseq targeted DNA panels enables efficient coverage of regions high in GC content Comprehensive coverage of GC-rich regions QIAseq Targeted NGS for Cancer Research, 10.10.2016
- 31. Sample to Insight 31 830 kb region was enriched from 20 ng of NA12878 DNA with Comprehensive Cancer Panel. Library was constructed for sequencing on a MiSeq, with 2600x read depth. The panel achieved a uniformity of 99.5% at 0.2x of mean coverage, and 98% at 0.3x of mean coverage. Unmatched uniformity QIAseq Targeted NGS for Cancer Research, 10.10.2016
- 32. Sample to Insight 32 Benefits of QIAseq targeted DNA panels QIAseq Targeted NGS for Cancer Research, 10.10.2016 Feature Benefit Low DNA input (as low as 20 ng DNA) Preserve sample High primer multiplexing capability (up to 10,000 primers) Detect a large number of DNA variants Single pool of primers Easier sample handling SPE-based target enrichment Flexibility in primer design Small amplicons (average size 150 bp) Generate relatively small library fragments to maintain compatibility with fragmented DNA (FFPE and ctDNA samples) High sample multiplexing (up to 384 samples) Increased sample throughput to decrease sequencing costs Automation-friendly workflow Streamline operations for high throughput Molecular barcode-aware variant caller Confidently call low-frequency mutations Suite of complementary data analysis tools Save resources Affordable per-sample cost Save $$$ All required reagents (including beads) in 2 kits Simplified logistics & ordering
- 33. Sample to Insight 33 This online GenomeWeb seminar focused on the design of a large cohort study for assessing breast cancer risk and how using an innovative digital sequencing approach is able to solve the previously unmet challenges of this type of NGS study design. Fergus J. Couch, PhD Professor and Chair Division of Experimental Pathology, Department of Laboratory Medicine and Pathology, Mayo Clinic https://genomeweb.webex.com/genomeweb/lsr.php?RCID=30c8e3fe698b7feca1ed79ac117fbed0 Application: Large cohort study for breast cancer risk QIAseq Targeted NGS for Cancer Research, 10.10.2016 Sequencing 60,000 samples
- 34. Sample to Insight RS 34 Sample to Insight: Integrated universal targeted NGS workflow QIAseq Targeted NGS for Cancer Research, 10.10.2016 To overcome NGS challenges Sample Insight • Turnaround time, and limited amounts of DNA • Uniformity of enrichment • Coverage of GC-rich regions • Platform- dependent challenges • Data processing & variant calling • Isolation of high-quality DNA samples • Quantification of amplifiable (not total) amounts of DNA • Clinical & biological interpretation of data Sample QC Library QC Variant confirmation Sample isolation Library construction & targeted enrichment NGS run Data analysis Interpretation
- 35. Sample to Insight 35 Barcode-aware variant caller has been developed Caller is available on the cloud In conjunction with molecular barcodes incorporated in the workflow, the caller can confidently call low-frequency variants (down to 1% variant allele frequency, “VAF”) Variant caller will do the following: • Mapping • Alignment • Molecular barcode counting • Variant / calling • Variant / annotation – variants based on public databases Data analysis with barcode-aware variant caller – overview QIAseq Targeted NGS for Cancer Research, 10.10.2016 For QIAseq targeted DNA panels
- 36. Sample to Insight Data analysis with barcode-aware variant caller – overview FASTQ or BAM files are uploaded into cloud-based data analysis portal The following inputs are needed (by customer): • Set up file • Panel used • File lanes o 1-lane (MiSeq/HiSeq/NextSeq concatenated) o 4-lane (NextSeq individual lane files) Inputs
- 37. Sample to Insight 37 Data analysis with barcode-aware variant caller – overview QIAseq Targeted NGS for Cancer Research, 10.10.2016 Outputs Summary file • Stats o Specificity o Uniformity o Molecular barcode counts • Variants o Frequency o Annotations VCF
- 38. Sample to Insight Actionable solid tumor Disease- specific Comprehensive Detection Discovery Multiplexing Target size Custom & extended PanelsApplicationsSpecifications Clinical research Translational & discovery research Whole Exome Seq Whole Genome Seq Targeted DNA sequencing: robust detection, limited discovery RS 38 Why choose targeted DNA sequencing? QIAseq Targeted NGS for Cancer Research, 10.10.2016 Detect known variants & discover novel variants
- 39. Sample to Insight Thank you for attending today’s webinar! Contact QIAGEN Call: 1-800-426-8157 Email: techservice-na@qiagen.com BRCsupport@QIAGEN.com QIASeq.NGS@QIGAEN.COM QIAWebinars@QIAGEN.COM Questions? 39 Thank you for attending QIAseq Targeted NGS for Cancer Research, 10.10.2016
Hinweis der Redaktion
- The concept of one-size-fits all for disease management is not effective. With this approach, some patients show positive effects, while others show no effects or adverse effects. The concept of precision medicine – delivering the right drug, to the right patient, at the right time and dose – is more effective. It builds on the idea of utilizing genetic information for disease management.
- There are several DNA variants that can be used for precision medicine. Three are shown here.
- Out of all the DNA variants, only a handful are actionable – which means we can use the information from these variants for disease management. For example, the presence of the V600E mutation in BRAF is seen in a large number of melanoma patients. While this variant causes the development and progression of the disease, its presence is favorable for melanoma patients because the variant (mutation) can be targeted by an approved therapy (vemurafenib).
- How many actionable mutations are there? Only a handful. Shown here is a typical process of filtering variants to build a list of actionable mutations. Starting with millions of common variants, we end up with 10s–100s of actionable variants.
- As an example, in lung cancer we see the following mutations accumulate in lung cancer patients. We can use the information on the type of mutations for disease management. For instance, patients who harbor the EGFR L858R mutation respond very well to tyrosine kinase inhibitors – while among patients who harbor an additional mutation in KRAS, patient response to the same class of drugs drops.
- There are three main sequencing levels which can be used to detect actionable mutations. The table here shows how these three levels differ on several parameters. Targeted DNA sequencing and analysis is extensively used in clinical research.
- What are the benefits of targeted DNA sequencing? What to include in a targeted panel? There are many approaches. One approach, highlighted by Dr. Carl Morrison, is to include genes for which actionable information exists. In other words, examine targets upon which you can act.
- With targeted DNA sequencing, one shrinks the genome to a number of targets that are crucial to the disease being examined. In this example, the user is interested in only the three genes shown.
- The reason we chose a PCR approach for target enrichment is to take advantage of the high specificity and uniformity that PCR provides, as shown in this report. Here, the authors compared different enrichment approaches and show that the specificity and uniformity of PCR surpasses those of hybridization approaches.
- QIAGEN has built a Sample to Insight workflow to overcome some of the main challenges associated with NGS.
- A variant identified in a sample represents one of two events: a true variant or a false variant. False variants can be introduced at any step during the workflow, including sequencing reactions. This results in the inability to accurately and confidently call rare variants (those present at 1% of the sample). Due to PCR duplicates generated in amplification steps, all DNA fragments look exactly the same – and there is no way to tell whether a specific DNA fragment is a unique DNA molecule or a duplicate of a DNA molecule. With molecular barcodes, since each unique DNA molecule is barcoded before any amplification takes place, unique DNA molecules are identified by their unique barcodes – and PCR duplicates carrying the same barcode are removed, thereby increasing the sensitivity of the panel.
- We also offer 2 approaches to build your own unique panel that fits your own needs and requirements. The Mix-n-Match approach gives you access to our 570 wet bench-tested gene designs, which is a very unique offering in the market. In case your genes of interest are not part of Mix-n-Match, you can use our Custom panels to build a fully customized panel.
- The QIAseq DNA panels use a proprietary buffer mixture to efficiently sequence both regular and GC-rich regions within the genome in a single reaction. Two examples are shown here: CEBPA and CCND1. Complete coverage of exonic regions within those two genes is achieved.