NGS: Digital RNAseq & Library Prep Seminar. Next-Generation Sequencing Lunch & Learn

Transcription

1 NGS: Digital RNAseq & Library Prep Seminar Next-Generation Sequencing Lunch & Learn Samuel Rulli, Ph. D Global Product Manager QIAseq Targeted RNA Panels 1

2 Targeted sequencing with UMIs QIAseq mirnaseq Kit UMIs Gel free library prep Complete mirnome Dec mirna DNA Biomarkers QIAseq Targeted DNA Panels Unique molecular index Mutation /SNP analysis CNV Insertions / deletions 2ng Fresh DNA mrna/ lncrna QIAseq Targeted RNA Panel UMIs Gene expression 100pg / 10 cells All kits utilize SPE (single primer extension) Fusion QIAseq Targeted RNAscan Panels Unique molecular index Known fusion genes (validation) Unknown partners (discovery) 2

3 QIAseq Library Products for Illumina Sequencers Nucleic acid extraction NGS Library preparation Sequencing & QIAGEN Bioinformatics Whole Genome Sequencing (WGS) Hybrid Capture Sequencign QIAseq FX DNA Library Kit FX fragmentation step eliminates shearing 2.5 hour workflow from gdna to library Tunable enzymatic fragmentation for any size probes with higher genomic complexity than Nextera DNA Amplicon/ PCR sequencing QIAseq 1-Step Amplicon Library Kit 1-tube, room temperature library prep cfdna for Liquid Biopsy or NIPT QIAseq cfdna Complete Solution QIAamp Circulating Nucleic Acid Isolation Optimized protocol specific for cfdna Low Input ChIP-seq, cfdna, LCM QIAseq Ultralow Input Library Kit Ultra-efficient chemistry for pg+ DNA Barcoded adapters for 96-plex sequencing Single Cell Genomics QIAseq Single Cell FX Lib Kits PCR-free MDA Amplification Sorted single cells or 6pg DNA input 3

4 QIAseq Library Products for Illumina Sequencers Nucleic acid extraction NGS Library preparation Sequencing & QIAGEN Bioinformatics Whole Genome Sequencing (WGS) Hybrid Capture Sequencign QIAseq FX DNA Library Kit FX fragmentation step eliminates shearing 2.5 hour workflow from gdna to library Tunable enzymatic fragmentation for any size probes with higher genomic complexity than Nextera DNA Amplicon/ PCR sequencing QIAseq 1-Step Amplicon Library Kit 1-tube, room temperature library prep cfdna for Liquid Biopsy or NIPT QIAseq cfdna Complete Solution QIAamp Circulating Nucleic Acid Isolation Optimized protocol specific for cfdna Low Input ChIP-seq, cfdna, LCM QIAseq Ultralow Input Library Kit Ultra-efficient chemistry for pg+ DNA Barcoded adapters for 96-plex sequencing Single Cell Genomics QIAseq Single Cell FX Lib Kits PCR-free MDA Amplification Sorted single cells or 6pg DNA input 4

5 Today s Agenda Targeted Sequencing 1. Principles of unique molecular indexes (UMIs) 2. Single primer extension (SPE) versus PCR for library construction 3. UMI and SPE in action gene expression analysis 4. DNA variant analysis and novel gene fusions discovery with UMIs & SPE 5. Single Cell Capture & NGS applications with QIAscout and QIAseq Single Cell FX kits 5

6 Section 1: Principal of unique molecular Index (UMIs) PCR duplicates and amplification bias are major issues in current RNAseq workflows, as they result in biased and inaccurate gene expression profiles Gene A Sample 1 Ratio of original state of genes 4 Ratio of genes based on reads [reads (ratio)] 12 (2) Gene A Sample (1) mrna cdna Raw reads 6

7 Section 1: Principal of unique molecular Index (UMIs) Targeted RNAseq still is a Read based approach to understanding gene expression. How do we go from Reads to counting transcripts? 7

8 Section 1: Principal of unique molecular Index (UMIs) Targeted RNAseq still is a Read based approach to understanding gene expression. How do we go from Reads to counting transcripts? 8

9 Section 1: Principal of unique molecular Index (UMIs) Molecular indexes allow the counting of original transcript levels instead of PCR duplicates, thereby enabling digital sequencing and resulting in unbiased and accurate gene expression profiles. Tag each gene with UMIs Count unique molecules, not reads Gene A Sample 1 Ratio of original state of genes 4 Ratio of genes based on UMIs 4 Ratio of genes based on reads [reads (ratio)] 12 (2) Gene A Sample (1) mrna cdna UMI reads 9

10 Section 1: Principal of unique molecular Index (UMIs) Each capture event is archived with a UMI 12 random bases 16.7 million indexes Strategy for gene expression uses UMI-gene specific primer Strategy for DNA variant and fusion gene is slightly different, but same principle 10

11 Today s Agenda Targeted Sequencing 1. Principles of unique molecular indexes (UMIs) 2. Single primer extension (SPE) versus PCR for library construction 3. UMI and SPE in action gene expression analysis 4. DNA variant analysis and novel gene fusions discovery with UMIs & SPE 5. Summary/Questions? 11

12 Section 2: Single Primer Extension Single Primer Extension 5 3 DNA or cdna 2 Primer Amplification PCR amplification PCR amplification captures information

13 Inefficient sequencing of GC-rich regions by PCR amplification Leads to lack of comprehensive coverage in those regions Normal GC content High GC content PCR Amplification using suboptimal chemistry under uniform conditions Coverage Regions with normal GC content are covered efficiently Regions with High GC content are not covered efficiently 13

14 Complete sequencing of GC-rich regions by SPE Leads to lack of comprehensive coverage in those regions Normal GC content High GC content PCR Amplification following SPE under uniform conditions Coverage Regions with normal GC content are covered efficiently Regions with High GC content are covered as well 14

15 SPE coverage of GC-rich regions CEBPA GC content Coverage CCND1 GC content Coverage 15

16 Section 2: Single Primer Extension Advantages of SPE: 1.) Only need 1 region for primer design unlocks entire transcriptome, genome and fusion genes - 50% primers lowers cost & allows for greater content during multiplexing 2.) Able to adopt to G/C rich and hard to PCR regions - sequence everything 3.) Uniform reaction - uniform library construction uniform sequencing better results 4.) strategy works very well on FFPE, fragmented and low quality samples Disadvantages: 1.) Extra step in library construction - may add 1 hour to total workflow 16

17 Today s Agenda Targeted Sequencing 1. Principles of unique molecular indexes (UMIs) 2. Single primer extension (SPE) versus PCR for library construction 3. UMI and SPE in action gene expression analysis 4. DNA variant analysis and novel gene fusions discovery with UMIs & SPE 5. Summary/Questions? 17

18 Section 3: UMI and SPE in Action Gene expression example Small Molecules Signal Transduction Application cells Experiment is to identify novel compounds that modulate known signal transduction pathways treated cells RNA 18

19 Section 3: UMI and SPE in Action Gene expression example Small Molecules Signal Transduction Application cells treated cells Cells are treated with different chemical inhibitors. Isolate RNA from cells Build library with QIAseq Targeted RNA Panels Human Signal Transduction 421 targets/ 10ng total RNA RNA 19

20 Section 3: UMI and SPE in Action Gene expression example 6 hours GSP1, GSP2 never see other, thereby minimizing primer dimers 20

21 Section 3: UMI and SPE in Action Gene expression example leave no scientist behind. 21

22 Section 3: UMI and SPE in Action Gene expression example Included in Panel Kit Library Quant Index Kit Kit Included in cloud 22

23 Section 3: UMI and SPE in Action Gene expression example Included in Panel Kit Library Quant Index Kit Kit CLC Biomedical workbench with MT plugin 23

24 Section 3: UMI and SPE in Action Gene expression example Requirement Species coverage Biological replicates Short reads for FFPE, and Exosomal RNA QIAseq Targeted RNA Panels Human Catalog, Extended, Virtual, Custom panels Mouse + Rat Custom Essential for robustness of experimental design (and statistics!) Average amplicon 97 bps ; range bases Depth of sequencing High enough to infer accurate statistics determined by UMI- ~2-5 reads per UMI is enough. Stranded library prep Not required, amplicons do not overlap lncrna Type of reads (paired or Unpaired?) mrna and lncrnas Not necessary, 150 base single reads more than enough for accurate data QIAseq was designed against database containing lncrna and mrna. Assay are specific for lncrna or mrna. Currently 54,881 genes from Ensembl version 81 24

25 Section 3: UMI and SPE in Action Gene expression example Free circulating nucleic acids RNA and DNA from dead cells shed into the bloodstream, can contain cancer-related mutations. Exosomes Tiny microvesicles found in body fluids that transport RNA between cells. Circulating tumor cells Tumor cells shed from a tumor into the bloodstream carrying genetic information. Tissue samples Fresh tissue or archived FFPE samples Qiagen comprehensive sample isolation portfolio compatible with QIAseq RNA Compatible with 100pg (10 cells) to 25ng RNA 25

26 Section 3: UMI and SPE in Action Gene expression example 26

27 Section 3: UMI and SPE in Action Gene expression example Flexible experiment design for any Catalog Panel options: Comprehensive Panels (available for 12, 96 or 384 samples) Cancer Transcriptome (395) Inflammation & Immunity Transcriptome (475) Signal Transduction PathwayFinder (406) Stem Cell & Differentiation Markers (293) Molecular Toxicology Transcriptome (370) Angiogenesis & Endothelial Cell Biology (340) Apoptosis & Cell Death (264) ECM & Adhesion Molecules (421) 27

28 Section 3: UMI and SPE in Action Gene expression example Flexible experiment design for any Extended Panels Add 25 of your favorite targets (mrnas or lncrnas) to QIAGEN s comprehensive panel lnc13 GAS5 MEG3 PTCSC3 TERC ADAMT S9 GAS6- AS1 MIR31 HG CAHM GNAS MIR7-3HG ZFAS1 DLEU2 LINC PTCSC1 NEAT NAMA DLX6 GACA T1 LINC What is the role of tumor suppressor lncrnas? Find out! 28

29 Section 3: UMI and SPE in Action Gene expression example Flexible experiment design for any Catalog Panel options: QIAseq Targeted RNA Virtual Panels (available for 12, 96 or 384 samples) Each Panel contains 84 genes + controls and house keeping genes. Choose from over 180 panels! Pathways Diseases mirna Targets 29

30 QIAGEN Automating Sample disruption Purification RNA Quality Control qpcr setup NGS library quantifiaction Real time PCR + HRM High-throughput PCR Fragment Analysis Bench top assay setup Mediumthroughput Pyrosequencing Hybrid capture Low-throughput Integrated assay setup GeneReader NGS 30

31 QIAGEN Automating Purification RNA Quality Control Cells in 96 well plates RNA Integrity (96 samples done automatically while at lunch) RNA isolation of 96 samples RNA quantification (16 samples/ 90 secs) 31

32 QIAGEN Automating Purification RNA Quality Control Assay setup Detection and analysis Cells in 96 well plates RNA Integrity (96 samples done automatically while at lunch) Library quantification Library Integrity RNA isolation of 96 samples Library quantification Setup by qpcr RNA quantification (16 samples/ 90 secs) 32

33 QIAseq Targeted RNA Application Data Small Molecules Signal Transduction Application cells HEK293T Cells were treated with 90 different chemical inhibitors. The 421 Signal Transduction Gene QIAseq Panel was interrogated. treated cells RNA In one day we went from total RNA to sequence ready libraries for 96 samples. The final libraries were quantified, normalized, and pooled. Prior to loading onto a NextSeq, the denatured libraries were diluted to the appropriate input concentration to obtain to generate suitable clusters on the NextSeq. Indexed libraries The parameters of the NextSeq sequencing run were; single 151 bp read, with a Custom Sequencing Primer (included in kit). Normalized, pooled libraries 33

34 Primary Data Analysis for QIAseq Targeted RNA Sequencing QIAseq Targeted RNA Data Analysis automated workflow Read Mapping Primer Trimming UMI Count Read Mapping Identify the possible position of the read within the reference genome Align the read sequence to reference sequences Primer Trimming Remove the primer sequences from the reads Go get coffee UMI Counting 34

35 Small Molecule Application Data Primary Data Analysis for QIAseq Targeted RNA Sequencing 35

36 Small Molecule Application Data Primary Data Analysis for QIAseq Targeted RNA Sequencing QIAseq RNA Quantification - Read Details: Unique Captures per Target Gene Count Differential gene expression inter- and intra-samples 36

37 QIAseq secondary data analysis setup Analysis: What kinds of things get flagged? Low tag #, high gdna, poor normalizer performance 37

38 Secondary Data Analysis for QIAseq Targeted RNA Sequencing Scatter plot and clustergram (HDAC Sample compared to Control) 38

39 Secondary Data Analysis for QIAseq Targeted RNA Sequencing Changes in gene expression due to chemical perturbation and quantified by QIAseq RNA NGS were characterized. 39

40 Ingenuity IPA analysis HDAC Mechanistic Network in HEK293T Cells Treated with Trichostatin A HDAC is predicted to be inhibited by Trichostatin A and drives a mechanistic network with 18 other regulators. Cell cycle NHR, proliferation Transcriptional activator 40

41 Where can you run this? QIAseq sample multiplexing guidelines on NGS platforms 41

42 Unparalleled efficiency and flexibility vs PCR An example: 96 samples, 421 genes Parameter QIAseq targeted RNA panels RT-PCR Material required One pool of primers well plates Run time 14 hours for NextSeq run 310 hours (2 hours per plate) Hands-on time 3 hours (for 96 samples) 105 hours (one hour per plate) Sample 10 ng each sample 4000 ng each sample 42

43 Today s Agenda Targeted Sequencing 1. Principles of unique molecular indexes (UMIs) 2. Single primer extension (SPE) versus PCR for library construction 3. UMI and SPE in action gene expression analysis 4. DNA variant analysis and novel gene fusions discovery with UMIs & SPE 5. Summary/Questions? 43

44 Section 4: DNA variant analysis and novel gene fusions discovery QIAseq mirnaseq Kit UMIs Gel free library prep Complete mirnome Dec mirna DNA Biomarkers QIAseq Targeted DNA Panels Unique molecular index Mutation /SNP analysis CNV Insertions / deletions 2ng Fresh DNA mrna/ lncrna QIAseq Targeted RNA Panel UMIs Gene expression 100pg / 10 cells Fusion QIAseq Targeted RNAscan Panels Unique molecular index Known fusion genes (validation) Unknown partners (discovery) 44

45 Section 4: DNA variant analysis and novel gene fusions discovery DNA QIAseq Targeted DNA Panels Unique molecular index Mutation /SNP analysis CNV Insertions / deletions 2ng Fresh DNA mirna Biomarkers mrna/ lncrna Fusion 45

46 Section 4: DNA variant analysis and novel gene fusions discovery PCR and sequencing errors (artifacts) limit variant calling accuracy Traditional 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 positive), or 2. A true low-frequency mutations Variant calling based on non-unique reads does not reflect the mutational status of original DNA molecules Applies to a wide range of panels 46

47 Section 4: DNA variant analysis and novel gene fusions discovery Count and analyze single original molecules (not total reads) = digital sequencing Traditional 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 positive), or 2. A true low-frequency mutations Digital sequencing with UMIs * * * False variant is present in some fragments carrying the same UMI Traditional Targeted DNA Seq True variant is present in all fragments carrying the same UMI 47

48 Section 4: DNA variant analysis and novel gene fusions discovery Count and analyze single original molecules (not total reads) = digital sequencing Traditional 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 positive), or 2. A true low-frequency mutations Add UMIs before any amplification Digital sequencing with UMIs UMI * * False variant is present in some fragments carrying the same UMI True variant is present in all fragments carrying the same UMI 48

49 Section 4: DNA variant analysis and novel gene fusions discovery QIAseq Targeted DNA Panel Workflow 49

50 QIAseq targeted DNA panels List of panels Panel Variant (Cat) number Number of genes Number of primers Type of coverage Breast cancer panel DHS-001Z Colorectal cancer panel DHS-002Z Myeloid Neoplasms panel DHS-003Z Lung cancer panel DHS-005Z Actionable solid tumor panel DHS-101Z BRCA1 and BRCA2 panel DHS-102Z BRCA1 and BRCA2 Plus panel DHS-103Z Pharmacogenomics panel DHS-104Z Mitochondria panel DHS-105Z Chromosome M Inherited diseases panel DHS-3011Z Comprehensive cancer panel DHS-3501Z Types of coverage: 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 50

51 QIAseq targeted DNA panels 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, Colorectal cancer panel DHS-002Z 215, Myeloid Neoplasms panel DHS-003Z 436, Lung cancer panel DHS-005Z 318, Actionable solid tumor panel DHS-101Z 15, BRCA1 and BRCA2 panel DHS-102Z 16, BRCA1 and BRCA2 Plus panel DHS-103Z 25, Pharmacogenomics panel DHS-104Z 3, Mitochondria panel DHS-105Z 16, Inherited diseases panel DHS-3011Z 838, Comprehensive cancer panel DHS-3501Z 836, Uniformity and Specificity are defined based on NA12878 tests 51

52 Section 4: DNA variant analysis and novel gene fusions discovery DNA mirna Biomarkers mrna/ lncrna Fusion QIAseq Targeted RNAscan Panels Unique molecular index Known fusion genes (validation) Unknown partners (discovery) 52

53 Section 4: DNA variant analysis and novel gene fusions discovery QIASeq Targeted RNAscan is a RNA target enrichment method that allows verification of known fusions and discovery of novel fusions with next-generation sequencing (NGS). RPS6KB1-VMP1 ARFGEF2-SULF2

54 Section 4: DNA variant analysis and novel gene fusions discovery 54

55 QIAseq targeted RNAscan panels List of panels Panel Variant (Cat) number Number of primers Type of coverage Hematology panel FHS-001Z 156 Breakpoint Solid Tumor panel FHS-002Z 101 Breakpoint Lung cancer panel FHS-003Z 137 Breakpoint Oncology panel FHS-3001Z 950 Breakpoint

56 Summary: Biomarkers come in many flavors. Gene Expression Indels mirna expression Biomarkers Mutations Fusions Copy number variants

57 Summary: NGS can be used for all of them NGS Gene Expression Indels mirna expression Biomarkers Mutations Fusions Copy number variants 57

58 QIAseq solutions to detect all Biomarkers using NGS Gene Expression QIAseq targeted RNA panels QIAseq targeted DNA panels Indels mirna expression QIAseq mirna sequencing system Biomarkers QIAseq targeted DNA panels Mutations Copy number variants Fusions QIAseq targeted RNAscan panels QIAseq targeted DNA panels 58

59 QIAseq Library Products for Illumina Sequencers Nucleic acid extraction NGS Library preparation Sequencing & QIAGEN Bioinformatics Whole Genome Sequencing (WGS) Hybrid Capture Sequencign QIAseq FX DNA Library Kit FX fragmentation step eliminates shearing 2.5 hour workflow from gdna to library Tunable enzymatic fragmentation for any size probes with higher genomic complexity than Nextera DNA Amplicon/ PCR sequencing QIAseq 1-Step Amplicon Library Kit 1-tube, room temperature library prep cfdna for Liquid Biopsy or NIPT QIAseq cfdna Complete Solution QIAamp Circulating Nucleic Acid Isolation Optimized protocol specific for cfdna Low Input ChIP-seq, cfdna, LCM QIAseq Ultralow Input Library Kit Ultra-efficient chemistry for pg+ DNA Barcoded adapters for 96-plex sequencing Single Cell Genomics QIAseq Single Cell FX Lib Kits) PCR-free MDA Amplification Sorted single cells or 6pg DNA input 59

60 QIAscout Release Device: Interface with Microscope Objective on revolving turret 4 adapters cover a diameter range from mm Objective magnifications include 4x, 5x, and 10x Dimensions based on popular objectives from 4 major players Release device controlled by button on controller box 60

61 Interface with microscope

62 QIAscout raft Can be used as standard cell culture dish Microrafts are magnetic, dislodgeable and self-sealing Each array contains 12,000 microrafts Cells settle according to Poisson distribution 6000 cells means a 30% chance of having a singe cell in a microraft 62

63 Cells can be imaged directly on the array by brightfield or fluorescence CellTracker Green Hoechst 63

64 Numbered microrafts make it easy to isolate cells Numbered microrafts on an unused Array 2. Array with seeded cells in medium 3. Microraft dislodged with needle 3. 64

65 Introducing QIAscout 65

66 Qiagen s Single Cell Portfolio: The cutting edge of genomics Highest accuracy Superior coverage High diversity, high quality libraries Streamlined workflows Data analyis & visualization Biological interpretation WGA or WTA Library preparation Sequencing & QIAGEN Bioinformatics Single-cell whole genome sequencing REPLI-g Single Cell FX DNA Library Kit Solution for variant calling, ideal for applications where SNV & CNV is equally important Single-cell gene expression analysis by RNAseq REPLI-g Single Cell FX RNA Library Kit Streamlined workflow with reliable detection of even low-abundance transcripts Single-cell targeted sequencing REPLI-g Single Cell DNA or WTA Kit QIAseq Targeted DNA\RNA Panels High quality targeted sequencing from a single cell for DNA variants or RNA expression 66

67 QIAGEN s REPLI-g Technology: use MDA, not PCR! MDA: Multiple Displacement Amplification Alkaline denaturation Hexamer random primers Phi29 polymerase strand displacement (30 C) Displaced strand becomes a template for replication Gentle denaturation results in intact DNA-template Even priming events covering the whole genome immediate amplification across all regions Modified high-fidelity Phi29 polymerase (SensiPhi) o High enzyme processivity molecular weight product ( kb) o Proofreading activity 1000-fold higher accuracy than normal PCR-enzyme Multiple displacement events efficient amplification of DNA 67

68 MDA amplifies through gdna secondary structure regions Denatured gdna has a complex secondary structure Consists of regions of ssdna and dsdna that can form complicated hairpins and loops QIAGEN s MDA enzyme handles complex DNA structures generating extremely long amplicons 68

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70 QIAseq Library Products for Illumina Sequencers Products for Illumina Sequencers: Nucleic acid extraction NGS Library preparation Sequencing & QIAGEN Bioinformatics Whole Genome Sequencing (WGS) Hybrid Capture Sequencign QIAseq FX DNA Library Kit FX fragmentation step eliminates shearing 2.5 hour workflow from gdna to library Tunable enzymatic fragmentation for any size probes with higher genomic complexity than Nextera DNA Amplicon/ PCR sequencing QIAseq 1-Step Amplicon Library Kit 1-tube, room temperature library prep cfdna for Liquid Biopsy or NIPT QIAseq cfdna Complete Solution QIAamp Circulating Nucleic Acid Isolation Optimized protocol specific for cfdna Low Input ChIP-seq, cfdna, LCM QIAseq Ultralow Input Library Kit Ultra-efficient chemistry for pg+ DNA Barcoded adapters for 96-plex sequencing Single Cell Genomics (QIAseq Single Cell FX Lib Kits) PCR-free MDA Amplification Sorted single cells or 6pg DNA input 70

71 QIAseq FX: Gold-Standard WGS libraries in just 2.5 hours Purified gdna 1ng 1µg 1000bp QIAseq FX 2.5 hour workflow: - gdna Fragmentation - Barcoded Adapter Ligation - Library Amplification 250bp 450bp Library QC Hybrid Capture (Optional) Target Enrichment (IDT xgen, Agilent SureSelect) Illumina HiSeq Compatible with Hybrid Capture downstream target enrichment Flexible to generate any size sample DNA fragment 71

72 QIAseq FX the ideal balance of speed & high data quality Illumina Truseq Nano Fragmentase + NEB Ultra Illumina Nextera QIASeq FX Total /Hands-on 5 hr/ 1hr 3.5 hr/45 min 1.5 hr/ 15min 2.5 hr /20min Pros Cons Good sequencing performance Relies on Covaris; Long Protocol Fully Enzymatic Fast Workflow Library Complexity Application Flexibility Known Bias Poor Reproducibility Loss of Complexity Precise input requirements Illumina-specific kit Fragmentation (Covaris) Fragmentase Enzyme Tagmentation Fragmentation End Repair A-Tailing End Repair Cleanup Cleanup Adapter Ligation Cleanup End Repair A-Addition Library Amp Cleanup A-Additon Adapter Ligation Library Amp Adapter Ligation Cleanup Cleanup Library Amp Library Amp 72

73 Superior coverage distribution & gold-standard % Duplication Fraction of target genome Coverage Distribution Coverage depth (X) QIAseq FX (100 ng) Supplier N Enzymatic (100 ng) Covaris + Standard LP (100 ng) Tagmentation Supplier I (1 ng) Duplication Rate, 1ng Input QIAseq FX (1ng and 100ng) Supplier N Enzymatic (1ng) Covaris + Standard LP (1 ng) Tagmentation Supplier I (1 ng) 73

74 QIAseq FX: Gold-Standard WGS libraries in just 2.5 hours For high throughput Illumina NGS facilities or small labs sequencing the whole genome (or human whole exome) of any organism, including metagenomics Need even genomic coverage to minimize the total sequencing and avoid dropout regions without sacrificing a fast workflow that s easy to scale QIAseq FX provides gold-standard WGS libraries in just 2.5 hours Competitor products: Covaris Fragmentation + Any Kit: Slow, extra tube transfers, expensive Illumina Nextera: Strong sequence bias, very expensive Illumina Nextera XT: Strong sequence bias, only for small genomes Kapa HyperPlus: No adapters provided, customer must source separately QIAseq FX DNA Library Kit (24), with 24-plex adapter plate QIAseq FX DNA Library Kit (96), with 96-plex adapter plate 74

75 QIAseq Library Products for Illumina Sequencers Products for Illumina Sequencers: Nucleic acid extraction NGS Library preparation Sequencing & QIAGEN Bioinformatics Whole Genome Sequencing (WGS) Hybrid Capture Sequencign QIAseq FX DNA Library Kit FX fragmentation step eliminates shearing 2.5 hour workflow from gdna to library Tunable enzymatic fragmentation for any size probes with higher genomic complexity than Nextera DNA Amplicon/ PCR sequencing QIAseq 1-Step Amplicon Library Kit 1-tube, room temperature library prep cfdna for Liquid Biopsy or NIPT QIAseq cfdna Complete Solution QIAamp Circulating Nucleic Acid Isolation Optimized protocol specific for cfdna Low Input ChIP-seq, cfdna, LCM QIAseq Ultralow Input Library Kit Ultra-efficient chemistry for pg+ DNA Barcoded adapters for 96-plex sequencing Single Cell Genomics (QIAseq Single Cell FX Lib Kits) PCR-free MDA Amplification Sorted single cells or 6pg DNA input 75

76 QIAseq 1-Step Amplicon: Fast Library Prep for PCR Products 30 minute single-tube room-temperature library prep for amplicon targeted re-sequencing Purified PCR Products (1-500 ng) GeneRead V2 Targeted Panels Other Panels (ie. AmpliSeq) Other PCR reactions Single-tube room temperature library prep (30 min) Proprietary single-tube technology: Only 1 pipetting step Fastest Possible Library Prep: 30 minutes! Compatible with any PCR amplicons and gene panels from QIAGEN or other manufacturers library amplification (optional) (45 min) 76

77 QIAseq 1-Step Amplicon: Fast Library Prep for PCR Products For translational research, biomarker discovery and applied testing using a targeted DNA panel (QIAGEN, 3 rd party, or home brew) on Illumina sequencers QIAseq 1-Step Amplicon provides the fastest, easiest workflow from PCR product to sequencer-ready libraries Competitor products workflows 2.5 hours (Kapa) to 6 hours (Illumina TruSeq) Illumina TruSeq Nano: 5-6 hours QIAseq 1-Step Amplicon advantages: 30 minute PCR-free workflow with a single room temp reaction to set up 96-plex adapters included in 96 reaction kit Compatible with ANY multiplexed PCR or gene panels QIAseq 1-Step Amplicon Library Kit (12), no adapters QIAseq 1-Step Amplicon Library Kit (96), with adapters 77

78 QIAseq Library Products for Illumina Sequencers Products for Illumina Sequencers: Nucleic acid extraction NGS Library preparation Sequencing & QIAGEN Bioinformatics Whole Genome Sequencing (WGS) Hybrid Capture Sequencign QIAseq FX DNA Library Kit FX fragmentation step eliminates shearing 2.5 hour workflow from gdna to library Tunable enzymatic fragmentation for any size probes with higher genomic complexity than Nextera DNA Amplicon/ PCR sequencing QIAseq 1-Step Amplicon Library Kit 1-tube, room temperature library prep cfdna for Liquid Biopsy or NIPT QIAseq cfdna Complete Solution QIAamp Circulating Nucleic Acid Isolation Optimized protocol specific for cfdna Low Input ChIP-seq, cfdna, LCM QIAseq Ultralow Input Library Kit Ultra-efficient chemistry for pg+ DNA Barcoded adapters for 96-plex sequencing Single Cell Genomics (QIAseq Single Cell FX Lib Kits) PCR-free MDA Amplification Sorted single cells or 6pg DNA input 78

79 QIAseq Ultralow Input: Maximize Sub-Nanogram Samples Fragmented gdna 10pg 100ng End-polishing Barcoded Adapter Ligation ULTRA-EFFICIENT library prep for maximum library with minimum PCR SUPERIOR DATA including low % adapter and even G/C coverage WHOLE GENOME library complexity from subnanogram input IDEAL for oncology LCM, Liquid Biopsy cfdna, ChIP-Seq, and low input whole genome sequencing Reaction Cleanup Library Amplification (Optional for >10ng input) 79

80 The highest library yield and quality from as little as pg DNA Comparison of Input Requirements ULTRA-EFFICIENT chemistry balances yield with cycles of PCR SUPERIOR % adapter and G/C coverage WHOLE GENOME library complexity IDEAL for cfdna, ChIP-Seq, and low input whole genome sequencing 80

81 QIAseq Ultralow Input: Maximize Sub-Nanogram Samples Launch Date: July 2016 For Illumina sequencing of fragmented DNA, especially limited DNA sample types. Liquid Biopsy cfdna, Ancient DNA, Laser Capture Microdissection, FFPE Sequencing, ChIP-Seq The best possible yield and genome coverage from as little as pg of DNA The best option for low input when REPLI-g pre-amplification is not possible Compatible with Covaris mechanical shearing (up to 100ng DNA) Competitor products: Illumina TruSeq Nano (100ng): Slow, extra tube transfers, expensive Kapa HyperPrep (1ng): No adapters provided, must source separately NEB Ultra II (500pg): 24-plex, tube-format adapters offered separately QIAseq Ultralow Input Library Kit (12), without adapters QIAseq Ultralow Input Library Kit (96), with 96 plex adapters 81

82 QIAGEN Recommended Workflows for Exome Hybrid Capture Exome Sequencing from Human gdna Exome Sequencing from circulating cfdna Exome Sequencing from gdna using Covaris Sample Type Human gdna Circulating cell-free DNA Human gdna Fragmentation Library Prep QIAseq FX DNA Library Kit Cat no QIAseq FX DNA Library Kit Cat no None cfdna is naturally fragmented QIAseq Ultralow Input DNA Library Kit Cat no Covaris QIAseq Ultralow Input DNA Library Kit Cat no Exome Capture Hybrid Capture Probes such as: IDT xgen Exome Research Panel v1.0 Protocol: Hybridization capture of DNA libraries using xgen Lockdown Probes and Reagents Amp GeneRead DNA I Amp Kit (100) Cat no QIAseq FX IDT xgen Exome Hybrid Capture (5.5 hours) GeneRead I Amp Kit QIAseq Library Quant QC QIAseq Ultralow Input IDT xgen Exome Hybrid Capture (5.5 hours) GeneRead I Amp Kit QIAseq Library Quant QC Fragmentation (Covaris) QIAseq Ultralow Input IDT xgen Exome Hybrid Capture (5.5 hours) GeneRead I Amp Kit QIAseq Library Quant QC 82

83 QIAseq Library Products for Illumina Sequencers Products for Illumina Sequencers: Nucleic acid extraction NGS Library preparation Sequencing & QIAGEN Bioinformatics Whole Genome Sequencing (WGS) Hybrid Capture Sequencign QIAseq FX DNA Library Kit FX fragmentation step eliminates shearing 2.5 hour workflow from gdna to library Tunable enzymatic fragmentation for any size probes with higher genomic complexity than Nextera DNA Amplicon/ PCR sequencing QIAseq 1-Step Amplicon Library Kit 1-tube, room temperature library prep cfdna for Liquid Biopsy or NIPT QIAseq cfdna Complete Solution QIAamp Circulating Nucleic Acid Isolation Optimized protocol specific for cfdna Low Input ChIP-seq, cfdna, LCM QIAseq Ultralow Input Library Kit Ultra-efficient chemistry for pg+ DNA Barcoded adapters for 96-plex sequencing Single Cell Genomics (QIAseq Single Cell FX Lib Kits) PCR-free MDA Amplification Sorted single cells or 6pg DNA input 83

84 QIAGEN s REPLI-g Technology: use MDA, not PCR! MDA: Multiple Displacement Amplification Alkaline denaturation Hexamer random primers Phi29 polymerase strand displacement (30 C) Displaced strand becomes a template for replication Gentle denaturation results in intact DNA-template Even priming events covering the whole genome immediate amplification across all regions Modified high-fidelity Phi29 polymerase (SensiPhi) o High enzyme processivity molecular weight product ( kb) o Proofreading activity 1000-fold higher accuracy than normal PCR-enzyme Multiple displacement events efficient amplification of DNA 84

85 MDA amplifies through gdna secondary structure regions Denatured gdna has a complex secondary structure Consists of regions of ssdna and dsdna that can form complicated hairpins and loops QIAGEN s MDA enzyme handles complex DNA structures generating extremely long amplicons 85

86 Qiagen s Single Cell Portfolio: The cutting edge of genomics Highest accuracy Superior coverage High diversity, high quality libraries Streamlined workflows Data analyis & visualization Biological interpretation WGA or WTA Library preparation Sequencing & QIAGEN Bioinformatics Single-cell whole genome sequencing REPLI-g Single Cell DNA Library Kit (48) Cat no Q3 2016: QIAseq FX Single Cell DNA Kit Superior solution for variant calling, ideal for applications where SNV & CNV is equally important Single-cell gene expression analysis by RNAseq REPLI-g Single Cell RNA Library Kit(24) Cat no Q32016: QIAseq FX Single Cell RNA Kit Streamlined workflow with reliable detection of even low-abundance transcripts REPLI-g Single Cell Kit Single-cell targeted sequencing & QIAseq Targeted DNA Panels High quality targeted sequencing from a single cell for DNA variants 86

87 QIAseq Library Products for Illumina Sequencers Products for Illumina Sequencers: Nucleic acid extraction NGS Library preparation Sequencing & QIAGEN Bioinformatics Whole Genome Sequencing (WGS) Hybrid Capture Sequencign QIAseq FX DNA Library Kit FX fragmentation step eliminates shearing 2.5 hour workflow from gdna to library Tunable enzymatic fragmentation for any size probes with higher genomic complexity than Nextera DNA Amplicon/ PCR sequencing QIAseq 1-Step Amplicon Library Kit 1-tube, room temperature library prep cfdna for Liquid Biopsy or NIPT QIAseq cfdna Complete Solution QIAamp Circulating Nucleic Acid Isolation Optimized protocol specific for cfdna Low Input ChIP-seq, cfdna, LCM QIAseq Ultralow Input Library Kit Ultra-efficient chemistry for pg+ DNA Barcoded adapters for 96-plex sequencing Single Cell Genomics (QIAseq Single Cell FX Lib Kits) PCR-free MDA Amplification Sorted single cells or 6pg DNA input 87

88 QIAseq ccf DNA Workflow Designed for any NGS-based cfdna research Up to 5 ml plasma can be used for cfdna extraction using QIAamp Mini Columns. 24 samples are processed in less than 2 h ng of cfdna can be directly used from up to 56.5 µl eluant volume. Quantifcation of cfdna prior to library prep is not necessary. Library prep includes end-polishing and adapter ligation and takes < 1 h. Optional HiFi amplification can be performed for < 10 ng cfdna before samples undergo sequencing. 88

89 QIAseq ccf DNA Workflow 89

90 QIAseq sequencing summary Unique molecular indexes remove bias due to technique and give improved data Applications for DNA variants, Gene expression, Fusion gene, mirna Complete workflow from start to data analysis On-Line based platform CLC-Bio solution (gene expression, more to follow) Leverage Qiagen content know-how for NGS Disease and pathway specific collections Extended panels where you add your targetes to our Panels Customizable solutions Library Preparation kits and applications Circulating cell-free DNA Single Cell Genomics (DNA/RNA) Genomic DNA (WGS/HC) enzymatic fragmentation in 1 tube Ultra-low input sequencing (10pg) 1-step amplicon sequencing 90