New Frontiers of Genetic Profiling Achieve Higher Sensitivity and Greater Insights with Molecular Barcodes, Long Read Capture and Optimized Exomes

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1 New Frontiers of Genetic Profiling Achieve Higher Sensitivity and Greater Insights with Molecular Barcodes, Long Read Capture and Optimized Exomes Jennifer Jones, PhD Senior Field Application Scientist September 27th, 2017

2 New Frontiers of Genetic Profiling Agenda SureSelect Portfolio Overview SureSelect XT HS Superior Performance SureSelect XT Low Input What is it? Introduction of Clinical Research Exome v2 Long Read SureSelect Capture on Nanopore 2

3 New Frontiers of Genetic Profiling Agenda SureSelect Portfolio Overview SureSelect XT HS Superior Performance SureSelect XT Low Input What is it? Introduction of Clinical Research Exome v2 Long Read SureSelect Capture on Nanopore 3

4 Target Enrichment: Focus on what Matters Key Goals of Target Enrichment Focus on a subset of the genome / transcriptome gdna Achieve greater depth in targeted regions Discover more mutations, SNPs and Indels Lower sequencing costs and obtain faster time to result SureSelect Enriched Targets 4

5 SureSelect Technology: The Best Performance Core Technology Ultra-Long RNA Baits (120-mer) Binding strength RNA:DNA Allele 1 > DNA:DNA SNP Benefits Better Sensitivity Detect more SNP, Indels, CNV, fusions Better Workflow Shorter Hybridization Better Allelic Balance Equal representation of both alleles Allele mer bait Indel Allele 2 25bp deletion Deletion

6 SureSelect Technology Simple Target Enrichment Workflow Library prep Hyb set-up Hybridization Baits: - crna probes - Long (120bp) - Biotin labeled - User-defined Capture Wash / Elution / Amp

7 Complete Workflow Solutions from Sample to Data Most Complete Enrichment Solution 1. Create a design 2. Qualify samples 3. Enrich libraries 4. Quantify and qualify libraries 5. Analyze and Interpret SureDesign human or nonhuman organisms TapeStation DIN or NGS FFPE QC SureSelect Workflows 2100 BioAnalyzer or 4200 TapeStation SureCall single sample, trio or pair analysis 7

8 Complete Workflow Solutions from Sample to Data Most Complete Enrichment Solution 1. Create a design 2. Qualify samples 3. Enrich libraries 4. Quantify and qualify libraries 5. Analyze and Interpret SureDesign human or nonhuman organisms TapeStation DIN or NGS FFPE QC SureSelect Workflows 2100 BioAnalyzer or 4200 TapeStation SureCall single sample, trio or pair analysis 8

9 SureSelect Library Prep for Every Application 9

10 SureSelect Library Prep for Every Application 10

11 SureSelect XT High performing workflow, trusted technology Post-capture pooling solution 1 sample = 1 hybridization Provides maximum complexity Efficient Workflow 16 hour hybridization, 1.5 day turn around time from sample to sequencing Available is standard 3µg or 200ng input Complete and Flexible Solution Solutions for custom design, QC and data analysis Manual or automated processing 11

12 SureSelect XT2 High performing workflow, trusted technology Pre-capture pooling solution Less pipetting, cost-efficient 8 samples per exome capture; 16 samples per custom capture Efficient Workflow Master mixed reagents 3X less tubes 16 hr hybridization, 1.5 day turn around time from sample to sequencing Complete and Flexible Solution Solutions for custom design, QC and data analysis Manual or automated processing Library Preps 8-16 Pooled libraries into single capture Prep for Sequencing 12

13 SureSelect Library Prep for Every Application 13

14 SureSelect QXT The Fastest Enrichment Workflow < 7 hours ~12.5* - 27** hours NO MECHANICAL SHEARING TRANSPOSASEbased Library Prep 90-minute Hybridization Sequence *MiSeq and **HiSeq systems *MiSeq, HiSeq systems DATA in hrs FAST, EASY and CONVENIENT 30 min hands-on time for library prep 3.5h overall hands-on time No special equipment required for fragmentation 50ng INTACT DNA SAMPLE INPUT 14

15 SureSelect Library Prep for Every Application 15

16 New Frontiers of Genetic Profiling Agenda SureSelect Portfolio Overview SureSelect XT HS Superior Performance SureSelect XT Low Input What is it? Introduction of Clinical Research Exome v2 Long Read SureSelect Capture on Nanopore 16

17 SureSelectXT HS Superior Performance 10 ng input FFPE optimized High sensitivity with molecular barcodes Streamlined workflow 17 PR For Research Use Only. Not for use in diagnostic procedures..

18 SureSelectXT HS Superior Performance 10 ng input FFPE optimized High sensitivity with molecular barcodes Streamlined workflow 18 PR For Research Use Only. Not for use in diagnostic procedures..

19 10 ng input Varying inputs with XT and XT HS 200 ng and 10 ng 100X Sequencing % NA12878 with V % 80.00% 70.00% 60.00% 50.00% 40.00% 30.00% 20.00% 10.00% 0.00% XT - 200ng XT HS - 200ng XT HS - 10ng % Dups % in Targeted Regions Uniformity (1/2 mean with upper tail): > 20 reads: 19 For Research Use Only. Not for use in diagnostic procedures..

20 10 ng input Varying inputs with XT and XT HS 200 ng and 10 ng 100X Sequencing % NA12878 with Kinome 90.00% 80.00% 70.00% 60.00% 50.00% 40.00% 30.00% 20.00% 10.00% 0.00% XT - 200ng XT HS - 200ng XT HS - 10ng % Dups % in Targeted Regions (not more <5%) Uniformity (1/2 mean with upper tail): > 20 reads: 20 For Research Use Only. Not for use in diagnostic procedures..

21 SureSelectXT HS Superior Performance 10 ng input FFPE optimized High sensitivity with molecular barcodes Streamlined workflow 21 PR For Research Use Only. Not for use in diagnostic procedures..

22 20X Coverage FFPE Optimized 20X Coverage with varying amounts of sequencing Decreasing sample quality Fresh Frozen FFPE 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% ddcq X 600X 700X 800X 900X 1000X For Research Use Only. Not for use in diagnostic procedures..

23 FFPE Optimized 10 ng FFPE CCP 100% 1000x sequencing depth; 10ng FFPE; CCP 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Breast and Kidney Larynx and prostate XT HS XT HS Percentage reads in targeted regions: 44.10% 24.81% Percent duplicate reads: 65.23% 71.48% Uniformity (1/2 mean with upper tail): 70.17% 78.64%...at least 20 reads: 95.57% 90.14% 23 For Research Use Only. Not for use in diagnostic procedures..

24 FFPE Optimized SNP concordance and sensitivity SNP sensitivity Lung FFPE vs matched FF SNP concordance Lung FFPE vs matched FF 100% 100% 98% 98% 96% 96% 94% 94% 92% 92% DIN: 4.8 ddcq: 0.2 XT HS with CCP 1000X seq depth 90% 5ng 10ng 50ng 100ng 200ng GT is variant HOM GT is variant HET 90% 5ng 10ng 50ng 100ng 200ng GT is variant HOM GT is variant HET 24 For Research Use Only. Not for use in diagnostic procedures..

25 SureSelectXT HS Superior Performance 10 ng input FFPE optimized High sensitivity with molecular barcodes Streamlined workflow 26 PR For Research Use Only. Not for use in diagnostic procedures..

26 SureSelectXT HS Molecular barcodes P7 Sample Index 8 bp sample index 10 bp molecular barcode MBC P5 27 For Research Use Only. Not for use in diagnostic procedures..

27 SureSelectXT HS Molecular barcodes Target region Baits Read pairs Final consensus reads 28 For Research Use Only. Not for use in diagnostic procedures..

28 Observed Frequency High sensitivity with molecular barcodes Expected vs Observed Allele Frequency 45% 40% R² = % 30% 25% 20% 15% 10% 5% HD 200, CCP 3000X seq depth 0% 0% 5% 10% 15% 20% 25% 30% 35% 40% 45% Expected Frequency 29 For Research Use Only. Not for use in diagnostic procedures..

29 SureSelect XT HS Allele Frequency (Consensus reads using MBC) High sensitivity with molecular barcodes Reduction in false calls 100.0% 80.0% 60.0% 40.0% 20.0% 0.0% 0.0% 20.0% 40.0% 60.0% 80.0% 100.0% Dedup using standard Start/Stop) 30 For Research Use Only. Not for use in diagnostic procedures..

30 Variant Call Number False Positive Reduction Rate High sensitivity with molecular barcodes Reduction in false calls % % 60% 50% % % 20% 0 HOM HET Low Frequency (<5%) Variant Allele Type 10% 0% HOM HET Low Frequency (<5%) XT (start/stop) XTHS (Concensus_MBC3) Variant Allele Type 32 For Research Use Only. Not for use in diagnostic procedures..

31 Variant Allele Frequency Variant Allele Frequency High sensitivity with molecular barcodes Reduction in false calls 2.0% 1.8% 1.6% 1.4% 1.2% 1.0% 0.8% 0.6% 0.4% 0.2% 0.0% Genome Position (Gb) 808 median coverage 218 variants detected VAF<2% 2.0% 1.8% 1.6% 1.4% 1.2% 1.0% 0.8% 0.6% 0.4% 0.2% 0.0% Genome Position (Gb) 906 median coverage 18 variants detected VAF<2% -92% Horizon cfdna reference and a custom 164 kb SureSelect panel courtesy of Dr LJ Barber and Dr M Gerlinger, Centre for Evolution and Cancer, The Institute of Cancer Research, London, UK 34 For Research Use Only. Not for use in diagnostic procedures..

32 SureSelectXT HS Superior Performance 10 ng input FFPE optimized High sensitivity with molecular barcodes Streamlined workflow 37 PR For Research Use Only. Not for use in diagnostic procedures..

33 Streamlined workflow Workflow improvements Protocol Step Approximate Time (manual processing of 8 samples) DNA QC Qubit (Intact) qpcr (FFPE) Covaris shearing BioA QC (optional) QC& pool libraries Repair, A-tailing, adapter ligation XT HS & XT Low Input XT 1.75 hours 3.25 hours Pre-capture PCR 1.1 hours 1.1 hours Hyb 1.8 hours 16.3 hours Capture & Wash 2.25 hours 2.25 hours Post-capture PCR 1.1 hours 1.1 hours STOP STOP STOP STOP Total: 8 hours 24 hours STOP STOP STOP STOP STOP Optional stopping points SPRI clean up steps 38

34 SureSelectXT HS Superior Performance 10 ng input FFPE optimized High sensitivity with molecular barcodes Streamlined workflow 39 PR For Research Use Only. Not for use in diagnostic procedures..

35 How do we compare to the competition? 10 ng FF with CCP % 90.00% Equivalent performance for most metrics Higher % on-target 80.00% 70.00% 60.00% 50.00% 40.00% 30.00% 20.00% 10.00% 0.00% Competitor K XT HS Competitor N Competitor K XT HS Competitor N FF Breast FF Prostate %duplicates %on-target Uniformity (1/2 mean with upper tail) 20X base coverage 100X base coverage 40 For Research Use Only. Not for use in diagnostic procedures..

36 How do we compare to the competition? 10 ng FFPE (mid quality) with CCP % Higher % on target Higher 100X coverage 90.00% 80.00% 70.00% 60.00% 50.00% 40.00% 30.00% 20.00% 10.00% 0.00% Competitor K XT HS Competitor N Competitor K XT HS Competitor N FFPE Breast FFPE Kidney %duplicates %on-target Uniformity (1/2 mean with upper tail) 20X base coverage 100X base coverage 41 For Research Use Only. Not for use in diagnostic procedures..

37 How do we compare to the competition? 10 ng FFPE (poor quality) with CCP % 90.00% Higher % on target Higher 20X coverage Higher 100X coverage 80.00% 70.00% 60.00% 50.00% 40.00% 30.00% 20.00% 10.00% 0.00% Competitor K XT HS Competitor N Competitor K XT HS Competitor N FFPE Larynx FFPE Prostate %duplicates %on-target Uniformity (1/2 mean with upper tail) 20X base coverage 100X base coverage 43 For Research Use Only. Not for use in diagnostic procedures..

38 New Frontiers of Genetic Profiling Agenda SureSelect Portfolio Overview SureSelect XT HS Superior Performance SureSelect XT Low Input What is it? Introduction of Clinical Research Exome v2 Long Read SureSelect Capture on Nanopore 44

39 SureSelect XT Low Input What is it? 10 ng input FFPE optimized High sensitivity with molecular barcodes Streamlined workflow 45 PR For Research Use Only. Not for use in diagnostic procedures..

40 SureSelect XT Low Input What is it? 10 ng input FFPE optimized High sensitivity with molecular barcodes Streamlined workflow 192 sample indexes plate format 46 PR For Research Use Only. Not for use in diagnostic procedures..

41 SureSelect XT HS and Low Input 47 For Research Use Only. Not for use in diagnostic procedures..

42 Product configuration Kit format XT HS 16 rxn kit A = index 1-16 B = index rxn kit index 1-32 (x3) XT Low Input 96 rxn kit A = index 1-96 B = index * Sample index will be provided in individual tubes *Sample index will be provided in plates 48 For Research Use Only. Not for use in diagnostic procedures..

43 Product configuration Pre and Post PCR reagents Pre-PCR Post-PCR Library prep reagents Sample index Hyb Reagents Box 1 Hyb Reagents Box 2 49 For Research Use Only. Not for use in diagnostic procedures..

44 SureSelect XT HS & Low Input Summary SureSelectXT HS outperforms the competition: 1. Higher % on target 2. Higher 20X coverage 3. Higher 100X coverage 10 ng input FFPE optimized High sensitivity with molecular barcodes Streamlined workflow 192 sample indexes plate format 50 PR For Research Use Only. Not for use in diagnostic procedures..

45 New Frontiers of Genetic Profiling Agenda SureSelect Portfolio Overview SureSelect XT HS Superior Performance SureSelect XT Low Input What is it? Introduction of Clinical Research Exome v2 Long Read SureSelect Capture on Nanopore 51

46 Automation TE Kits Library Prep Kits SureSelect Technology Most Complete Enrichment Solution DNA RNA XT XT2 QXT Stranded RNA RNA capture Stranded RNA RNA-Seq Methyl- Seq (V6, CREv1/2, Human, Non-human) (Inherited Disease, Kinome) Whole Genome Panels (Kinome) Custom Solutions for Copy Number OR 52

47 Three pillars that guide Agilent exomes Performance Content Flexibility 53

48 Agilent SureSelect Exomes New! SureSelect Clinical Research Exome V2 (CREv2) SureSelect All Human Exon V6 SureSelect Focused Exome Comprehensive exome optimized for rare & inherited disorders Comprehensive exome for translational and clinical research Targeted exome with optimized coverage of only the disease associated genes 54

49 What s New? CRE V1 54 Mb total capture size Uses Human All Exon V5 (launched in 2012) as a base CRE V Mb total capture size Uses Human All Exon V6 (launched in June 2015) as a base 5109 genes identified as disease relevant Catalogue pricing for plusenabled content 55

50 Performance, Content, Flexibility in CREv2 Performance Content Flexibility Enhanced coverage of disease associated genes Optimized content including non-coding regions associated with disease Design customizability to further enhance your exome 56

51 CRE v2 Sequencing Performance with SureSelect XT and QXT Library Prep Kits Performance Data averaged from 8 different HapMap DNA samples 57

52 CREv2 provides high SNP and Indel concordance in targeted regions Samples SNP Concordance Indel Concordance Hom Het Sample % 99.41% 97.15% Sample % 99.29% 96.63% Sample % 99.36% 96.95% Sample % 99.40% 97.48% Sample % 99.48% 97.19% Sample % 99.29% 96.5% Sample % 99.46% 97% Sample % 99.42% 97.32% SNP concordance calculated using HapMap data Indel concordance calculated using dbsnp data 58

53 CREv2 provides enhanced coverage of disease-associated genes Performance 5,109 disease-associated genes 100x average sequencing depth; 67.3Mb design; 6.5Gb sequencing 59

54 Normalized coverage Avg_Cov Normalized coverage Avg_Cov Agilent exomes provide uniform coverage regardless of GC content Agilent CREv2 Pearson s r = Vendor ID Pearson s r = Low GC as.factor(bin) High Low as.factor(bin) GC High 0 0 Smaller deviation from the mean across GC bins in CREv2 All exomes sequenced to the same average sequencing depth Exons were divided into deciles based on GC to calculate normalized coverage 60

55 CREv2 provides consistent coverage in high and low GC regions 41% GC 25% GC Agilent CREv2 Vendor ID Low coverage of AT-rich exon in Vendor ID exome 45% GC 29% GC Agilent CREv2 Vendor ID Low coverage of AT-rich exon in Vendor ID exome All exomes sequenced to the same average sequencing depth 61

56 CREv2 provides the most comprehensive coverage of diseaseassociated regions Content In our comparison, the Agilent V6 exome performs better than the NimbleGen s MedExome both in terms of efficiency and evenness of coverage distribution. With its larger target size, it is also more comprehensive, and therefore the better choice in research projects that aim to identify novel disease-associated genes. * CRE v2: Optimized coverage of disease-associated genes Plus Coverage of splice sites & deep intronic regions Coverage of other non-coding regions.associated with disease Curated in collaboration with Dr. Madhuri Hegde, Emory University *: Altmuller J, et al. Biol Chem 2016, 397(8):

57 CREv2 provides the most comprehensive coverage of diseaseassociated regions 63

58 Many Genes Single Gene Genetic causes of rare diseases can be complex Wildtype CFTR Mutation in CFTR Cystic Fibrosis 1 gene Cl- Leukodystrophy 30 genes Healthy neuron Damaged neuron Retinitis Pigmentosa 77 genes Rods & cones in healthy retina Rods & cones in RP 66

59 Example of CREv2 providing more disease-associated regions in two rare diseases ClinVar Pathogenic/Likely Pathogenic Leukodystrophy Variants covered ClinVar Pathogenic/Likely Pathogenic Retinitis Pigmentosa Variants covered Agilent CREv2 98.1% 95.3% Competitor ID 90% 87.9% Competitor R 90.7% 94.6% 67

60 CREv2 provides superior disease relevant content associated with Leukodystrophy Pathogenic variant associated with leukodystrophy only detectable by Agilent CREv2 but not competitor ID exome Pathogenic variant in 5 UTR of GJC2 A>G SNV Agilent CREv2 Vendor ID 68

61 Mutations that cause retinitis pigmentosa frequently occur in noncoding regions Agilent CREv2 Vendor ID Vendor R Agilent CREv2 Vendor ID Vendor R 69

62 Build your own exome with SureSelect customization capability Flexibility Unmatched flexibility in customization of content and formats. Use existing designs as a base to optimize the exome for your research needs 70

63 Mutation spectrum in rare diseases spans small and large changes CNVs account for 10-15% pathogenicity in rare diseases Nuclear receptor binding SET domain protein 1 (NSD1) 75% point mutations 25% deletions/duplications Duchenne Muscular Dystrophy (DMD) 35% point mutations 65% deletions/duplications SNVs/Indels Copy Number Variation Cystic Fibrosis (CFTR) 90% point mutations 10% deletions/duplications Familial Adenomatous Polyposis (APC) 80% point mutations 20% deletions/duplications 71

64 OneSeq: One NGS Assay for All Variants (CNVs, LOH, SNVs & Indels) ClinGen disease associated regions Gene A ClinGen disease associated regions Gene B 1) Evenly spaced genome-wide baits 2) High density baits in ClinGen disease associated regions Copy number & LOH 3) User-defined baits in exonic regions SNVs & indels 72

65 CRE V2 can be combined with any OneSeq backbone High Res Low Res CNV genome-wide 300 kb 1 Mb CNV in ClinGen regions kb 1 MB LOH 5 Mb 10 Mb Design size 12 Mb 2.7 Mb 73

66 Agilent provides the most comprehensive solution for rare and inherited disorders Sample QC 74

67 New Frontiers of Genetic Profiling Agenda SureSelect Portfolio Overview SureSelect XT HS Superior Performance SureSelect XT Low Input What is it? Introduction of Clinical Research Exome v2 Long Read SureSelect Capture on Nanopore 75

68 Long Read SureSelect Capture on Nanopore Procedure modifications: Probe design: stringency, probe spacing Input DNA amount: 3-4 ug Genomic DNA shearing: 2 kb+ long-range PCR: both pre-capture and post-capture Target Regions: 2.4 Mb from two full genes, p16/cdkn2a and SMAD4 Sequencers: Nanopore MinION and Illumina MiSeq 76

69 Long Read SureSelect Capture on Nanopore 77

70 Long Read SureSelect Capture on Nanopore Nanopolish to call SNV 80

71 Long Read SureSelect Capture on Nanopore Sniffles to call Structural variations 81

72 Long Read SureSelect Capture on Nanopore 84

73 For Research Use Only. Not for use in diagnostic procedures. 85