Introducing combined CGH and SNP arrays for cancer characterisation and a unique next-generation sequencing service. Dr. Ruth Burton Product Manager

Transcription

1 Introducing combined CGH and SNP arrays for cancer characterisation and a unique next-generation sequencing service Dr. Ruth Burton Product Manager

2 Today s agenda Introduction CytoSure arrays and analysis software The CytoSure Haematological Cancer +SNP Array Probe design Analysis The NGS Sequencing Service from OGT Analysis Pipeline Working with cancer samples Summary

3 OGT - provides advanced clinical genetics solutions - develops innovative molecular diagnostics Founded by Ed Southern in 1995 >70 people OGT Begbroke: Corporate offices and highthroughput labs OGT Southern Centre: Biomarker discovery

4 OGT s key businesses IP Licensing 40 licence relationships Diagnostic Biomarkers Genomic- and protein-based diagnostics Technologies For Molecular Medicine Clinical and Genomic Solutions Cytogenetics products and genomic services

5 Today s agenda Introduction CytoSure arrays and analysis software The CytoSure Haematological Cancer +SNP Array Probe design Analysis The NGS Sequencing Service from OGT Analysis Pipeline Working with cancer samples Summary

6 The OGT acgh design process All possible human genome probes Selection based on specificity, T m, GC, etc. Oligome TM database Further selection based on OGT probe rating and desired coverage and content acgh arrays Design & hyb two different acgh arrays Optimised acgh design Selection of best performing probes based on experimental results Optimised array design for catalogue and custom products

7 CytoSure cytogenetics range Arrays ISCA and Syndrome Plus Chromosome X Aneuploidy DMD* Molecular Testing Arrays and Panels ISCA UPD Oligome custom arrays Labelling kits, reagents and automation

8 CytoSure cytogenetics range Arrays ISCA and Syndrome Plus Chromosome X Aneuploidy DMD* Molecular Testing Arrays and Panels ISCA UPD New! ISCA +SNP Arrays New! Haematological Cancer +SNP Arrays Oligome custom arrays Labelling kits, reagents and automation New! CytoSure Interpret Software v4

9 Today s agenda Introduction CytoSure arrays and analysis software The CytoSure Haematological Cancer +SNP Array Probe design Analysis The NGS Sequencing Service from OGT Analysis Pipeline Working with cancer samples Summary

10 CytoSure Haematological Cancer +SNP Array (8 x 60k) For: Chronic Lymphocytic Leukaemia (CLL), Multiple Myeloma (MM), Myeloproliferative Neoplasms (MPN) and Myelodysplastic Syndromes (MDS) Allows confident detection of copy number changes and loss of heterozygosity on a single array Amplifications and deletions are shown alongside SNP data

11 CytoSure Haematological Cancer +SNP Array (8 x 60k) For: Chronic Lymphocytic Leukaemia (CLL), Multiple Myeloma (MM), Myeloproliferative Neoplasms (MPN) and Myelodysplastic Syndromes (MDS) Allows confident detection of copy number changes and loss of heterozygosity on a single array Amplifications and deletions are shown alongside SNP data An optimised design allowing identification of key genomic aberrations Targets regions known to be important predictors, plus good backbone coverage

12 CytoSure Haematological Cancer +SNP Array (8 x 60k) For: Chronic Lymphocytic Leukaemia (CLL), Multiple Myeloma (MM), Myeloproliferative Neoplasms (MPN) and Myelodysplastic Syndromes (MDS) Allows confident detection of copy number changes and loss of heterozygosity on a single array Amplifications and deletions are shown alongside SNP data An optimised design allowing identification of key genomic aberrations Targets regions known to be important predictors, plus good backbone coverage Cancer-specific tracks enable fast and easy data generation and interpretation of cytogenetic results Includes CLL regions from the Mitelman database, Cancer Gene Census Genes and Haematology Regions from Atlas of Genetics and Cytogenetics in Oncology and Haematology

13 CytoSure Haematological Cancer Array +SNP targeted design

14 Today s agenda Introduction CytoSure arrays and analysis software The CytoSure Haematological Cancer +SNP Array Probe design Analysis The NGS Sequencing Service from OGT Analysis Pipeline Working with cancer samples Summary

15 What are the challenges? Step 1: Selecting the SNPs 407,787 human SNPs Select most informative SNPs (minor allele frequency of ) Elect candidate SNPs using OGT probe selection algorithms Microarray Design Reassess probe design criteria Test the design with multiple samples with known genotypes Analyse to determine functional probe designs

16 What are the challenges? Step 1: Selecting the SNPs ~7,500 SNPs, each targeted by 2 optimised probes in duplicate, incorporated into 8x60k acgh Cancer design of ~30,000 copy number probes Validation on further set of known genotype samples Validation of SNP and CNV detection against clinical samples

17 Step 2: Final design One probe is designed to target each allele. The difference between the intensities of these two probes indicates the genotype of the sample. This difference in intensity is not always consistent between Allele 1 Allele 2 SNPs and probe designs.

18 Today s agenda Introduction CytoSure arrays and analysis software The CytoSure Haematological Cancer +SNP Array Probe design Analysis The NGS Sequencing Service from OGT Analysis Pipeline Working with cancer samples Summary

19 Developing the Software New! CytoSure Interpret Software v4 Fast, accurate and simple analysis of acgh data

20 Developing the Software New! CytoSure Interpret Software v4 Fast, accurate and simple analysis of acgh data Comprehensive data annotation with direct links to external databases and online resources

21 Developing the Software New! CytoSure Interpret Software v4 Fast, accurate and simple analysis of acgh data Comprehensive data annotation with direct links to external databases and online resources Robust relational database allowing sophisticated data querying and filtering

22 Developing the Software New! CytoSure Interpret Software v4 Fast, accurate and simple analysis of acgh data Comprehensive data annotation with direct links to external databases and online resources Robust relational database allowing sophisticated data querying and filtering Improved SNP calling algorithm

23 CytoSure Interpret Software and SNP Analysis For each SNP: Compare signal intensities of the 2 alleles Apply weighting factors Assign genotype based on user-definable threshold Normal chromosome: ~50% Homozygosity

24 CytoSure Interpret Software and SNP Analysis For each SNP: Compare signal intensities of the 2 alleles Apply weighting factors Assign genotype based on user-definable threshold Normal chromosome: ~50% Homozygosity

25 CytoSure Interpret Software and SNP Analysis Visualisation Gain Loss

26 CytoSure Interpret Software and SNP Analysis Visualisation Genotype Assignment Gain Chromosome 9 Homozygosity above userdefinable threshold Loss Percentage Homozygosity

27 CytoSure Interpret Software and SNP Analysis Visualisation Significance score Whole Genome Regions of LOH

28 CytoSure Interpret Software and the Haematological Cancer Array +SNP

29 CytoSure Interpret Software and the Haematological Cancer Array +SNP Whole chromosome Whole genome

30 CytoSure Interpret Software and the Haematological Cancer Array +SNP QC metrics

31 CytoSure Interpret Software and the Haematological Cancer Array +SNP Detailed view

32 Today s agenda Introduction CytoSure arrays and analysis software The CytoSure Haematological Cancer +SNP Array Probe design Analysis The NGS Sequencing Service from OGT Analysis Pipeline Working with cancer samples Summary

33 Microarray Service High-throughput with outstanding quality High-throughput processing and analysis service allowing screening of >2000 samples / week in an automated workflow Scalable system same process for pilot study or large study >40 QC checks on each sample ensure high-quality data

34 Delivering Expert Project Design Step 1: Selection of most appropriate genomic regions for your project and budget Whole exome Pre-designed, validated whole exome capture probes Coding regions are most likely candidates for many disorders Custom genomic regions Expert custom design of capture probes for your regions of interest Flexibility to focus on regions of clinical significance or GWAS regions

35 Bait Design Problems Facing Users Potential sources of bias Regions of interest too short Bait thermodynamic behaviour GC content Melting Temperature Risk of Design Errors OGT s extensive experience in designing probes for microarrays allows us to minimise bias and ensure evenness of coverage giving the best chance to identify mutations Optimised coverage = improved capture

36 OGT custom bait design helps overcome GC issues OGT Catalogue Exome Kit In a region with 70% GC content OGT custom bait design achieved a maximum read depth of 50x. Catalogue Exome Capture Kit does not capture any reads in this region.

37 Custom baits improve coverage at region boundaries OGT 1KG OGT custom bait design gives increased read depth around edges of target regions.

38 Custom approach ensures variant detection OGT 1KG Even at more than 50x coverage, whole exome sequencing does not accurately identify all SNPs. OGT custom baits design compared with 1000 Genomes whole exome capture data.

39 Customer Report Design Parameters Depth of Coverage On target / Off target Regions not covered and why not Bait Details Singletons GC distribution T m distribution Library Design Baits generated 1. Data 2. Draft Design 3. Singleton Baits 4. Bait Thermodynamics 5. Report

40 Today s agenda Introduction CytoSure arrays and analysis software The CytoSure Haematological Cancer +SNP Array Probe design Analysis The NGS Sequencing Service from OGT Analysis Pipeline Working with cancer samples Summary

41 The Analysis Challenge Sequencer Hard drive with ~4Gb per exome Publication NGS Raw data Mapping Annotation Filtering Reporting

42 Processing pipeline Read QC (FastQC) Alignment (bwa/grch37) Re-alignment around indels (GATK) Variant calling (GATK UG) Base quality recalibration (GATK) Duplicate marking (Picard) Variant filtration (VQSR/hard) Variant annotation (modified VEP) Report generation (XSLT/JS) Additional analysis steps

43 Why Mark Duplicates and Realignment around Indels? 3 incorrect calls within 40bp!

44 SNP and Indel classification as standard We check and annotate every detected SNP and Indel against all human Ensembl genes and dbsnp dbsnp is the variant known? Does it affect any of the following? Promoter region or UTR Splice sites or intronic region Synonymous or non synonymous mutation Frameshift mutation Stop codon

45 Today s agenda Introduction CytoSure arrays and analysis software The CytoSure Haematological Cancer +SNP Array Probe design Analysis The NGS Sequencing Service from OGT Analysis Pipeline Working with cancer samples Summary

46 Cancer analysis Projects on familial susceptibility and paired / normal analysis NCI Istituto Nazionale dei Tumori (Milan) Developed a subtractive approach to detect tumour specific mutations Important considerations: Sequence quality Changes in copy number

47 Cancer analysis our approach Subtractive implementation Call genotype in both cancer and normal sample Discard reversions to reference genotype in cancer sample Threshold by depth of coverage at both sites Threshold by genotype likelihood at both sites Easy to implement, returns results in seconds Comparison to SomaticSniper For positions with depth >20, 90% of OGT calls are a high-quality subset of quality SomaticSniper calls OGT s calls are more stringent ~700 somatic changes compared to ~2500 in Wilm s tumour test samples Why?

48 OGT and SomaticSniper concordance

49 More calls with SomaticSniper

50 Depth of coverage with SomaticSniper

51 Cancer specific report

52 Today s agenda Introduction CytoSure arrays and analysis software The CytoSure Haematological Cancer +SNP Array Probe design Analysis The NGS Sequencing Service from OGT Analysis Pipeline Working with cancer samples Summary

53 CytoSure excellence from sample to result Optimized targeted array content for analysis of CLL, MM, MPN and MDS samples Reliable detection of CNV and SNPs on a single array Effortless data analysis easy-to-use, class-leading software Expert, high-throughput sequencing service from bait design to bespoke data analysis with an 8 week turnaround time The complete solution for cytogenetics arrays (product or custom), software, labelling, reagents, automation, services

54 OGT collaborators and customers

55 Thank you

56 CytoSure: For research use only This product is provided under an agreement between Agilent Technologies, Inc., and OGT. The manufacture, use, sale or import of this product may be subject to one or more of U.S. patents, pending applications, and corresponding international equivalents, owned by Agilent Technologies, Inc. The purchaser has the non-transferable right to use and consume the product for RESEARCH USE ONLY AND NOT for DIAGNOSTICS PROCEDURES. It is not intended for use, and should not be used, for the diagnosis, prevention, monitoring, treatment or alleviation of any disease or condition, or for the investigation of any physiological process, in any identifiable human, or for any other medical purpose. This document and its contents are Oxford Gene Technology IP Limited. All rights reserved. OGT, CytoSure, Genefficiency, Oligome,and Oxford Gene Technology are trademarks of Oxford Gene Technology IP Limited.