Genomic Technologies For Cancer Research

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Genomic Technologies for Cancer Researchwww.illumina.com/applications/cancer.html

Table of ContentsI. Introduction: Genomic Technologies for Cancer Research3II. Approaches for Detecting Somatic Mutations4Targeted Sequencing Solutions for Somatic Mutation Detection4Exome Sequencing4Focused Sequencing Panels4Custom Targeted Sequencing4Whole-Genome Sequencing Solutions4Data Analysis Tools for Somatic Variant Detection5III. Evaluating Germline Mutations in Cancer6Targeted Sequencing to Detect Common Germline Mutations7Microarray-Based Approaches7IV. Structural Variant Detection in Cancer7DNA and RNA Sequencing for Translocation Detection8Copy Number Variation Arrays8V. Investigating Gene Regulation in Cancer8DNA–Protein Interactions8DNA Methylation9RNA Sequencing9Targeted RNA Sequencing9Small RNA Sequencing10Data Analysis Tools for the Study of Gene Regulation11VI. Summary11For Research Use Only. Not for use in diagnostic procedures.

I. Introduction: Genomic Technologies for Cancer ResearchHETEROGENEITYPROGRESSIONIn recent years, genomic technologies have emerged as invaluable tools in cancer research (Figure 1). Internationalprojects such as the International Cancer Genome Consortium (ICGC)1 and The Cancer Genome Atlas (TCGA)2, taskedwith mapping the biology of dozens of tumor types, would not have been possible without these tools. Next-generationsequencing (NGS) and high-density microarrays are used to study the biology of cancer. Both provide the cancerresearch community with a growing body of knowledge that may lead to more effective drug design, better patienttreatment options, and more accurate prognoses.3NormalNeoplastic ChangesTumorTreatment ResponseRecurrenceSomatic MutationsGermlineMutationsGene Expression &Epigenetic ChangesAdditional MutationsChromosomal AbnormalitiesFigure 1: The Tumor Progression Pathway—Genomic technologies are helping researchers achieve a deeper understanding of the tumorprogression pathway. Much of the research thus far has focused on the study of basic tumor biology and the identification of variants in germlineDNA that influence cancer risk and susceptibility. However, there has been a shift over the past few years to more translational study designs,linking genetic information with phenotypes to understand the clinical significance.4,5NGS is suited to the study of cancer biology. Paired tumor-normal (T/N) whole-genome sequencing (WGS) or T/N whole-exome sequencing (WES)have emerged as ideal methods for the discovery of somatic mutations. NGS-based RNA sequencing (RNA-Seq) has revolutionized gene expression studies by enablingresearchers to measure relative expression changes across the whole genome in a single experimentand to identify novel transcripts. RNA-Seq has emerged as a leading method for identifying gene fusions, a critical class of somaticdriver mutations in tumor cells. Chromatin immunoprecipitation sequencing (ChIP-Seq), bisulfite sequencing, or methylation-targetingmicroarrays, can be used to investigate the role of epigenetic factors in the biology of tumorigenesis.The adoption of these genomic approaches in cancer research has led to a deeper understanding of tumor biologyand is establishing the foundation necessary to support the long-term goals of personalized medicine. Researchersare also using NGS and microarray-based genotyping of germline DNA to identify inherited variations that influencecancer susceptibility. While NGS is an excellent tool for assessing the germline status of known cancer predispositiongenes and identifying novel loci, microarrays are ideal for large-scale population studies, where large sample numbersare required to identify weakly predisposing genes.6This primer describes the broad portfolio of genomic technologies offered by Illumina that are directly applicable tocancer research.For Research Use Only. Not for use in diagnostic procedures.–3–

II. Approaches for Detecting Somatic MutationsDepending on the experimental goals, desired throughput, and available budget, researchers can choose acomprehensive, whole-genome approach or a focused, targeted approach to somatic variant detection (Table 1). WGSoffers a hypothesis-free method for discovering somatic changes across the genome, while targeted sequencing allowsresearchers to focus their investigation on specific regions of interest based on a priori criteria. For a given budget,targeted sequencing enables higher coverage across specific regions of interest, enabling the detection of low frequencysubclones within heterogeneous tumors or detection of rare somatic variants.Targeted Sequencing Solutions for Somatic Mutation DetectionTargeted sequencing offers several key advantages compared to whole-genome approaches: Narrows the scope of a sequencing project significantlyReduces the overall data analysis burdenLowers the cost of sequencing per sampleReduces the turnaround timeThese advantages also enable researchers to perform deep sequencing, which is critical for identifying raremutations or subclonal detection in heterogenous tumor samples. The trade-off with targeted sequencing is thatit can miss key mutations, such as those found in intergenic or previously unknown regions. Therefore, choosingbetween the comprehensive view offered by WGS or the focused power of targeted sequencing depends on thespecific research goals and available resources.Exome SequencingMany oncogenic variants are found within exons (protein coding regions), which comprise approximately 1% ofthe genome.7 Exome sequencing offers a cost-effective, efficient approach for analyzing T/N cohorts. T/N exomesequencing has been the preferred method for large projects, such as The Cancer Genome Atlas (TCGA), becauseit offers an attractive combination of efficient processing time and price and because it focuses on easy-to-interpretgenomic regions.Focused Sequencing PanelsFocused cancer panels are predesigned targeted sequencing panels with content selected by leading oncologyexperts and offer high levels of sensitivity for low-frequency somatic variants. For sequence-specific content,Illumina offers a choice of 2 different chemistries: Amplicon-based targeted sequencing involves a preliminary amplification step using predesigned primermixtures that target specific regions of interest. The amplified targets are then purified and sequenced. Target-capture involves a preliminary DNA capture or enrichment step using predesigned capture probesconjugated to magnetic beads.Illumina focused sequencing panels are compatible with various tissue types including solid or liquid tumorsamples, and fresh-frozen or formalin-fixed, paraffin-embedded (FFPE) tissue.Custom Targeted SequencingFor researchers or consortia interested in designing their own targeted sequencing panel, probe design softwareand custom targeted sequencing kits are available for both amplicon-based and target-capture approaches. Bothcustom capture probes or custom amplicon primers can be designed and ordered through Illumina DesignStudio Software. For more on DesignStudio, visit www.illumina.com/designstudio.For Research Use Only. Not for use in diagnostic procedures.–4–

Whole-Genome Sequencing SolutionsThrough WGS, researchers can compare T/N sample pairs to identify somatic mutations in coding and noncodingregions across the entire genome. As a hypothesis-free approach, WGS is well suited for the discovery of noveldriver mutations.For researchers who prefer a T/N WGS service, the Illumina Genome Network (IGN) Cancer Analysis Serviceperforms medium- to large-cohort T/N sequencing studies for researchers looking for a cost-effective solution.To learn more about IGN Cancer Analysis Services, visit www.illumina.com/ign.Data Analysis Tools for Somatic Variant DetectionWhether performing whole-genome, exome, or targeted sequencing, Illumina offers seamless workflow solutionsthat include library preparation kits, sequencing platforms, and data analysis software packages (Figure 2).Figure 2: Illumina Seamless Workflow Solutions—Illumina sequencing solutions are fully integrated, DNA-to-data solutions from librarypreparation to final data analysis. Prepare T/N paired libraries with optimized kits for whole-genome, exome, transcriptome, or targetedsequencing. Perform sequencing using the MiniSeq , MiSeq , NextSeq , or HiSeq sequencing systems. Access Illumina data analysis tools foralignment, variant calling, T/N comparative analysis, or expression profiling.Data analysis, the final step in the workflow, is essential to experimental success and must be tailored to theresearch question at hand. For example, it is important that the variant calling method properly model the complexitiesof multiple cancer subclones versus normal sample contamination. Illumina primary data analysis and somaticvariant detection software solutions are easily accessible through on-instrument analysis software, such as MiSeqReporter Software and Local Run Manager, or through BaseSpace Sequence Hub, the Illumina cloud-basedgenomic computing environment. Data are streamed from the MiniSeq, MiSeq, NextSeq, or HiSeq Systems directlyand seamlessly into BaseSpace Sequence Hub, which offers a suite of apps tailored to various data analysis needs.These tools are packaged into a user interface designed to be accessible to any user, regardless of bioinformaticsexperience. Simple prompts guide users through the entire process, starting from selecting the files generated by thesequencer to data filtration and analysis.For somatic variant detection, the BaseSpace Tumor Normal App can be used to report single-nucleotidepolymorphisms (SNPs), indels, copy number variants (CNVs), and structural variations found only withinthe tumor sample. For amplicon-based sequencing panels, MiSeq Reporter with Somatic Variant Caller,or the TruSeq Amplicon BaseSpace App can be used. Depending on the panel used, Illumina also offersVariantStudio, an annotation and filtering software that allows customers to create customized reports ofvariant data. For a comprehensive view of Illumina informatics solutions for variant detection, visit www.illumina.com/informatics.html.For Research Use Only. Not for use in diagnostic procedures.–5–

Table 1: Illumina DNA and RNA Sequencing Solutions in CancerProductKey Features/AdvantagesGenomic ContentDNA/RNA InputSequencing DepthData Analysis Tools FFPE-compatible Minimal DNA input Detect variants as low as 5% allelicfrequency 1 day library prep44 kb250 amplicons15 genesa20 ng 500 minimum coverage MiSeq Reporter BaseSpace TruSightTumor 15 App VariantStudio Identify somatic mutationsin myeloid malignancies Detect variants as low as5% allele frequency 1.5 day library prep141 kb568 amplicons54 genesb50 ng500 for 95%of amplicons 35 kb212 amplicons48 genes150 ng(250 ngfor FFPE)1000 meancoverage40 ng DNA,40 ng RNA 250 minimum coverage1385 genes10 ng total RNA(20–100 ngFFPE RNA)3M readsper sample507 genes10 ng total RNA(20–100 ngFFPE RNA)3M readsper sampleCustom10 ngCustom MiSeq ReporterEnrichment Workflow BaseSpace EnrichmentCore Apps4–650 kb10 ng total RNA(10-50 ngfor FFPE)Custom MiSeq Reporter TruSeqAmplicon Workflow BaseSpace TruSeqAmplicon Core AppTargeted SequencingTruSight Tumor 15TruSight Myeloid PanelTruSeq AmpliconCancer Panel FFPE-compatible Targets mutational hotspots infrequently mutated cancer genes 1 day library prepTruSight Tumor 170 FFPE-compatible Low DNA and RNA input Comprehensive panel detectsSNVs, amplifications and fusionscontributing to tumorigenesisTruSight RNAPan-Cancer PanelTruSight RNAFusion Panel FFPE-compatible Focused on oncology-specificcoding regions Optimized for degraded samples orlimited starting materials FFPE-compatible Detects known and novelgene fusions Optimized for degraded samples orlimited starting materials533 kb, 166 genes12,000 probes (DNA)358 kb, 55 genes8000 probes (RNA) MiSeq Reporter TruSeqAmplicon Workflow BaseSpace TruSeqAmplicon Core App MiSeq Reporter TruSeqAmplicon Workflow BaseSpace TruSeqAmplicon Core App BaseSpace TruSightTumor 170 App BaseSpace RNA-SeqAlignment App Local Run ManagerRNA Fusion Module BaseSpace RNA-SeqAlignment AppCustom Targeted SequencingNextera RapidCapture Custom Kits Custom genomic content 0.5–25 Mb of target sequence 1.5 day library prepTruSeq CustomAmplicon Low Input FFPE-compatible 16–1536 amplicons 1 day library prepExome SequencingTruSeq Exome LibraryPrep Kit Includes coding exons 2.5 day library prep45 Mb214,405 exons100 ng100 Normal/130 Tumor BaseSpaceEnrichment AppsTruSeq Rapid ExomeLibrary Prep Kit Includes coding exons 2.5 day library prep45 Mb214,405 exons50 ng100 Normal/130 Tumor BaseSpaceEnrichment AppsNextera Rapid CaptureCustom ExpandedExome Kits Includes coding exons, untranslatedregions, miRNA 1.5 day library prep62 Mb201,121 exons50 ng100 Normal/130 Tumor BaseSpaceEnrichment Apps Greatly reduced library bias High coverage of challengingregions such as high GC/ATregions, promotors, and repetitiveregionsWhole-genome1–2 µgMinimum40 Normal/60 Tumor BaseSpace CoreWGS or T/N Apps Optimized for low-quantityDNA samples Greatly reduced library bias Improved coverage uniformityWhole-genome100–200 ngMinimum40 Normal/60 Tumor BaseSpace CoreWGS or T/N AppsWhole-Genome SequencingTruSeq DNA PCR-FreeLibrary Prep KitTruSeq Nano DNALibrary Prep Kita. Genes informed by guidelines published by organizations such as the National Comprehensive Cancer Network (NCCN)8 and European Society forMedical Oncology (ESMO)9, and late-stage pharmaceutical research.b. Designed by recognized experts in blood cancers, the panel targets loci with known association to acute myeloid leukemia (AML), myelodysplasticsyndrome (MDS), myeloproliferative neoplasms (MPN), chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia (CMML), and juvenilemyelomonocytic leukemia (JMML).For Research Use Only. Not for use in diagnostic procedures.–6–

III. Evaluating Germline Mutations in CancerGenomic technologies can be used to detect germline mutations associated with cancer (Table 2). Array-basedstudies can be used for the identification of weakly predisposing variants, because they offer a cost-effectivemethod for genotyping large numbers of samples with an experimental design similar to a genome-wideassociation study (GWAS). Targeted sequencing studies, however, allow researchers to focus on previouslyidentified genes known or suspected to play a role in cancer susceptibility.Targeted Sequencing to Detect Common Germline MutationsTargeted sequencing panels enable researchers to rapidly sequence known or suspected cancer-related genes forcommon germline mutations. As with all sequencing panels, the targeted sequencing approach provides a rapidand cost-effective alternative to single-gene testing, and allows sequencing to higher coverage levels comparedto WGS.Microarray-Based ApproachesMicroarrays are a powerful, high-throughput method for researching cancer risk. Current microarray formats supportup to 24 samples per BeadChip for genotyping studies with large sample sizes. Illumina high-density BeadChips aredesigned to interrogate hundreds of thousands of SNPs associated with breast, colorectal, lung, ovarian, and prostatecancers in addition to traits associated with ancestry and pharmacogenetics. Custom content can be added ontoexisting BeadChips, giving researchers the option to investigate their specific variants of interest.Table 2: Illumina Sequencing and Microarray-Based Solutions for Evaluating Cancer PredispositionProductKey Features/AdvantagesGenomic ContentDNA InputSequencing DepthData Analysis Tools255 kb 4000 probes94 genesa, b50 ng20 MiSeq ReporterEnrichment Workflow orBaseSpace Enrichment Apps 500,000 SNPs frombreast, colorectal,lung, ovarian, andprostate cancerc200 ngNAGenomeStudio GenotypingModule3072–1,000,000attempted bead types200 ngNAGenomeStudio GenotypingModuleTargeted SequencingTruSight CancerSequencing Panel Provides comprehensivecoverage of genes associatedwith cancer predisposition 1.5 day library prepIllumina MicroarraysInfinium OncoArray-500KBeadChip 120,000 custom markerspossible 24 samples per microarrayInfinium iSelect HDCustom GenotypingBeadChips Interrogate virtually any SNPfrom any species Interrogate SNPs, CNVs,or indels 24 samples per microarraya. The content was designed by Professor Nazneen Rahman, a recognized expert in the field of genetic susceptibility, and targets 94 genes associated withinherited cancers, plus 284 SNPs found through GWAS studies.b. Researchers can also add on content to TruSight Cancer to create a custom panel using the Illumina DesignStudio tool.c. Developed in collaboration with leading experts from the OncoArray Consortium,10 an international collaboration of leading experts from the NCIsupported Genetic Association and Mechanisms in Oncology (GAME-ON) consortium, Genome Canada, Genome Quebec, and Cancer Research UK.IV. Structural Variant Detection in CancerMany cancer types carry large structural aberrations, such as CNVs, translocations, and inversions, that provideinsight into cancer etiology. It is essential to use techniques that identify these aberrations accurately and efficiently.Illumina offers both array and sequencing-based approaches to identify gross chromosomal changes (Table 3).For Research Use Only. Not for use in diagnostic procedures.–7–

DNA and RNA Sequencing for Translocation DetectionTranslocations are important driver mutations in many cancer types, and can be detected using whole-genome T/Napproaches (Table 1), along with the BaseSpace Tumor Normal App. However, detecting structural variants withtargeted DNA sequencing approaches or microarrays can be difficult. Many researchers have turned to RNA-basedsequencing approaches, which allow detection of expressed fusion genes as an alternative approach.Copy Number Variation ArraysAccurate profiling of chromosomal aberrations, such as amplifications, deletions, rearrangements, and copy-neutral lossof heterozygosity (LOH) events, is crucial for the investigation of cancer genetics. Whole-genome SNP microarrays, withenriched coverage for genes of known cytogenetic relevance, are an ideal tool for the detection of CNVs, copy-neutralLOH, low-level mosaicism, and sample heterogeneity.Table 3: Illumina Sequencing and Array-Based Solutions for Structural Variant DetectionProductsKey Features/AdvantagesGenomic ContentDNA/RNA InputData Analysis Tools1385 target genes10 ng total RNA20–100 ng FFPE RNA BaseSpace RNA-SeqAlignment App10 ng total RNA20–100 ng FFPE RNA Local Run ManagerRNA Fusion Module BaseSpace RNA-SeqAlignment App40ng DNA,40 ng RNA BaseSpace TruSightTumor 170 AppRNA SequencingTruSight RNAPan-Cancer PanelTruSight RNAFusion PanelTruSight Tumor 170 FFPE-compatible Focused on oncology-specificcoding regions Optimized for degraded samples orlimited starting material Ideal for gene-fusion detection FFPE-compatible Focused on oncology-specificcoding regions Optimized for degraded samples orlimited starting material Detects known and novel gene fusionpartners FFPE-compatible Low DNA and RNA input Comprehensive panel detects SNVs,amplifications and fusions that contributeto tumorigenesisTruSeq RNA Access LibraryPreparation Kit FFPE-compatible Detects known and novel gene fusions Optimized for degraded samples orlimited starting materialTruSeq Stranded Total RNALibrary Preparation Kit FFPE-compatible Precise detection of strand orientation Whole-transcriptome analysis507 target genesDNA: 533 kb,12,000 probes, 166 genesRNA: 358 kb,8000 probes, 55 genes21,415 target genesaCoding and multiple forms ofnoncoding RNA (eg, miRNA,snRNA, lincRNA, snoRNA,and more)b10 ng total RNA20–100 ng FFPE RNA0.1–1 µg total RNA BaseSpaceSequence HubApps for RNA BaseSpace CoreApps for RNAIllumina MicroarraysInfinium CytoSNP-850KBeadChip FFPE-compatible More than 850,000 markers Higher det

For Research Use Only. Not for use in diagnostic procedures. Table of Contents I. Introduction: Genomic Technologies for Cancer Research 3 II. Approaches for Detecting Somatic Mutations 4 Targeted Sequencing Solutions for Somatic Mutation Detection 4 Exome Sequencing 4 Focused Sequencing Panels 4 Custom Targeted Sequencing 4 Whole-Genome Sequencing Solutions 4

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