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The world leader in serving science Finding high quality associations in RNA expression profiling from precious samples Geoff Scopes, PhD. Applications Specialist, Microarray
The world leader in serving science
Finding high quality associations in RNA expression profiling from precious samples
Geoff Scopes, PhD. Applications Specialist, Microarray
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Which RNA Are You Interested In?
~70–90% of genome transcribed
Total RNA ~100K transcripts
mRNA (~3%)
ncRNA (~7%)
rRNA (~90%)
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Practical considerations for RNA Expression studies
Data analysisSample collection & preparation RNA analysis
FFPE, Liquid biopsy
Isolation Kit? Stabilisation?
Gene expression?Transcriptome?Splice variant?ncRNA?
Answering the question? Value vs RNA Biology?
Pre-analytical Analytical Post-analytical
For Research Use only. Not for use in diagnostic procedures.
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Pre-analytical Phase: Routine Samples Are Difficult to Analyze
Blood
• Which blood collection tube to use?
• Consistent collection?
• Variable transportation conditions and time?
• Globin reduction?
• Sample collection and FFPE preparation?
• Extent of chemical modifications?
• Extent of degradation?
• Low yields?
FFPEblocks
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Gene Expression Solutions
Targets25K 101001K20K
qPCR
NGS
Microarray
NGS
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Ion Total RNA-Seq Kit
Clariom™ D Assay
Ion AmpliSeq™
Transcriptomeand
Clariom S™ Assay
Ion AmpliSeq™
panelsOpenArray™
platesTaqMan®
array cardsTaqMan®
array platesSingle-tube
assays
Discovery Verification Routine testing
The world leader in serving science
Microarray Portfolio
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Genome-Wide Analysis: DNA and RNA Applications
miRNA Arrays
Expression arrays
DNA copy number arrays
Allele Specific Copy Number
Non-coding RNA Expression
Applications
Gene Expression
Loss of Heterozygosity (LOH)
Alternative Splicing
Gene regulation
OncoScan™ FFPE Express
CytoScan®
HD Array
Clariom Assays: Human, Mouse, Rat
GeneChip ® miRNA 4.0 Array
Solutions
Genotyping arraysIndel genotyping
SNP genotyping
Clinical Sample
Axiom® Genotyping Solution
Axiom PharmacoScan Array
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Microarray Workflows
GCS 3000 Sample Prep
Overnight Hybridization Wash & Stain Scan
Day 1 Day 2 Day 348 samples/day (max)1.5 hrs hands on time
GeneTitan Sample Prep(Biomek FXp)
Array processing (Hyb, Wash, Stain, Scan)
Day 1 Day 2 Day 3192 samples/day (max)0.5 hrs hands on time
TAC analysis
TAC analysis
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Probe Tiling and Design Strategies for Expression Products
Genomic locus
mRNA transcripts{3’ IVT Array
11 probes
Gene ArrayMedian ~25 probes
HTA & ClariomD ArraysMedian >100 probes
ClariomS Arrays10 probes
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• Genomic locus
Whole-Transcript Arrays Detect a Variety of Transcript Isoforms
• Presumed Standard Transcript AAAA
• Non-polyadenylated messages
• Degraded Samples
• Truncated transcripts
• Alternate polyadenylation Sites AAAA
• Genomic deletions AAAA
• Alternative 5’ start site AAAA
• Alternative splicing AAAA
• Transcripts with undefined 3’ end
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GeneChip Probe Sets
• The power of the probe set• Each transcript represented by multiple independent 25mer probes• For Clariom D arrays, there are typically 6 probes per exon, and the
median number of probes per gene/transcript greater than 100• Combined together, multiple probes generate robust and reliable
expression signals with high sensitivity and specificity• The probe set is unique to Affymetrix Microarrays
• High densities (up to 7M probes), made possible with photolithography, adapted from the semi-conductor industry
• Other microarrays rely on a single probe per transcript• 25mer oligonucleotides are highly sequence specific
• Differentiate between sequences with >90% identityMultiple 25mers combine the benefits of nucleotide level specificity and sensitivity (at least 500bp of transcript typically covered)
The world leader in serving science
Clariom: The Next Generation of Transcriptome profiling solutions for Human, Mouse and Rat
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Why Should We Care About Alternative Splicing and ncRNA?
lncRNA
• Attractive as biomarkers• Specific disease gene expression• Very stable in body fluids
• 615% growth in publications0
200
400
600
800
1,000
1,200
2007 2008 2009 2010 2011 2012 2013 2014 2015
lncRNA and disease publications
500
700
900
1,100
1,300
2007 2009 2011 2013 2015
Splicing and disease publicationsAlternative splicing• ~95% human genes undergo
alternative splicing• Attractive as biomarkers
• Mutations affecting splicing cause distinct disease gene expression
For Research Use only. Not for use in diagnostic procedures.
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• Breast cancer
• Oral and oropharyngeal cancers
• Ovarian cancer
• Papillary thyroid cancer
• Gastric cancer
• Head and neck squamous cell carcinoma
• Lung cancer
• Lymphoma
Human Diseases Associated With Alternative Splicing and ncRNA
• Melanoma
• Giant cell tumors of bones
• Renal and urothelial cancers
• Wilms tumor
• Fragile X syndrome
• Facioscapulohumeral muscular dystrophy (FSHD)
• Myotonic dystrophy
• Prader-Willi syndromeFor Research Use only. Not for use in diagnostic procedures.
>300 human diseases
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Biomarker Detection and Utility – Desirable Attributes
Robustness
Ease of use
Accuracy Rapid results
Fits in budget
Broad range of samples
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Seeing the Bigger Picture
RNA-Seq
Microarray
New discovery potential• Novel RNA transcripts• Not a priori
Flexibility for different studies• Read depth• Targeted analysis
High data density
Robust, validated technology High-throughput• Large research studies
Clinically amenable• CE-marked, IVD cleared technology
Standardized data analysis• Simple, quick data analysis
Transcriptomics• Robust rare transcript detection• Discovery of biomarkers, pathways
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Broad Range of Samples
Blood, Fresh frozen tissue, cell line
WT Plus
50-500 ng
No rRNA or globin depletion
Fresh and Archival FFPE, Small Sample
WT Pico
100 pg – 10 ng
No rRNA or globin depletion
30 ng
45 min assay
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Ease of Use, Rapid: Simple & Fast
WT Plus/Pico Kit Clariom microarrays (human, mouse, rat or custom)
Transciptome Analysis Console (TAC)Software
Isolate Prepare Measure Interpret
Isolate well characterized subpopulations of cells• Nasal / Bronchial/ Blood/ Tonsil • Flow cytometry• Antibody pull down
Prepare sample from as little as 100 pg total RNA (~10 cells)
Automated protocols for Beckman BioMek robot
Transcriptome analysis Analyze and visualize gene expression patterns, pathways, and ncRNA and target gene interactions
Sample to Result ~3-4 days
For Research Use only. Not for use in diagnostic procedures.
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Accurate and Reproducible?
Affymetrix Microarrays provide precise gene expressionquantitation regardless of transcript abundance
PNAS (2011): 108(9): 3707-3712 Xu et al.
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Sequence Depth Required at Exon-level at Least 10-fold Higher than Gene-level
GENE coverage is not the same as EXON coverage!
Exon 4 Exon 5 Exon 6 Exon 7 Exon 8
Exon 1 Exon 2 Exon 3 Exon 4 Exon 5 Exon 6
Exon 1 Exon 2 Exon 3
Exon 7 Exon 8 Exon 9 Exon 10
Exon 9 Exon 10
Average exons per gene 10.Average exon Size 150bp
10X gene coverage
10X exon coverage
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Clariom D: Deep and Broad Coverage of the Transcriptome
Example: COCLA2Colorectal Cancer Associated 2 Gene
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Precision at the Gene Level
RNA-Seq Mapping to Refseq using novoalignRPM is calculated as (# of reads mapped to the target)/(# total reads mapped/10^6)CV is calculated as SD/Mean using non-logged RPM.
15M 30M 60M
120M 240M 480MMapped Reads
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Precision at the Exon Level – Where we Need to be Accurate to See Splicing Events!
15M 30M 60M
120M 240M 480MMapped Reads
RNA-Seq Mapping to Refseq using novoalignRPM is calculated as (# of reads mapped to the target)/(# total reads mapped/10^6)CV is calculated as SD/Mean using non-logged RPM.
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Comparison to deep RNA-Seq
• Accuracy for RNA-Seq is read-depth dependent
• Clariom D Transcriptome Assays deliver accurate results equivalent to ~720 M reads• By evaluating a linear tissue mixture model in
which RNA from two samples are mixed in known proportions, the accuracy of expression was evaluated across all measured exons.
Clariom™ D Assay
For Research Use only. Not for use in diagnostic procedures.
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HTA Arrays offer more stable detection of alt. splicing events vs RNAseq1. Higher variability was observed in RNA-
seq compared to HTA.
2. RNA-seq tended to detect more abundant (long) genes, active at the extracellular matrix, while HTA showed more genes active within the nucleus.
3. Reproducibility between platforms for differential exon /junction analysis was low. RNA-seq tended to identify more differentially used exons in the 3′ end of a gene (3’ exons are longer), while HTA found significantly spliced exons in the middle.
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• Both platforms performed equally well for protein-coding RNAs, however stochastic variability was higher for sequencing data than for microarrays. This reduced the number of differentially expressed genes and genes with predictive potential for RNA-Seq compared to microarray data.
• Analysis of this variability revealed a lack of reads for short and low abundant genes; lncRNAs, being shorter and less abundant RNAs, were found especially susceptible to this issue.
• A major difference between the two platforms was uncovered by analysis of alternatively spliced genes. Investigation of differential exon abundance showed insufficient reads for many exons and exon junctions in RNA-Seq while the detection on the array platform was more stable. Nevertheless, 207 genes that undergo alternative splicing were identified and were consistently detected by both techniques.
• Conclusions: Despite the fact that the results of gene expression analysis were highly consistent between Human Transcriptome Arrays and RNA-Seq platforms, the analysis of alternative splicing produced discordant results.
• They concluded that modern microarrays can still outperform sequencing for standard analysis of gene expression in terms of reproducibility and cost.
Results and Conclusions
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Success with Clariom D application in Oncology
2017: Clariom D Transcriptomic Analysis Reveals Immune-Related Pathways Overexpressed in Regressing Tumors
“RNA was extracted from FFPE samples using the RecoverAll Total NA kit.RNA expression was assessed using the human Clariom D Pico assay. Arrays were analyzed using the SST-RMA algorithm in the Affymetrix Expression Console Software. Expression was determined by using the Transcriptome Analysis Console,”
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GEO – Gene Expression Omnibus growing content in public domain
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• Deep Sequencing (High sensitivity)• 300-700 million reads/sample• Discovery (RNA isoforms & ncRNA.)
Clariom assays are a cost effective alternative to RNA-seq
Clariom S Assay
Clariom S array, human
Clariom D Assay
Targeted Sequencing• ~10 million reads/sample (<1,000 targets)
Shallow Sequencing (Low sensitivity)• 30-100 million reads/sample. • Discovery (Gene & Exome)
For Research Use only. Not for use in diagnostic procedures.
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Deepest Coverage from 100 pg of Starting Material
Content summary HumanGenes >134,700Transcripts >542,500Exons >948,300Exon-exon splice junctions >484,900Total probes >6,765,500Probes targeting exons >4,781,200Probes targeting exon-exon splice junctions
>1,984,300
Probe length (bases) 25Probe feature size 5 μmBackground probes Antigenomic set
Sources
EnsemblVega
NONCODE
lncRNAWiki
UCSC Genes
AceView
miTrans-criptome
RefSeqMGC
Consensus CDS
RNA Central
circBase
Human Body Map
lincRNAdb
Publica-tions
Clariom™ D Pico assays, humanContent from 15 data sources
Expand the potential to find new, actionable biomarkers, quickly
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Gene-level Expression of Well-annotated Genes to Act Quickly on Your Discoveries
Sources
EnsemblVEGA
NONCODE
lncRNAWiki
UCSC Genes
AceView
miTrans-criptome
RefSeqMGC
Consensus CDS
RNA Central
circBase
Human Body Map
lincRNAdb
Publica-tions
Clariom™ S assays, humanContent from 15 data sources
Obtain a gene-level view of changes in the transcriptomequickly and easily
Generate expression profiles from >20,000 well-annotated genes in three days
Choose a format that suits your throughput needs, processing up to 192 samples per day
Find answers. Move on.
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Explore mRNA/miRNA Interactions with miRNA 4, Clariom D and TAC
Visualize other miRNA that target a gene: Selecting a gene related to your miRNA and TAC will provide the network of additional miRNA that interact with that gene.
Visualize the network of relationships between miRNA and genes: select another miRNA that interacts with your gene will expand you network of interactions to include common genes as wells as the genes unique to the miRNA.
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Affymetrix® miRNA 4.0 Array
• Comprehensive content – Provides complete coverage of miRBase v20, including probes to interrogate snoRNA and scaRNA
• Highest confidence in measurements – Nine replicates for every transcript on the array
• Unique content – Independently measure pre- and mature miRNA in same experiment
• High sensitivity – Detects 94 percent of miRNA transcripts at 1.0 amol
• High reproducibility – 0.95 reproducibility (inter- and intra-lot)• Easiest workflow – 45-minute target labeling assay with no
pre-amplification or purification steps• Low sample input – As little as 130ng total RNA
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Whole Transcript Expression Arrays
Arabidopsis
Rice
Soybean
Medicago
C. elegans
Drosophila
Canine
Feline
Cynomologus
Rhesus
Zebra Finch
Bovine
Ovine
Porcine
Equine
Chicken
Guinea Pig
Rabbit
Marmoset
Zebrafish
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Data to Insights in Minutes: Transcriptome Analysis console
• Identify what genes are differentially expressed (LIMMA)
• Visualize global expression patterns
• Filter results to pathways
• Find other impacted pathways
• Investigate relationships between miRNA as well as coding & non-coding RNA
• Identify changes in alternative-splicing patterns
• Design validation experiments
Take control of your analysis
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• Signal intensity low in liver and high in muscle
• Exon (PSR)
• Junction probes
• Splicing index for liver vs. muscle is low
• Simple visualization shows down regulation of this inclusion event in liver
Examine Splice Variants
Similar splice junction resolution with RNA-seq requires >100M reads
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Go Deep Into the Transcriptome with Clariom D Assays
Get all the data you need. Now. • Obtain a deep and broad view of the transcriptome—gene- and exon-level analysis of
coding and ncRNA isoforms in just 3 days• Get coverage of >540,000 transcripts from 15 data sources• Don’t miss rare and low expressing transcripts
Precious samples? Get it right the first time. • Extract robust sample data from as little as 100 pg total RNA• Utilize a wide range of challenging samples types including whole blood and FFPE
tissue• No globin or rRNA removal step required
Data to insight in minutes. • Take control of your analysis with free software designed for the biologist• Quickly identify statistically significant expression changes• Visualize and analyze alternative splicing patterns
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Questions?
