HaloPlex
For research use only 1
Revolutionize Your
Clinical Research
Olle Ericsson, PhD, Marketing Director DNA sequencing
HaloPlex
For research use only
Complete Clinical Research Solution
• SureDesign – High confidence custom designs
• Haloplex – Fast, easy and high performance workflow
• SureCall – From raw-data to mutation report
HaloPlex
For research use only
Complete Clinical Research Solution
• SureDesign – High confidence custom designs
• Haloplex – Fast, easy and high performance workflow
• SureCall – From raw-data to mutation report
HaloPlex
For research use only
SureDesign
Create your design in only 10 minutes
https://earray.chem.agilent.com/suredesign
1. Input gene
ID/name/coordinate
2. Define regions of interest
(eg. Exons, UTRs, etc)
3. Click “Start Design”
4. Receive design report in 10
minutes
HaloPlex
For research use only
SureDesign v1.1
HaloPlex advanced design wizard
1. Custom designs based on HaloPlex Exome probes
2. FFPE optimized designs
3. Improved coverage of pseudogenes
4. Improved design visualization
1. Direct link to UCSC browser to review target regions and individual amplicons
2. Preview sequencing data from exome probes in SureCall
5
www.agilent.com/genomics/suredesign
HaloPlex
For research use only
Exon 1 Exon 2
All HaloPlex exome probes overlapping with defined targets will be selected Exome probes do not exist for non-coding regions no probes will be selected for these regions
SureDesign v1.1
Use amplicons from HaloPlex Exome
1. Define target regions
2. Use all exome probes for coding regions
3. Combine with custom design for non coding regions
HaloPlex
For research use only
Review Your Designs in Progress
Direct links to UCSC Genome Browser
• Link out to tiling results from Design Summary
• Review amplicon structure and coverage in high-resolution
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HaloPlex
For research use only
Exome probes - Beta-program Visualize sequencing data and QC performance report in SureCall
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HaloPlex
For research use only
SureDesign v1.1 - Optimize for FFPE
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For FFPE optimized designs 1. The design will include shorter fragments (50-400bp) 2. Both target polarities will be selected
HaloPlex
For research use only
Target Region Target Region
Normally fragments in the range of 100-400bp are used
For FFPE samples shorter fragments (50-400bp) are included
Standard design
Designed for FFPE compatibility
Exon Exon
FFPE design
HaloPlex
For research use only
One probe is targeting the sense and one the anti-sense strand of the target DNA
Sense Strand
AntiSense Strand
DNA TARGET FRAGMENT
Probes are designed for both target fragment polarities • Improves capture efficiency • Eliminate strand specific FFPE artifacts
Designed for FFPE compatibility
HaloPlex
For research use only
FFPE design optimization results
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Standard FFPE optimized
Coriell DNA
FFPE DNA
Fraction of target region covered at 10% of mean
HaloPlex
For research use only
Pseudogene designs
- Probe Stringency alteration
13
1. Maximize Specificity - Requires probe with only one unique hit in the genome 2. Balanced – Allows selection of probes with 2 hits in the genome. 3. Maximize Coverage – Allows up to 5 hits in the genome.
HaloPlex
For research use only
Pseudogene Designs
- Probe Stringency Alteration
14
Balanced probe selection – 100% coverage of MYH6
Standard probe selection – 96.6% coverage of MYH6
HaloPlex
For research use only
Complete Clinical Research Solution
• SureDesign – High confidence custom designs
• Haloplex – Fast, easy and high performance workflow
• SureCall – From raw-data to mutation report
HaloPlex
For research use only
FOLLOW-UP CLINICAL RESEACH
From Discovery to Clinical Research
All Solutions currently
available are based on
Multiplex PCR and complex data analysis
Challenges: Fast turnaround Time
Flexibility in panel size
Simple workflow
High Coverage and Specificity
High Throughput
Data Analysis Solution
HaloPlex
For research use only
The advantages with Traditional PCR
High Coverage
High Specificity
Fast
Cost Effective
Simple
Well established
HaloPlex
For research use only
The problems with Multiplex PCR
Primer Cross reactivity
Limited in # target regions
Long optimization
Hard to change, no flexibility
Dropouts
Artifacts
HaloPlex
For research use only
HaloPlex Target Enrichment
Macro scale PCR
Physical separation
Hundreds of parallel reactions
Micro scale PCR
Physical separation
Thousands of parallel reactions
Nano scale PCR
Molecular separation
Millions of parallel amplifications
HaloPlex
For research use only Page 20
HaloPlex Target Enrichment System
HaloPlex
For research use only
HaloPlex Workflow
A Simple & Fast 4-step protocol
Sample is fragmented using restriction
enzymes
2. Hybridize
probes
3. Purify and
ligate targets
4. Amplify targeted
fragments
Probe library is added and hybridized
to the targeted fragments making
them form a Halo shape.
Probe/Fragment hybrids are retrieved
with magnetic streptavidin beads. The
circular molecules are then closed by
ligation
Only circular DNA targets are
amplified. Sample barcodes are
introduced. Final product is ready for
sequencing
1. Digest DNA 1
2
3
4
HaloPlex
For research use only
HaloPlex Other PCR based solutions
TARGET TARGET
With HaloPlex each target base is covered by multiple amplicons (different start and stop sites)!
With other multiplex PCR based technologies, each target base is
covered by only one amplicon (same start and stop sites)
If a variant occurs – it can be checked by multiple amplicons
with HaloPlex
If a variant occurs, it is hard to know if it is a real mutations and
not a PCR artifact
DNA variant DNA variant
If an unknown mutation appears in a restriction site, it may affect
one or two fragments but all others will be present
If an unknown mutation appears in a primer site it causes a
complete dropout in the target region
Amplicon redundancy
HaloPlex
For research use only
High Coverage and Uniformity Across Target Sizes
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Covered by design
% Bases on Target
% Bases covered 1x
% Bases covered 20x
% Bases covered
100x
% Bases covered at
10% of
mean
% Bases covered at
20% of
mean
4.4Kb
21Kb
60Kb
120Kb
280Kb
396Kb
Exome average – 90% of 37 Mbp covered 10X usign 4 Gbp of sequencing
HaloPlex
For research use only
Complete Clinical Research Solution
• SureDesign – High confidence custom designs
• Haloplex – Fast, easy and high performance workflow
• SureCall – From raw-data to mutation report
HaloPlex
For research use only
Agilent SureCall Software Resolves data analysis bottleneck
An easy-to-use software for end-to-end data analysis from
alignment to categorization of mutations
• Easy to Implement
• Easy to Use with a
Streamlined Workflow
• Faster Time-to-Results
• Cost-Effective
SureCall makes NGS data analysis easier, faster, cheaper
Page 25
HaloPlex
For research use only
Steps included in SureCall
Report generation
Categorization Table output Visualization Links to external
databases
Post alignment processing
Identify mutations Mutation impact analysis QC reports
Read alignment
Adapter removal BAM file generation
Sequence generation
De-multiplexing Base caller, quality assessment
Sample
DNA isolation Target enrichment
Not approved for use in diagnostic
Adapted from: Frederick National Laboratory for Cancer Research
Page 26
HaloPlex
For research use only
Simple 3-step workflow-based data analysis
Not approved for use in diagnostic
1. Import Samples
2. Describe Samples
3. Run Analysis
Page 27
HaloPlex
For research use only
Sample review - Triage View
Not approved for use in diagnostic
genome
viewer
mutation
table
Page 28
HaloPlex
For research use only
Reports
Not approved for use in diagnostic
1-page mutation report
QC report
Make your own report
Page 29
HaloPlex
For research use only
Complete Clinical Research Solution
• SureDesign
• Optimize for FFPE samples and pseudogenes
• Leverage 2.5 million all exon probes
• Haloplex
• Single day, easy workflow
• High target coverage and amplicon redundancy
• SureCall
• From raw-data to categorized mutation report
HaloPlex
For research use only
HaloPlex Cancer Research Panel
Catalog kit
10Kb design 47 genes targeting COSMIC mutations
o Designed with relevant cancer targets
Designed for FFPE compatibility o Includes shorter fragments
o Design with both polarities
Analyze data in SureCall
Gene List
ABL1 JAK2
AKT1 JAK3
ALK KIT
AR KRAS
ATM MAP2K1
BRAF MAP2K4
CDKN2A MET
CSF1R NOTCH1
CTNNB1 NPM1
EGFR NRAS
ERBB2 PDGFRA
ERBB4 PIK3CA
FANCA PIK3R1
FANCC PTEN
FANCF RET
FANCG RUNX1
FGFR1 SMAD4
FGFR2 SMO
FGFR3 SRC
FLT3 STK11
HRAS TP53
IDH1 VHL
IDH2 WT1
MAP2K2 http://www.sanger.ac.uk/genetics/CGP/cosmic
HaloPlex
For research use only
HaloPlex Cancer Research Panel
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HaloPlex
For research use only
HaloPlex Cancer Research Panel Coverage
33
HaloPlex
For research use only
Yong Yi
Associate Marketing Director
Agilent Next Generation Sequencing
Solutions for Gene Regulation
HaloPlex
For research use only
Complete Solutions for Gene Regulation Studies Looking at the complete picture
Gene Regulation
RNA • Novel Gene Discovery
• Gene Expression
DNA • Human Exome
Methylation • DNA Methylation
Investigating only one may prevent identification of a novel aberration which could be the real driving force behind a disease/phenotype of interest.
HaloPlex
For research use only
SureSelect - Most Complete Workflow Solutions From Sample to Analysis
MX3000 SureCycler 8800
Agilent NGS
Automation
2100 Bioanalyzer 2200TapeStation
GeneSpring
NGS
HaloPlex
For research use only
SureSelect RNA-Seq Whole Transcriptome and Targeted RNA-Seq
NGS Gene Regulation Solutions
HaloPlex
For research use only
SureSelect Strand Specific RNA Library Prep
Better Performance and Highly Strand-Specificity
High Strand Specificity and Fast Workflow
Strand specific for deeper understanding of gene regulation
Exceptional RNA-Seq Performance with Low RNA Input
Greater uniformity across the entire transcriptome and higher
complexity
Complete Gene Regulation Solutions from Agilent
Targeted RNA-Seq, Methyl-Seq, GE arrays, qPCR, GeneSpring NGS
Automation for high throughput sites
HaloPlex
For research use only
SureSelect Strand Specific RNA Library Prep
Strand-Specificity Increases Understanding of Gene Regulation
Identify Overlapping Transcripts
Discern transcripts nested in other genes
Enable Easier Analysis of Data
Increase alignable reads and more accurate annotation
Antisense Expression Can Play a Key Role
Discover antisense transcripts and measure antisense expression
HaloPlex
For research use only
SureSelect Strand Specific RNA Library Prep
High Strand Specificity and Outstanding RNA-Seq Performance
– Strand-specificity provides greater understanding of gene regulation
– High uniformity and library complexity
*Top 1000 Expressed Transcripts. 50ng of Human UHRR samples Data normalized to 20 million reads. 2x100bp sequencing
95
96
97
98
99
100
Agilent - Replicate 1
Agilent - Replicate 2
Agilent - Replicate 3
300
320
340
360
380
400
420
440
Agilent - Replicate 1
Agilent - Replicate 2
Agilent - Replicate 3
Mean per Base Coverage Strand Specificity
HaloPlex
For research use only
*200ng of Human UHRR samples Data normalized to 20 million reads. 2x100bp sequencing
SureSelect Strand Specific RNA Library Prep
Outstanding RNA-Seq Performance and High Strand Specificity
– Improved performance (Top 1000 Expressed Transcripts)
300
350
400
450
500
550
Agilent Strand-Specific TruSeq Stranded
Mean per Base Coverage
30% greater coverage compared to other strand specific kits
HaloPlex
For research use only
SureSelect Strand Specific RNA Library Prep
Outstanding RNA-Seq Performance and High Strand Specificity
– Less 5’-3’ transcript bias
0
0.2
0.4
0.6
0.8
1
1.2
1.4
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8
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92
96
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0
No
rmal
ize
d c
ove
rage
Normalized Distance Along Transcript (5’-3’)
Agilent
TruSeq Stranded
HaloPlex
For research use only
SureSelect Strand Specific RNA Library Prep
Excellent Correlation to Gene Expression Microarrays
– Ideal for follow up and companion studies
R=0.903 R=0.894
RN
ASe
q:
log2
fo
ld c
han
ge B
rain
/UH
RR
Microarray: log2 fold change MAQC_B/MAQC_A
200 ng 50 ng
Microarray: Human catalog 60K array.
Microarray: log2 fold change MAQC_B/MAQC_A
RN
ASe
q:
log2
fo
ld c
han
ge B
rain
/UH
RR
HaloPlex
For research use only
SureSelect Targeted RNA-Seq
High Performance for Whole Transcriptome and Targeted RNA-Seq
– RNA Kinome kit - targets transcripts of over 600 kinase and kinase related genes
60
70
80
90
100
Breast-T Human RNA kinome
Breast-N Human RNA kinome
Per
cen
t
% on Target
Strand Specificity
*Total RNA input: 200 ng
HaloPlex
For research use only
Demonstrate ability to capture gene fusions using targeted RNA-Seq
• Target set of 913 human kinase genes (~3.77Mb)
– target ABL1 transcripts
– exclude BCR baits
– look for BCR-ABL1 fusions
– compare whole transcriptome vs. targeted RNA Capture
BCR-ABL1 cDNAs
Fragmentation
ABL1-baits
BCR-ABL1 fusion genes
present in the
transcriptome
SureSelect baits
developed against BCR
transcripts - No baits designed to
BCR
SureSelect ABL1 baits
pull down both ABl1
fragments and BCR-
ABL1 fusions
Efficiency of SureSelect baits capture both BCR and BCR-ABL1 fusions
HaloPlex
For research use only
SureSelect Targeted RNA-Seq – Discovery More
KCL-22-SR (cell line) Targeted Non-targeted
Fusions 72 Not reported
ABL specific fusions 45 0
Alternatively Spliced Variants 79 3
Non-Synonymous SNPs 156 19
Whole Transcriptome vs. Targeted RNA Capture
Comparable amount of filter passed reads ~40 Million reads
• 88% of captured cDNAs mapped to the target regions using SureSelect
• ~7% of cDNAs from whole transcriptome sample mapped to target regions
Increased depth enables researchers to Discover More!
> 45X enrichment
HaloPlex
For research use only
SureSelect Human Methyl-Seq
NGS Gene Regulation Solutions
HaloPlex
For research use only
Target Known Methyl Regions
9-18x more efficient with greater depth per region
Less than 3% of the genome is reported to vary
it’s methylation state
• Focus your research with SureSelect Methyl-Seq
Whole Genome
Bisulfite Sequencing SureSelect Human
Methyl-Seq
3-6 lanes for one sample
90-180Gb per sample
3 samples per lane
10Gb per sample
HaloPlex
For research use only
Differentially Methylated Regions (DMR)
• CpG islands (e.g. 4~8 % tissue-specific differentially methylated regions or T-DMR)
• CpG island shores (~2kb away from islands, e.g. 76% of T-DMRs in shores)
• CpG island shelves (~4kb away from islands)
Irizarry RA et al. Nature Genetics 2009
HS3ST4 : heparan sulfate D-glucosaminyl 3-O- sulfotransferase 4
HaloPlex
For research use only
SureSelectXT Human Methyl-Seq The First Comprehensive Methylation Discovery System
DESIGN CONTENT - 84 Mb Design,
3.7M CpGs
CpG islands
Cancer, Tissue-specific DMRs
GENCODe promoters
DMRs or regulatory features in:
CpG Islands, shores and shelves ±4kb
DNAseI hypersensitive sites
Refseq Genes
Ensembl Regulatory Features
Discovery Tool
• Probes independent of methylation state
• Determine methylation state of all
methyl sites in region
Comprehensive Design
• CpG Islands
• Promoter regions
• DMRs (Differentially Methylated
Regions)
HaloPlex
For research use only
SureSelect vs. Whole-Genome Bisulfite Sequencing
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10
20
30
40
50
60
70
80
90
100
1X 5X 10X 15X 20X 25X 30X
Coverage of at least...
Coverage vs. % CpGs covered in targeted regions (~3.7 million CpGs)
Per
cen
t C
pG
s co
vere
d
SureSelect Methyl-Seq
Whole-genome sequencing
10Gb input (raw)
4Gb (filtered)
180Gb input (raw)
91 Gb (filtered)
HaloPlex
For research use only
Good Concordance between WGBS & SureSelect Methyl-Seq
R = 0.927
Excellent concordance with whole genome bisulfite sequencing data.
HaloPlex
For research use only
Proteomics
Metabolomics
SureSelect QC
DNA-Seq
RNA-Seq
ChIP-Seq
Methyl-Seq
miRNA-Seq
Multi-Omic Analysis
Canonical Pathways
Computational Network Discovery
mRNA & miRNA expression arrays
Exon arrays
SNP arrays for GWAS & CNV
GeneSpring 12.5 Analysis Software
HaloPlex
For research use only
MeDip-Seq & Reduced Representation BiSulfite Sequencing
(RRBS)
• Cannot target specific regions (i.e. DMRs in Shelf and Shore regions) • Biased towards methylated regions, Repeat sequences & CpG-rich sequences • Can miss under-methylated regions • Difficult to design since knowledge of methylation state for the target region is
needed
Limitations
HaloPlex
For research use only
More Coverage of CpGs Compared to Infinium 450K Array
SureSelect Human Methyl-Seq
Infinium 450K
480,000 CpGs
3,700,000 CpGs
HaloPlex
For research use only
SureSelect Human All Exon V5
HaloPlex
For research use only
SureSelect Human All Exon V5 and V5+UTRs
– Updated to the latest genomic databases,
including CCDS, GENCODE, RefSeq,
TCGA
– Less sequencing than other exomes, only
4Gb for V5 and 6Gb for V5+UTRs
– Low overall costs, less sequencing and
greater sample throughput
– 16 hr (overnight) hybridization protocol
– Fastest turnaround time for projects
Ready for Sequencing the Next Day
The Best Performance
HaloPlex
For research use only
The Best Performance Outstanding coverage with less sequencing
30%
40%
50%
60%
70%
80%
90%
100%
% Reads On-Target +/-100bp % bases >10x cov. % bases >20x cov.
SureSelect V5 (4Gb)
Nimblegen Exome (4Gb)
Nextera Exome (4Gb)
HaloPlex
For research use only
The Fastest Workflow From sample to sequencing the next day
Library Preparation
6 hours
Capture
16 hours
Wash and PCR
4 hours
Overnight Hybridization Enables Sequencer Ready Samples the Next Day
HaloPlex
For research use only
The Fastest Workflow Less sequencing, faster time to result
Time to sequence 150 samples on a HiSeq based on recommended sequencing amounts
0 5 10 15 20 25 30 35
Nextera Exome
Nimblegen V3
SureSelect V5
Days
HaloPlex
For research use only
HaloPlex Exome Early Access
HaloPlex
For research use only
HaloPlex Exome
HaloPlex
Panels HaloPlex
Fast
HaloPlex
Large
HaloPlex
Agilent Innovation for Target Enrichment
HaloPlex
For research use only
HaloPlex Exome
Fast and simple workflow
Minimal pipetting steps and no library prep
Generate results in 3 days
Only 3 days from sample to data
Low DNA input
Only 200ng of DNA required
Cover all coding content in the genome
Designs against the most recent databases
HaloPlex
For research use only
Comprehensive Human Exome Design
What if I could
research all
diseases at the
same time?
Target Size 37 Mb
# Genes ~21,500
# Targeted Exons ~357,000
Exome Content
Databases
CCDS, RefSeq,
GENCODE, miRBase,
TCGA, UCSC, VEGA
HaloPlex Exome Content
HaloPlex
For research use only
The greatest coverage with less sequencing Only 4Gb of sequencing to achieve 80% at 20X coverage
HaloPlex Exome – Premium Exome Performance
30%
40%
50%
60%
70%
80%
90%
100%
% bases covered at 10X
% bases covered at 20X
HaloPlex Exome
Nextera Exome
HaloPlex
For research use only
Complete Solutions for Gene Regulation Studies Looking at the complete picture
Gene Regulation
RNA • Novel Gene Discovery
• Gene Expression
DNA • Human Exome
Methylation • DNA Methylation