agilent target enrichment solutions for ngs · haloplex for research use only sureselectxt2 -...
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HaloPlex
For research use only
Agilent Target Enrichment Solutions for NGS
Winfried van Eyndhoven Genomics FAS, BeNelux/Iberia
HaloPlex
For research use only
Agenda
4 Whole Methylome Sequencing
NGS Target Enrichment Solution 1
5 Custom SureSelect & HaloPlex Panels
Targeted RNA Sequencing 6
7 Automation
Whole Exome Sequencing V4 & V4+UTR 3
2 QC workflow
HaloPlex
For research use only
Targeted Resequencing vs. Whole Genome
Required Throughput = Genome Size x Average Coverage
v
Target = 50Mb x 100 = 5Gb
Target = 5Mb x 100 = 500Mb
Target = 0.5Mb x 100 = 50Mb
Target =50Kb x 100 = 5Mb
HaloPlex
For research use only
Library Preparation
0.5µg
Hybridization / Capture 24 hours
Baits:
- cRNA probes
- Long (120bp)
- Biotin labeled
- User-defined (eArray)
- SurePrint synthesis
Bead Separation
Wash / Elution / Amp
Basic SureSelect
Target Enrichment Workflow
HaloPlex
For research use only
Key Benefits – Agilent SureSelect
PROVEN Technology
COMPLETE solution
FLEXIBLE solution
High PERFORMANCE
HaloPlex
For research use only
SureSelect Portfolio
What do
you want to
capture?
DNA RNA
Human Human
Non
Human Non
Human
Animal
Models Bovine
All Exon
*
Zebrafish
All Exon
*
Dog
All Exon
*
Mouse
All Exon
Custom
Capture
Custom
Capture
Kinome
RNA
X
Chrom
Whole
Exome
All Exon
V4 *
(51Mb)
All Exon
50Mb
Custom
Capture
Kinome
DNA
Custom
Capture
All Exon
V5*
(71Mb) * NEW
HaloPlex
For research use only
SureSelect All Exon – also non-Human Species
All Exon Bovine:
64Mb design. The library is based on University of Maryland build 3.1 against NCBI
exons as well as predicted exons, UTRs, and miRNAs. Worked with USDA on
design.
All Exon Canine:
54Mb design. UCSC tracks from CanFam2 for ensembl as well as Refseq, human
protein alignments, and spliced ESTs that lie outside of ensembl.
All Exon Zebrafish:
96Mb design. Zv9 library is in Emsembl and is annotated with gene and exon
coordinates. Worked with Sanger on design.
All Exon Mouse:
49.6Mb design. Exon definition derived from Ensembl + RefSeq, designed against
mm9 reference from UCSC
Note: All Exon Bovine, Canine and Zebrafish are being provided in Early Access Format
HaloPlex
For research use only
SureSelectXT – Complete Workflow Solution
SureSelectXT/XT2
Target Enrich. Library
Prep
Herculase
enzyme
HaloPlex
For research use only
APPENDIX – SURESELECTXT2
SureSelectXT2
A more Efficient Workflow:
Pre-Capture Pooling
Streamlined workflow
Pool More, Pippete Less
HaloPlex
For research use only
SureSelectXT2 - Pre-Capture Pooling
Pre-capture pooling of:
8 Samples for All Exon Kits
16 Samples for Custom Kits
MasterMixed Reagents:
Streamlined workflow – 3x less tubes
Less Pipetting, no gel size-selection
96 indexes:
Scalable
Availability:
Illumina NGS Platforms
Low Starting DNA input
1ug DNA only
Individual
Library Preps
Sample Pooling
Pooled Capture
Multiple Capture
Pooling
Prep for
Sequencing
Pre-Capture Pooling
HaloPlex
For research use only
Key Benefits – Agilent SureSelect
PROVEN Technology
COMPLETE solution
FLEXIBLE solution
High PERFORMANCE
HaloPlex
For research use only
SureSelect kits are platform agnostic!
Illumina GAIIx
HiSeq 2000 SOLiD 4
5500
454 FLX
GS Junior
Pacific
Biosciences*
Ion Torrent
PGM
MiSeq*
* -coming soon!
From High throughput to desktop sequencers
NEW!
NEW!
SureSelect and HaloPlex
For research use only
DISCOVERY FOLLOW-UP CLINICAL
The NGS Applications Workflow
Whole Exome
Whole Genome
GWAS
Follow-up Exome
Follow-Up WGS
Follow-up GWAS
Clinical Research
Panels
HaloPlex
For research use only
SureSelect Custom Designs
For all sizes and needs
1Kb-
500kb
10Mb-
20Mb
5Mb-
10Mb
500kb-
5Mb
HaloPlex
For research use only
Different paths to create your custom design
eArray Web Portal -you
create the design
Design Services We create the
design for you
Your Custom
SureSelect Kit A bait library to capture
your regions of interest
Genomic
Coordinates Regions you want
to capture
NEW!
HaloPlex
For research use only
Key Benefits – Agilent SureSelect
PROVEN Technology
COMPLETE solution
FLEXIBLE solution
High PERFORMANCE
HaloPlex
For research use only
SureSelect: Enabling Scientific Discovery
… and many more to
come
>200 Publications…
HaloPlex
For research use only
Unlocking the Cancer Genome
Breast and Ovarian
Leukemia
Renal
Pancreatic
Colorectal
Melanoma
Hepatocellular
HaloPlex
For research use only
Key Benefits – Agilent SureSelect
PROVEN Technology
COMPLETE solution
FLEXIBLE solution
High PERFORMANCE
HaloPlex
For research use only
Performance of a Target Enrichment technology
• Mappability
•% on-target
• Uniformity
• Coverage (% bases at 20x)
MOST IMPORTANT
• SNPs!
• Indels!
• CNVs!
• Allelic Balance
Longer probes will
perform better!
Overall
Performance Mapping
Metrics Sensitivity
Metrics
Operational
Excellence
• Automation?
• # indexes
• Compatibility
• Protocol
Don’t forget to look at the whole picture!
HaloPlex
For research use only
SureSelect Baits
Better sensitivity
Feature Benefits
Ultra-Long RNA Baits
Only 24hr hybridization compared to 72hrs for
competitor
Capture small and large genetic variations:
SNP, InDels, CNV, fusions
Low # PCR cycles, less artifacts
Better Allelic Balance - Better Uniformity
Low input DNA needed
Binding strength
RNA:DNA DNA:DNA >
HaloPlex
For research use only
Really Long RNA Baits Tolerate Mismatches (even long ones)
Longer baits = Better Sensitivity
Allelic Balance
post-capture
Allele 1 Allele 2 - SNP
0.5 0.5
120-mer
bait
Smaller Probes will poorly hybridize!
Allele 1 Allele 2 – 25bp deletion
0.5 0.5
SureSelect and HaloPlex
For research use only
25bp deletion
7bp deletion
10bp deletion
11bp deletion
SureSelect – Proven high sensitivity Capture not only SNPs and Indels, but also CNVs!
Large deletions and duplications
HaloPlex
For research use only
Agenda
4 Whole Methylome Sequencing
NGS Target Enrichment Solution 1
5 Custom SureSelect & HaloPlex Panels
Targeted RNA Sequencing 6
7 Automation
Whole Exome Sequencing V4 & V4+UTR 3
2 QC workflow
SureSelect and HaloPlex
For research use only
Get the best data and know it
PCR: Agilent Surecycler
Automation:
Agilent NGS Automation
qPCR: Agilent Mx3005P
QC: Agilent Bioanalyzer
QC: Agilent Tapestation
HaloPlex
For research use only
Quality Control steps in SureSelect workflow
Shear Genomic
DNA
Repair Ends
3’-dA Addition
Adapter
Ligation
PCR
Prepped
Library
SureSelect
Oligo Capture
Library Library
Hybridization
Magnetic
Bead Capture
PCR
Sequencer
SureSelectXT
Library Prep
SureSelect
Target Enrichment
Automated
Bead Capture
QC
QC
QC
HaloPlex
For research use only
Typical vs. Atypical Post-Shearing Profile: Bioanalyzer DNA 1000 Assay
Typical
• Sample volume • Too much DNA • Wrong buffer • Bubbles
Atypical
HaloPlex
For research use only
Agenda
4 Whole Methylome Sequencing
NGS Target Enrichment Solution 1
5 Custom SureSelect & HaloPlex Panels
Targeted RNA Sequencing 6
7 Automation
Whole Exome Sequencing V4 & V4+UTR 3
2 QC workflow
HaloPlex
For research use only
Most Updated Content
Better Performance
Complete Workflow
Human All Exon V4 and V4+UTR kits
Discover More, Faster!
NEW!
HaloPlex
For research use only
30
SureSelect All Exon V4 and V4+UTR Choose the design that is right for your research
Coding
Exons
Human
All Exon V4
(51Mb)
Sequence 4Gb
Coding
Exons +
UTRs
Human
All Exon
V4+UTR
(71Mb)
Sequence 6Gb
HaloPlex
For research use only
SureSelect All Exon V4 and V4+UTR Most updated content; best performance, complete workflow
Improved design algorithm: captures difficult regions
better
Designed against the most updated databases
Coding content only (V4) or with UTRs (V4+UTR’s)
Improved
Design
Better Uniformity and % Target
Less Sequencing: 4Gb for V4 / 6Gb for V4+UTR
Improved Sample Throughput per Run
Better
Performance
Only 1x24hr Hyb – Achieve your results faster!
Complete Workflow with SureSelectXT
Better
Workflow
HaloPlex
For research use only
32
SureSelect All Exon V4 and V4+UTR Better Performance, achieve results faster with 20
less sequencing
0,00%
10,00%
20,00%
30,00%
40,00%
50,00%
60,00%
70,00%
80,00%
90,00%
100,00%
% Reads On-
Target
% Reads On-
Target ±
100bp
% bases >1x
cov.
% bases
>10x cov.
% bases
>20x cov.
50Mb (5Gb)
V4 - 51Mb (4Gb)
V4+UTR - 71Mb (6Gb)
Improved
% on-target
Improved Coverage, with less sequencing
HaloPlex
For research use only
33
SureSelect All Exon V4 and V4+UTR Improved Uniformity – Less Sequencing Needed
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3
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10
0
15
30
45
60
75
90
105
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165
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285
300
New SureSelect All Exon V4/V4+UTR
More Uniform Coverage – Less Sequencing Needed
Higher Sample throughput, Faster time to Results!
All Exon 50Mb
HaloPlex
For research use only
Agenda
4 Whole Methylome Sequencing
NGS Target Enrichment Solution 1
5 Custom SureSelect & HaloPlex Panels
Targeted RNA Sequencing 6
7 Automation
Whole Exome Sequencing V4 & V4+UTR 3
2 QC workflow
HaloPlex
For research use only
• Hypomethylation
Under methylated regions
Can lead to genomic instability – Changes in the chromosome structure
• Hypermethylation
Over methylated regions
Leads to transcriptional repression of genes
In tumor suppressor gene (TSG i.e. p16 and Sox17) – Leads to cancer
DNA Methylation: Role in Cancer
Robertson K. Nature Genetics, 2005
HaloPlex
For research use only
Methylation Effects on Other Diseases
Fragile X Syndrome
Rett Syndrome
Autistic Patients and their Parents
Schizophrenia and Bipolar Disorder
Bipolar Disease
Dementia
Alcoholism
Suicide
Alzheimer’s Disease
Imprinting disorders
Prader-Willi syndrome
Angelman syndrome
Beckwith-Wiedemann syndrome
• Epigenetic modifications in disease extends beyond cancer
• Many epigenetic changes are associated in mental diseases
Epigenetics Market Trend 2011, SelectBioSciences
HaloPlex
For research use only
Genomic locations of CpG dinucleotides
CpG islands
• High frequency of CpG dinucleotides
– > 500bp & GC content >55% & observed/expected CpG ratio > 0.65
• In or near about 40% of promoters of mammalian genes
Promoters
• 75% transcription start site have CpG-rich regions
• 88% active promoters are associated with CpG-rich sequences
HaloPlex
For research use only
Common Targets for DNA Methylation
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
•Discovery Tool - Probes are not methylation
state dependent so you do not need to have
prior knowledge of the methylation states of
the regions that you want to target
•Comprehensive design - Not limited to CpG
Islands. Comprehensive targeting key
methylation regions: CpG Islands, Promoters
and DMRs
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
The First Comprehensive Methylation Discovery System
HaloPlex
For research use only
DNA
Shearing, End
Repair & ‘A’
addition
SureSelect
Hyb (24hr)
SureSelect Methyl-Seq Protocol
mAdapter
ligation
Bisulfite
treatment
me me me me
me me me me
A A
Library
Quant and
PCR
Sequence
Bisulfite treatment is performed after
hybridization to maximize sample complexity
No PCR before Bisulfite treatment
to preserve the Methylation state
HaloPlex
For research use only
Whole genome data vs. SureSelect Methyl-Seq
R = 0.927
Excellent concordance with whole genome bisulfite sequencing data.
SureSelect vs. Whole-genome bisulfite sequencing
Agilent SureSelect™ Platform
Enabling Products for the Next-
Generation Sequencing Workflow
Page 42
0
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
Highly sensitive and accurate methylation detection after SureSelect target enrichment demonstrated DNA methylation differences between HCT116 human colon cancer cells and its methyltransferase double-knockout (DNMT1-/- and DNMT3b-/-).
Proof of concept (HCT116 vs.
Methyltransferase DKO)
HaloPlex
For research use only
Proof of concept (TIMP3: Metalloproteinase inhibitor 3)
HaloPlex
For research use only
SureSelectXT Human Methyl-Seq
The First Comprehensive Methylation Discovery System
•Covers more individual CpGs compared to Methylation
microarrays
•Increases throughput, reduces costs compared to Whole
Genome Bisulfite Sequencing
•Reveals methylated regions undetected by RRBS and Me-Dip
HaloPlex
For research use only
Methylation RNA DNA
SureSelect: Complete “Omics” Solution
DNA: Genetic variation
RNA: Gene Expression
Methylation: Effects on Gene Expression
HaloPlex
For research use only
Agenda
4 Whole Methylome Sequencing
NGS Target Enrichment Solution 1
5 Custom SureSelect & HaloPlex Panels
Targeted RNA Sequencing 6
7 Automation
Whole Exome Sequencing V4 & V4+UTR 3
2 QC workflow
HaloPlex
For research use only
Custom content from <200Kb up to 34Mb Capture only what
you want
>1,000 samples/run with 96 indexes! Higher sample
throughput per run
Up to 10 times less expensive than exome sequencing – save on enrichment and on sequencing
Cost effective
Faster time from sample to results
Results in hours with desktop sequencers
Easier Data Analysis
Why use SureSelect Custom panels?
HaloPlex
For research use only
All 54 known
deafness genes
The Power of Smaller Panels
21 breast and ovarian
cancer genes
Detects fusions for 90
human tyrosine
kinases Exons – Chrom 7
437 Genes related to
Mendelian Disorders
3.19Mb
Custom Design
HaloPlex
For research use only
ANNOUNCING HaloPlex Next Gen PCR Target Enrichment System
HaloPlex
For research use only
HaloPlex – Next Generation of PCR
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
HaloPlex – Simplicity of Next Generation PCR
SIMPLE
Library-Prep free Target
Enrichment in less than a day
All in a single tube!
96 indexed samples ready for
sequencing
Simple Design Wizard
Any Sequencer, Any Targets
(up to 500kb) A whole lab in a single tube!
HaloPlex
For research use only
HaloPlex – Performance of Next Generation PCR
PREMIUM
PERFORMANCE
Low DNA Input
High Coverage
Uniform Amplification
High Specificity
Sensitive variant detection
*Ion Torrent compatibility coming soon
HaloPlex
For research use only
HaloPlex Design Wizard
Create your design in only 10 minutes
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
Gene
Panel 1
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
A simple workflow providing superior data quality
HaloPlex Next Gen PCR Summary
Single tube protocol, ideal for desktop sequencing
Customize gene panels easily using online Design Wizard
Superior coverage enabling comprehensive mutation detection
HaloPlex
For research use only
Agenda
4 Whole Methylome Sequencing
NGS Target Enrichment Solution 1
5 Custom SureSelect & HaloPlex Panels
Targeted RNA Sequencing 6
7 Automation
Whole Exome Sequencing V4 & V4+UTR 3
2 QC workflow
HaloPlex
For research use only
The caveats of analyzing whole
transcriptomes through NGS RNA-Seq
Expensive:
o The expenditure necessary to obtain sufficient sequencing coverage for several research
and potential clinical applications is still prohibitive
o Calling genotypes with high confidence requires coverage levels of at least fivefold to 20-
fold, that can only be obtained with several million reads
Difficult to scale-up
o Limited sample throughput with current NGS systems and sequencing requirements
Data analysis
o Still a challenge
Overall coverage per region may not be high
enough
HaloPlex
For research use only
RNA Target Enrichment
Enrich your sample for a subset of transcripts of interest
to…
Get deeper coverage per transcript
Discover splice junctions, fusions, SNPs, allelic expression
Quantify gene expression with sensitivity similar to qPCR
Have more cost-effective and faster experiments
Easier data analysis
HaloPlex
For research use only
SureSelect RNA Target Enrichment Protocol
Start with <1ug of total RNA or mRNA*
Similar process to DNA Target enrichment,
except working with a
cDNA NGS library
Complete protocol takes ~ 3-4 days
Protocols available for Illumina and SOLiD,
for individual or multiplexed samples
*see protocol for details and recommendations
HaloPlex
For research use only
RNA Target Enrichment in Cancer
RNA Target Enrichment of 467 Cancer Genes (~all Tyrosine
Kinases + Genes from Cancer Gene Census);
Overall >800 target transcripts
Sequencing Libraries: K-562 chronic myeloid leukemia (CML) cell
line cDNA libraries: with and without target enrichment
HaloPlex
For research use only
What only RNA enrichment Reveals!!!
Feature Without RNA Target
Enrichment
With RNA Target
Enrichment
Avg. Coverage 14.4x 606x
Fold-Enrichment N/A 42x
Regions with 20x cov. 13% 63%
Common SNPs 76 257
Novel Variants 4 16
Splice-Junctions 2958 4720
Alternatively spliced-
genes 52 177
Gene Fusions 2 6
HaloPlex
For research use only
Performance of SureSelect RNA Target Enrichment
Enri
che
d li
bra
ry
Unenriched library
Gene Expression Levels
R2=0.885
High correlation to unenriched RNA library = enrichment process doesn’t create any bias – reliable gene expression results!
HaloPlex
For research use only
Agenda
4 Whole Methylome Sequencing
NGS Target Enrichment Solution 1
5 Custom SureSelect & HaloPlex Panels
Targeted RNA Sequencing 6
7 Automation
Whole Exome Sequencing V4 & V4+UTR 3
2 QC workflow
HaloPlex
For research use only
Automation of Agilent SureSelectXT for NGS
Increase Throughput and
Reproducibility
Improve Efficiency
Process up to 192 Samples
Per Week
Validated and ready to run
SureSelectXT
NEW! SureSelectXT AUTO
kits in 96, 480 or 960
formats
HaloPlex
For research use only
Automation of Next-Gen Sequencing Using SureSelect
Shear Genomic
DNA
Repair Ends
3’-dA Addition
Adapter
Ligation
PCR Enrichment
Prepped
Library
SureSelect
Oligo Capture
Library Library
Hybridization
Magnetic
Bead Capture
PCR
QA
Sequencer
SureSelectXT
Library Prep
SureSelect
Target Enrichment
Automated
Bead Capture
Automated
Purification
Automated
Purification
Automated
Purification
Automated
Purification
Automated
Purification
Automated
Purification
HaloPlex
For research use only
High Performance and Reproducibility
High Sensitivity and Specificity
HaloPlex
For research use only
Comparison of Timing and Throughput of
Manual vs. Bravo SureSelect Sample Processing
Up to 10x
throughput per
week vs. manual
processing!
HaloPlex
For research use only
Agenda
4 Whole Methylome Sequencing
NGS Target Enrichment Solution 1
5 Custom SureSelect & HaloPlex Panels
Targeted RNA Sequencing 6
7 Automation
Whole Exome Sequencing V4 & V4+UTR 3
2 QC workflow
HaloPlex
For research use only
Thank You!