HaloPlex
For research use only 1
eSeminario
Introducción a captura
de secuencias con
Haloplex en NGS
Amparo Dávila Especialista Producto Genómica para Iberia 4 abril 2013
HaloPlex
For research use only
• Key Benefits 1
• How it Works 2
• Design a panel 3
• Recent Advancements 4
• Performance And Applications 5
• New Cancer Panel 6
• Data Analysis 7
Agenda – HaloPlex
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
FOLLOW-UP CLINICAL
From Discovery to Clinical Research
All Solutions currently
available are based on
Multiplex PCR Needs:
Fast turnaround Time
Flexibility in panel size
Simple workflow
High Coverage and Specificity
High Throughput
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
Dropouts
Artifacts
HaloPlex
For research use only
HaloPlex – Simplicity of Next Generation PCR
SIMPLE
Library-Prep free Target
Enrichment in as little as 6
hours
All in a single tube!
Simple SureDesign
No capital equipment, simple
to implement
A whole lab in a single tube!
HaloPlex
For research use only
HaloPlex – Simplicity of Next Generation PCR
FLEXIBLE
Any targets (sizes can be up
to 5Mb depending on platform)
96 indexed samples ready for
sequencing
Compatible with Ion Torrent
and Illumina Platforms! Any sequencer, Any targets
HaloPlex
For research use only
HaloPlex – Performance of Next Generation PCR
PREMIUM
PERFORMANCE
Low DNA Input (200ng)
High Coverage
Uniform Amplification
High Specificity
Sensitive variant detection
HaloPlex
For research use only
• Key Benefits 1
• How it Works 2
• Design a panel 3
• Recent Advancements 4
• Performance And Applications 5
• New Cancer Panel 6
• Data Analysis 7
Agenda – HaloPlex
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
HaloPlex Workflow
A Simple & Fast 4-step protocol
2013-04-04
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
Each DNA sample is fragmented in eight
double-digest reactions 1. Digest DNA
Define region of interest (ROI)
Genome digested in silico
Fragments chosen for enrichment
Design Wizard - In Silico digestion
and HaloPlex probe design
Molecular Concept
HaloPlex
For research use only
Each DNA sample is fragmented in eight
double-digest reactions 1. Digest DNA
Amplicon tiling improves coverage
by design
Amplicon redundancy reduces risk
of loosing completeness if a probe
fails; protects against primer site
mutations
Specificity of the restriction
enzymes add specificity to the
capture
Amplicon tiling
Molecular Concept
HaloPlex
For research use only
2. Hybridize probes
Fragments are mixed with custom
HaloPlex probes and a Primer Cassette.
Hybridization overnight.
Biotin
Same hybridization requirement as in
regular PCR – high specificity!
Both “primers” on the same oligo – no
cross-reactivity!
Sample barcode is integrated (96 barcodes
available)
Correctly hybridized fragments will form a
nicked dsDNA structure with the HaloPlex
probe
Hybridization guides circularization
Molecular Concept
Red parts – complementary to the targeted fragment
Grey part – contains sequencer specific
motifs and the sample barcode
HaloPlex
For research use only
3. Purify and ligate
targets
Probe/Fragment hybrids are retrieved
with magnetic streptavidin beads. The
circular molecules are then closed by a
DNA ligase.
Biotin
Level of enrichment is increased by
removal of non-targeted fragments
Only perfectly hybridized fragments
will be ligated
Purification and DNA Ligation
Molecular Concept
HaloPlex
For research use only
4. Amplify targeted
fragments
Only circular DNA targets are
amplified. Sample barcodes are
introduced. Final product is ready for
sequencing
1) Bridge PCR primer 2) Sequencing primer
PCR is primed from the Primer
Cassette
Tailed primers add bridge PCR
motifs
Thousands of amplicons – one
primer pair
Molecular Concept
1
2
HaloPlex
For research use only
HaloPlex Technology Overview
Eight different fragments cover each target position. Amplicons are tiled over the region.
Genomic region Target exon
HaloPlex
For research use only
HaloPlex Technology Overview
Genomic region Target exon
A subset covering the target region is selected using HaloPlex probes
More than one different fragment cover the target region
HaloPlex
For research use only
HaloPlex Technology Overview
Genomic region Target exon
HaloPlex baits guide the circularization
HaloPlex
For research use only
HaloPlex Technology Overview
The selected subset is equipped with sequencing primer sequences by the
selector probes.
Genomic region Target exon
1) library bridge PCR primer
2)Sequencing primer+Sample
barcode
1 2 1
HaloPlex
For research use only
HaloPlex Technology Overview
The sequencing primers are used to sequence in from the fragment ends
(paired-end sequencing).
Genomic region Target exon
HaloPlex
For research use only
HaloPlex Technology Overview
Genomic region Target exon
0001111122322222333455422221111222111110000 Read coverage
HaloPlex
For research use only
HaloPlex Technology Overview
By tiling amplicons high target coverage is achieved
Genomic region Target exon
0001111122322222333455422221111222111110000
HaloPlex
For research use only
Pileup in Integrative Genome Viewer
2013-04-04
Outstanding coverage of targeted bases; highly specific coverage
HaloPlex
For research use only
HaloPlex Others (TruSeq, AmpliSeq, Fluidigm, RainDance)
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
What makes HaloPlex better than other PCR methods!
HaloPlex
For research use only
Streamlined Workflow
From Sample to Sequencing in a single day!
1 2 3
Design your gene
panel using the
HaloPlex Design
Wizard
Prepare samples in
less than 6 hours
with HaloPlex Kit
Sequence with a
desktop or high
throughput sequencer
~10 mins <6 hours <1 day
HaloPlex
For research use only
Consistent Results among Different Design Sizes
Extremely high Performance
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
HaloPlex
For research use only
• Key Benefits 1
• How it Works 2
• Design a panel 3
• Recent Advancements 4
• Performance And Applications 5
• New Cancer Panel 6
• Data Analysis 7
Agenda – HaloPlex
HaloPlex
For research use only
SureDesign
Agilent’s Next Generation Custom Design Software
•3 Easy Steps to Your Targeted Design
•Steamlined Workflow and Sleek User Interface
•Collaboration tools enabling sharing of pre-designed panels
•Both SureSelect and HaloPlex NGS platforms to choose from
www.agilent.com/genomics/suredesign
HaloPlex
For research use only
• Key Benefits 1
• How it Works 2
• Design a panel 3
• Recent Advancements 4
• Performance And Applications 5
• New Cancer Panel 6
• Data Analysis 7
Agenda – HaloPlex
HaloPlex
For research use only
NEW! HaloPlex for Ion Torrent
Featuring…
Paired-End
by Design!!!
Sequence Single-End…
Get Paired-End Coverage of your
targets!
HaloPlex
For research use only
How Paired-End by
Design Works
For each target, 2 probes are created.
One probe is for the sense strand and
the other for the antisense strand
After the PCR step with universal
primers…
Amplicon products now have
different ends next to the sequencing
primer.
When generating SINGLE-END reads,
reads will be generated from BOTH
ENDS of the target
Sense Strand AntiSense Strand
Seq primer
Seq primer
DNA TARGET
HaloPlex
For research use only
Why is HaloPlex Breaking Barriers?
Up to 5Mb capture
Page 34
Protocol time
(days)
Capture Size
(Mb) 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0
1
2
3
4
Short protocol, small capture size
Long protocol, large capture size
For the first time ever, a Target
Enrichment technology can at the
same time be FAST and allow
LARGE CAPTURE SIZE
Multiplex
PCR
Hybrid Capture
HaloPlex
For research use only
FFPE Samples Using HaloPlex
FFPE Challenges
• DNA quality issues (age, storage, fixation process)
• Sample degradation can impact sequencing quality (sensitivity, quality, allelic balance, etc)
HaloPlex with FFPE samples
• Validated protocol
• Adapted Design for FFPE
• Shorter probes
• Both DNA strands
• Quality Assesment protocol
HaloPlex
For research use only
HaloPlex Compatibilituy with FFPE Samples
Cancer Panel developed by OICR
19 Genes, ~60Kb size
• High Coverage
• Consistent Results
• FFPE Samples Compatibility
• Great variant calling sensitivity
POSTER FILE AVAILABLE – PLEASE
CONTACT US
HaloPlex
For research use only
• Key Benefits 1
• How it Works 2
• Design a panel 3
• Recent Advancements 4
• Performance And Applications 5
• New Cancer Panel 6
• Data Analysis 7
Agenda – HaloPlex
HaloPlex
For research use only
HaloPlex performs extremely well at larger sizes!
38
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Coverage by
design
Specificity Coverage 1x Coverage at
10% of mean
1.5 Mb
3.2 Mb
5.0 Mb
73,328 amplicons!
100,550 amplicons!
193,775 amplicons!
How many amplicons does
each design amplifies in the same tube???
HaloPlex
For research use only
Taking the Technology to the next level
Low DNA Input – down to 50ng with same performance
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
% reads ontarget
Cov 1x Cov 10x Cov 20x Cov 100x
Panel - 400kb (1,954 targets)
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
% reads ontarget
Cov 1x Cov 10x Cov 20x Cov 100x
Panel - 340kb (3,078 targets)
800ng
400ng
200ng
100ng
50ng
0%
20%
40%
60%
80%
100%
800ng 400ng 200ng 100ng 50ng
HET Concordance (HapMap)
400kb 340kb
0%
20%
40%
60%
80%
100%
800ng 400ng 200ng 100ng 50ng
HET Sensitivity (HapMap)
400kb 340kb
800 400 200 100 50
0.0
0.2
0.4
0.6
0.8
1.0
Agilent (82−1)
Sample Input (ng)
AB
800 400 200 100 50
0.0
0.2
0.4
0.6
0.8
1.0
Larry (45−1)
Sample Input (ng)
AB
0.0 0.2 0.4 0.6 0.8 1.0
02
46
81
01
2
AB
De
nsity
Sample Input
800 ng400 ng200 ng100 ng50 ng
0.0 0.2 0.4 0.6 0.8 1.0
02
46
81
01
2
AB
De
nsity
Sample Input
800 ng400 ng200 ng100 ng50 ng
Allelic Balance – 400kb Panel Allelic Balance – 340kb Panel
HaloPlex
For research use only
• Design Input:
– List of 78 genes
• Design Output:
– Total design coverage 95.5%
of 197391 bp (CCDS)
41
Sten Linnarsson
Dept of Medical Biochemistry and
Biophysics
Molecular Neurobiology
HaloPlex Cancer Panel Pilot Study – Compatibility with 100 Cells+WGA
HaloPlex
For research use only 4 April 2013 Una Kjällquist 42
6% 2%
95% 98%
0%
20%
40%
60%
80%
100%
Targets Bases
0 reads
1+ reads
3% 1%
97% 99%
0%
20%
40%
60%
80%
100%
Targets Bases
0 reads
1+ reads
1M cells, unamplified 100 cells, GenomiPhi
HaloPlex Cancer Panel Little Loss due to WGA
HaloPlex
For research use only
HaloPlex Cancer Panel Analysis of Cell populations in Leukemia
Detection of rare alleles
HaloPlex
For research use only
Application: GWAS follow up
Hypertension
Dr. Anders Dahm Department of Hematology Ullevaal University Hospital Oslo, Norway
Four linkage regions were identified in a GWAS
study of female hypertension patients.
Challenge: Target continuous regions in a large
patient cohort
HaloPlex
For research use only
High Uniform Coverage among all target regions
Design success in silico
Coverage by design:
150 kbp (93,2%)
Repeated bp in ROI: 38%
Assay success after sequencing
97.4% of sequencing reads on amplicons
96.6% or reads on target
>90% of target region sequenced
Targeted region: 4 regions of in total 160 kbp
• Comprehensive Coverage • Uniform Coverage • High Coverage
HaloPlex
For research use only 2013-04-04
Cystic Fibrosis CFTR gene
4443 bp, 27 exons
Covered by design: 100%
Covered by sequencing: 99.8%
Specificity (on target reads): 99.97%
All variants detected
Berivan Baskin, MD, PhD Clinical Molecular Geneticist Uppsala University
Peter Ray , PhD, FCCMC, FACMG Head, Molecular Genetics, The Hospital for sick children
Application – Clinical Research Panel
Cystic Fibrosis
HaloPlex
For research use only
Uniform Coverage among all target exons
Example of enrichment of all exons within CFTR gene, for 5 different samples:
• Comprehensive Coverage
• Uniform Coverage
• High Coverage • Enrichment of
exons of any size
HaloPlex
For research use only
Detection of all types of genetic variants
Effective detection of 4bp indel
after enrichment with HaloPlex:
• Top: heterozygote sample
• Bottom: homozygote sample
Effective detection of single
nucleotide variant (C/T) after
enrichment with HaloPlex:
HaloPlex
For research use only
• Key Benefits 1
• How it Works 2
• Design a panel 3
• Recent Advancements 4
• Performance And Applications 5
• New Cancer Panel 6
• Data Analysis 7
Agenda – HaloPlex
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
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
• Key Benefits 1
• How it Works 2
• Design a panel 3
• Recent Advancements 4
• Performance And Applications 5
• New Cancer Panel 6
• Data Analysis 7
Agenda – HaloPlex
Introduction to SureCall
• Agilent SureCall software was developed
for clinical researchers looking for a fast, accurate and
easy-to-use analysis tool for HaloPlex panels
• SureCall reduces the time to results from days to hours by
providing an end-to-end data analysis solution: from
alignment to categorization of mutations
• Agilent SureCall is free-of-charge for HaloPlex users
For Research Use Only. Not Approved for
Use in Diagnostic Procedures
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
For Research Use Only. Not Approved for
Use in Diagnostic Procedures
SureCall makes NGS data
analysis easier, faster,
cheaper
www.agilent.com/genomics/surecall
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
For Research Use Only. Not Approved for
Use in Diagnostic Procedures
Adapted from: Frederick National Laboratory for Cancer Research
Simple 3-step workflow-based data analysis
For Research Use Only. Not Approved for
Use in Diagnostic Procedures
1. Import Samples
2. Describe Samples
3. Run Analysis
Sample review - Triage View
For Research Use Only. Not Approved for
Use in Diagnostic Procedures
genome
viewer
mutation
table
What you can do from the Mutation Table
1. Suppress mutations
2. Add notes
3. Change categorization
4. Link out to external databases: OMIM, GeneCard, dbVar, NCBI, Ensembl,
and Uniprot
For Research Use Only. Not Approved for
Use in Diagnostic Procedures
1
2 3 4
Genome Viewer
For Research Use Only. Not Approved for
Use in Diagnostic Procedures
Go to next/
previous
variant
Zoom in/out Disable/enable
text in grey boxes
Allele fraction
+ genotype
Variants from
VCF
Coverage
from BAM
Regions and
amplicons
from BED
80 bp around variant,
color-coded by category
Links to external databases available from the
Mutation Table
For Research Use Only. Not Approved for
Use in Diagnostic Procedures
Right click on column
in SureCall Database
ID dbSNP
Gene Name OMIM
GeneCard
Position dbVar
Transcript NCBI
Transcript ID Ensembl
Uniprot Uniprot
All changes that are made are tracked in the
Audit Trail
For Research Use Only. Not Approved for
Use in Diagnostic Procedures
Data Search functionality
For Research Use Only. Not Approved for
Use in Diagnostic Procedures
Find other samples containing the same
mutation(s) stored in the system
Choose VCF file or folder of VCF files to add to
the VCF search index (for example a file which
contains known benign variants)
VCF = Variant Call Format = a specification for
storing gene sequence variations
Reports
For Research Use Only. Not Approved for
Use in Diagnostic Procedures
1-page
mutation
report
QC
report
Make your
own
report
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
Gracias por asisitir
Para más información: [email protected]
Promoción especial: 1304_WEBNGS_SP
Page 73
SureCycler 8800
Kit de 96 HaloPlex
Panel Cancer 25% OFF
HaloPlex
For research use only
Thank You!