eseminar: diseño de un nuevo test ngs para el análisis hereditario€¦ · diseño de un nuevo...
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HaloPlex
For research use only 1
eSeminar:
Diseño de un nuevo test
NGS para el análisis
rutinario de cáncer
hereditario
5 marzo 2014
Ponente Invitado
Dr. Alberto Acedo
Co-fundador y Dr. Técnico
AC-Gen Reading Life S.L.
Amparo Dávila
Especialista Producto Genómica para Iberia
Agilent Technologies
HaloPlex
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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
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The advantages with Traditional PCR
High Coverage
High Specificity
Fast
Cost Effective
Simple
Well established
HaloPlex
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The problems with Multiplex PCR
Primer Cross reactivity
Limited in # target regions
Long optimization
Hard to change
Dropouts
Artifacts
HaloPlex
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HaloPlex Workflow
A Simple & Fast 4-step protocol
2014-03-05
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
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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
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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
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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
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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
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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
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HaloPlex Technology Overview
The sequencing primers are used to sequence in from the fragment ends
(paired-end sequencing).
Genomic region Target exon
HaloPlex
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HaloPlex Technology Overview
Genomic region Target exon
0001111122322222333455422221111222111110000 Read coverage
HaloPlex
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HaloPlex Technology Overview
By tiling amplicons high target coverage is achieved
Genomic region Target exon
0001111122322222333455422221111222111110000
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)
What makes HaloPlex better than other PCR methods!
HaloPlex
For research use only
HaloPlex Others (TruSeq, AmpliSeq, Fluidigm, RainDance)
TARGET TARGET
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
What makes HaloPlex better than other PCR methods!
HaloPlex
For research use only
HaloPlex Others (TruSeq, AmpliSeq, Fluidigm, RainDance)
TARGET TARGET
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
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
Don’t limit yourself: thousands of amplicons
25
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 are amplified in a single tube?
HaloPlex
For research use only
Publications
Page 26
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
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
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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
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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
Thank You!
Muchas gracias por su atención
Dirección de contacto: [email protected]