Agenda Introduction How HaloPlex HS works 1 2 A flexible and accelerated solution 5 3 Performance data For Research Use Only Not for use in diagnostic procedures From Discovery to Clinical Research ID: 662940
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Slide1
HaloPlex
HS
Get to Know Your DNA. Every Single Fragment
.Slide2
Agenda
Introduction
How HaloPlex
HS
works
1
2
A flexible and accelerated solution
5
3
Performance data
For Research Use Only.
Not for use in diagnostic procedures.Slide3
From Discovery to Clinical Research
DISCOVERY
FOLLOW-UP
Whole
Exome
Whole Genome
GWAS
Follow-up
Exome
Follow-Up WGAS
Follow-up GWAS
Clinical Research Panels
CLINICAL RESEARCH
For Research Use Only.
Not for use in diagnostic procedures.Slide4
Requirements of clinical research
FOLLOW-UP
CLINICAL RESEACH
Clinical
r
esearch applications require:
Fast turnaround time
Flexibility in capture size
Simple workflowHigh coverageHigh accuracy in variant detection
Data analysis solutionFor Research Use Only. Not for use in diagnostic procedures.Slide5
The need for sensitivity and accuracy
For Research Use Only.
Not for use in diagnostic procedures.Slide6
What are low allele frequency variants implicated in?
Clonal evolution and pathogenesis
Tumor
subclonal heterogeneity Immunological diversity
What are low allele frequency variants?Variants present at a frequency below 3%Low allele frequency variants
Adapted from Stead
et al
(2013) Human Mutation 34: 1432-1438For Research Use Only. Not for use in diagnostic procedures.Slide7
Low allele frequency variants are difficult to detect by conventional NGS methods
Relatively high error rate of sequencers
(1 wrong base call in 100-1000 sequenced bases)
Kennedy et al (2014) Nature Protocols 9: 2586 - 2606Requires molecular barcodes for increased sensitivity and accuracy
Low allele frequency variants
For Research Use Only. Not for use in diagnostic procedures.Slide8
Basic molecular barcode analysis
Align reads
Group read pairs to designed probes based on read start-stop position
For each probe: group reads with identical molecular barcode sequence
Consolidate read information to one read per molecule (remove PCR duplicates)
T
T
T
A
A
C
G
Group reads with the same molecular barcode
Barcode family
T
Consensus read
True variant
Random error
Molecular barcode
Sample Index
For Research Use Only.
Not for use in diagnostic procedures.Slide9
Ability to identify unique progenitor DNA fragments (de-duplication)
Biases and errors from PCR amplification or sequencing steps can be detected.
Decreased error rate, increased accuracy for variant calling (low-input DNA)
Low allele frequency variant detection
CNV detection
Benefits of molecular barcode analysis
For Research Use Only.
Not for use in diagnostic procedures.Slide10
Agenda
Introduction
How HaloPlex
HS
works
1
2
A flexible and accelerated solution
5
3
Performance data
For Research Use Only.
Not for use in diagnostic procedures.Slide11
Introducing HaloPlex
HS
– High Sensitivity Next Gen PCR
Key Features :
More than a million unique 10nt molecular barcodes are incorporated into DNA library fragmentsRequires only 50ng starting DNA inputRapid workflow : From sample to sequencing-ready libraries in <6hr
Compatible with FFPE samples
More sensitive and accurate than other conventional NGS TE methods
For Research Use Only. Not for use in diagnostic procedures.Slide12
Key Benefits
For Research Use Only.
Not for use in diagnostic procedures.Slide13
Select the HaloPlex
HS
design workflow
Input gene ID/name/coordinateDefine regions of interest (
eg. Exons, UTRs, etc)
Click “
Start Design”Design report in 10 minutes
www.agilent.com/genomics/suredesign
SureDesign
– Create a custom design in minutes
For Research Use Only.
Not for use in diagnostic procedures.Slide14
How HaloPlex
HS
works
For Research Use Only.
Not for use in diagnostic procedures.Slide15
The HaloPlex
HS
Workflow
Improves design coverage Redundancy reduces risk of allele dropout if a probe fails; protects against primer site mutations
Specificity of the restriction enzymes add specificity to the captureAmplicon tiling
Each 50ng DNA sample is fragmented in eight double-digest reactions
1
For Research Use Only.
Not for use in diagnostic procedures.Slide16
HaloPlex
Others
TARGET
TARGET
With
HaloPlex
each target base is covered by up to eight
amplicons
(different start and stop sites)!
If an unknown mutation appears in a restriction site, it may affect one or two fragments but all others will be present
If
a variant occurs – it can be checked by multiple
amplicons
with
HaloPlex
With other multiplex PCR based technologies, each target base is covered by only one
amplicon
(same start and stop sites)
If an unknown mutation appears in a primer site it causes a complete dropout in the target region
If a variant occurs, it is hard to know if it is a real mutations and not a PCR
artifact
DNA variant
DNA variant
1
2
3
1
2
3
Basics of
HaloPlex
technology –
amplicon
redundancy
For Research Use Only.
Not for use in diagnostic procedures.Slide17
Genomic region
Target
0001111122322222333455422221111222111110000 Read coverage
Increased Confidence in Mutation Calling
Amplicon
redundancy provides excellent coverage.
For Research Use Only.
Not for use in diagnostic procedures.Slide18
DNA fragments are mixed with custom HaloPlex probes and primer cassettes containing the molecular barcodes.
2
Same dual hybridization requirement as regular PCR for high specificity
More than a million unique molecular barcodes are
available for incorporation, ensuring unique coverage
Both primers incorporated on the probe avoiding cross reactivity
Hybridization
For Research Use Only.
Not for use in diagnostic procedures.Slide19
Probe
/fragment
hybrids are
ligated and retrieved
with
streptavidin magnetic beads, followed by high stringency wash.
3
Ligation, capture and wash
Only perfectly hybridized fragments will be ligated
Ligated fragments are directly captured using streptavidin
For Research Use Only. Not for use in diagnostic procedures.Slide20
Only
fully circularized
DNA targets are
amplified on-bead.
4
PCR amplification
Thousands of different
amplicons
, one primer pair
On-bead PCR of ligated fragments simplifies workflow
Ready for sequencing in <6hr!
For Research Use Only. Not for use in diagnostic procedures.Slide21
Agenda
Introduction
How HaloPlex
HS
works
1
2
A flexible and accelerated solution
5
3
Performance data
For Research Use Only.
Not for use in diagnostic procedures.Slide22
HaloPlex
HS
Performance - High Uniformity and Specificity
Uniform coverage of targeted bases: >95% covered at 10% of average depthHigh Specificity: >80% on-target specificityimportant since deep sequencing is required for low frequency variant detection
For Research Use Only. Not for use in diagnostic procedures.Slide23
HaloPlex
HS
Performance – Excellent coverage even with FFPE samples
DIN
Excellent coverage of target bases (>90% covered at 100x) even with poor quality FFPE DNA. A custom cancer panel was used to enrich FFPE DNA of varying qualities as indicated by the DNA Integrity Number (DIN) provided by the 2200 Tapestation
System, where a DIN of 10 and 1 indicate intact
gDNA and completely degraded gDNA respectively.For Research Use Only.
Not for use in diagnostic procedures.Slide24
HaloPlex
HS
Performance – Detection down to 0.5% variant allele frequency
Detection of down to 0.5% allele frequency in HapMap dilutionsExpected allele frequency
HapMap cell lines, NA18507 and NA10831, were mixed to generate allelic fractions ranging from 0.5% - 5%. The close agreement between expected and observed frequency at various chromosomal positions demonstrates the high sensitivity of HaloPlexHS for low frequency variant detection. Data shown is representative of replicates
(sequencing depth = 2000x – 4000x)
For Research Use Only.
Not for use in diagnostic procedures.Slide25
HaloPlex
HS
Performance – Detection down to 0.5% variant allele frequency
Figure 1B
. Number of total unique reads covering the target regions. The unique reads covering each allele fraction at the various chromosomal positions are highlighted (green) Number of total unique reads covering the target regions.
The unique reads covering each allele fraction at the various
chromosomal positions are highlighted (green) For Research Use Only. Not for use in diagnostic procedures.Slide26
Simplify Data Analysis with
SureCall
For Research Use Only.
Not for use in diagnostic procedures.Slide27
Agenda
Introduction
How HaloPlex
HS
works
1
2
A flexible and accelerated solution
5
3
Performance data
For Research Use Only.
Not for use in diagnostic procedures.Slide28
HaloPlex
HS
- a flexible solution
Compatible with both ILM and ION PGM platforms
Create custom designs up to 5Mb (2.5Mb for ION)NGS Disease Research Panels are available in catalog or made-to-order format Multiplex up to 96 samples for ILM and 16 samples for ION
For Research Use Only.
Not for use in diagnostic procedures.Slide29
Accelerate Time to Results
1
2
Prepare DNA libraries in <6hr
Begin sequencing on a desktop sequencer
Analyze your data
Day 1
Prepare
Sequence
Analyze
Day 2
For Research Use Only.
Not for use in diagnostic procedures.Slide30
Summary
HaloPlex
HS
Target Enrichment System is a high sensitivity method for the accurate identification of low allele frequency variantsHaloPlexHS incorporates unique molecular barcodes into each DNA library fragment
HaloPlexHS performs with high coverage, on-target specificity and with a sensitivity that allow detection of alleles down to below 1% allele frequency (at least 0.5%)It is ideally suited for cancer research and studies that involve the detection of somatic variants in
heterogenous
samples For Research Use Only. Not for use in diagnostic procedures.