haloplex hs get to know your dna. every single fragment
TRANSCRIPT
HaloPlexHS
Get to Know Your DNA. Every Single Fragment.
Agenda
Introduction
How HaloPlexHS works
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A flexible and accelerated solution
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3 Performance data
For Research Use Only. Not for use in diagnostic procedures.
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.
Requirements of clinical research
FOLLOW-UP CLINICAL RESEACH
Clinical research applications require:Fast turnaround time
Flexibility in capture sizeSimple workflowHigh coverage
High accuracy in variant detectionData analysis solution
For Research Use Only. Not for use in diagnostic procedures.
The need for sensitivity and accuracy
For Research Use Only. Not for use in diagnostic procedures.
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: 1432-1438
For Research Use Only. Not for use in diagnostic procedures.
• 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 - 2606
Requires molecular barcodes for increased sensitivity and accuracy
Low allele frequency variants
For Research Use Only. Not for use in diagnostic procedures.
Basic molecular barcode analysis
1. Align reads
2. Group read pairs to designed probes based on read start-stop position
3. For each probe: group reads with identical molecular barcode sequence
4. Consolidate read information to one read per molecule (remove PCR duplicates)
T
T
T
A
A
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Group reads with the same molecular
barcode
Barcode family
TConsensus
read
True variant
Random error
Molecular barcode
Sample Index
For Research Use Only. Not for use in diagnostic procedures.
1. Ability to identify unique progenitor DNA fragments (de-duplication)
2. Biases and errors from PCR amplification or sequencing steps can be detected.
3. Decreased error rate, increased accuracy for variant calling (low-input DNA)
4. Low allele frequency variant detection
5. CNV detection
Benefits of molecular barcode analysis
For Research Use Only. Not for use in diagnostic procedures.
Agenda
Introduction
How HaloPlexHS works
1
2
A flexible and accelerated solution
5
3 Performance data
For Research Use Only. Not for use in diagnostic procedures.
Introducing HaloPlexHS – High Sensitivity Next Gen PCR
Key Features :
o More than a million unique 10nt molecular barcodes are incorporated into DNA library fragments
o Requires only 50ng starting DNA inputo Rapid workflow : From sample to sequencing-ready libraries in <6hro Compatible with FFPE sampleso More sensitive and accurate than other conventional NGS TE methods
For Research Use Only. Not for use in diagnostic procedures.
Key Benefits
• Uniquely tag DNA fragments with more than a million 10-nt molecular barcodes
• Confidently detect mutations present at below 1% frequency in genetically heterogeneous samples
Unparalleled Sensitivity
• Differentiation of true variants from PCR or formalin fixation artifacts by targeting both DNA strands
Superior Accuracy
• Complete target enrichment in less than 6hr from only 50ng of gDNA
• From raw data to categorized mutations in 3 steps using SureCall data analysis software
Accelerated Solution
For Research Use Only. Not for use in diagnostic procedures.
1. Select the HaloPlexHS design workflow
2. Input gene ID/name/coordinate
3. Define regions of interest (eg. Exons, UTRs, etc)
4. Click “Start Design”5. 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.
How HaloPlexHS works
For Research Use Only. Not for use in diagnostic procedures.
The HaloPlexHS 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 capture
Amplicon tiling
Each 50ng DNA sample is fragmented in eight double-digest reactions
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For Research Use Only. Not for use in diagnostic procedures.
HaloPlex Others
TARGET TARGET
1. With HaloPlex each target base is covered by up to eight amplicons (different start and stop sites)!
2. If an unknown mutation appears in a restriction site, it may affect one or two fragments but all others will be present
3. If a variant occurs – it can be checked by multiple amplicons with HaloPlex
1. With other multiplex PCR based technologies, each target base is covered by only one amplicon (same start and stop sites)
2. If an unknown mutation appears in a primer site it causes a complete dropout in the target region
3. If a variant occurs, it is hard to know if it is a real mutations and not a PCR artifact
DNA variant
DNA variant
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Basics of HaloPlex technology – amplicon redundancy
For Research Use Only. Not for use in diagnostic procedures.
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.
DNA fragments are mixed with custom HaloPlex probes and primer cassettes containing the molecular barcodes.
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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.
Probe/fragment hybrids are ligated and retrieved with streptavidin magnetic beads, followed by high stringency wash.
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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.
Only fully circularized DNA targets are amplified on-bead.
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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.
Agenda
Introduction
How HaloPlexHS works
1
2
A flexible and accelerated solution
5
3 Performance data
For Research Use Only. Not for use in diagnostic procedures.
HaloPlexHS Performance - High Uniformity and Specificity
Uniform coverage of targeted bases: >95% covered at 10% of average depth
High Specificity: >80% on-target specificity
• important since deep sequencing is required for low frequency variant detection
9.9kb 48.1kb 142.7kb 260kb 1.8Mb 4.8Mb0%
10%
20%
30%
40%
50%
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90%
100%
HaloPlexHS sequencing performance
On-target specificity 10% of ave. depth Coverage at 10X Coverage at 20x
For Research Use Only. Not for use in diagnostic procedures.
HaloPlexHS Performance – Excellent coverage even with FFPE samples
5.8 3.8 3.6 1.9 2.1 8.2FFPE Sample
1FFPE Sample
2FFPE Sample
3FFPE Sample
4FFPE Sample
5Cell line
NA18507
0%
10%
20%
30%
40%
50%
60%
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80%
90%
100%
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HaloPlexHS performance with FFPE samples
Specificity bp (%) Coverage at 20x (%)Coverage at 100x (%) Average depth Seq Region
Cove
rage
Sequ
enci
ng D
epth
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.
HaloPlexHS Performance – Detection down to 0.5% variant allele frequency
• Detection of down to 0.5% allele frequency in HapMap dilutionsExpect
ed a
llele
fr
equency
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)
1789
1700
5
5515
2040
1521
2907
7
5522
9255
5523
8268
5524
0461
5524
2609
9224
4422
5342
42
5649
4991
6923
3215
6923
3716
6886
3314
3616
5041
chr3 chr4 chr6 chr7 chr7 chr7 chr7 chr7 chr11 chr12 chr12 chr12 chr16 chr21
0.0%
1.0%
2.0%
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Detection down to 0.5% allele frequency
5% 2.5% 1% 0.5%
For Research Use Only. Not for use in diagnostic procedures.
HaloPlexHS Performance – Detection down to 0.5% variant allele frequency Chr. Pos. 5% 2.5% 1% 0.5%
chr3:178917005 58 18 18 10
1062 746 1052 1280
chr4:55152040 113 27 30 12
2102 1391 1912 2602
chr6:152129077 219 69 43 26
3989 2682 3816 5143
chr7:55229255 60 27 7 7
883 757 998 1285
chr7:55238268 170 61 35 17
3155 2194 3142 4132
chr7:55240461 102 45 34 14
2337 1766 2444 3028
chr7:55242609 33 9 4 10
592 400 554 666
chr7:92244422 78 31 29 14
1562 1306 1738 2264
chr11:534242 201 63 32 14
3629 2391 3656 4952
chr12:56494991 74 26 13 12
1584 1136 1508 1802
chr12:69233215 120 50 36 12
2518 1885 2666 3309
chr12:69233716 31 16 8 3
734 637 818 979
chr16:68863314 14 8 6 2
233 265 365 394
chr21:36165041 8 5 1 2
117 88 128 175
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 variouschromosomal positions are highlighted (green)
For Research Use Only. Not for use in diagnostic procedures.
Simplify Data Analysis with SureCall
For Research Use Only. Not for use in diagnostic procedures.
Agenda
Introduction
How HaloPlexHS works
1
2
A flexible and accelerated solution
5
3 Performance data
For Research Use Only. Not for use in diagnostic procedures.
HaloPlexHS - 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.
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.
Summary
• HaloPlexHS Target Enrichment System is a high sensitivity method for the accurate identification of low allele frequency variants
• HaloPlexHS 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.