1. HaloPlexHS Get to Know Your DNA. Every Single Fragment.
Presentation Title
4/24/2017
Confidentiality Label
HaloPlexHS Get to Know Your DNA. Every Single Fragment.

2. Agenda Introduction How HaloPlexHS works Performance data1
Introduction 2
How HaloPlexHS works 3
Performance data 5
A flexible and accelerated solution
For Research Use Only.
Not for use in diagnostic procedures.

3. From Discovery to Clinical Research
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.

4. Requirements of clinical research
FOLLOW-UP
CLINICAL RESEACH
Clinical research applications require:
Fast turnaround time
Flexibility in capture size
Simple workflow
High coverage
High accuracy in variant detection
Data analysis solution
For Research Use Only.
Not for use in diagnostic procedures.

5. The need for sensitivity and accuracy
For Research Use Only.
Not for use in diagnostic procedures.

6. Low allele frequency variants
What are low allele frequency variants?
Variants present at a frequency below 3%
What are low allele frequency variants implicated in?
Clonal evolution and pathogenesis
Tumor subclonal heterogeneity
Immunological diversity
Adapted from Stead et al (2013) Human Mutation 34:
For Research Use Only.
Not for use in diagnostic procedures.

7. Low allele frequency variants
Low allele frequency variants are difficult to detect by conventional NGS methods
Relatively high error rate of sequencers (1 wrong base call in sequenced bases)
Kennedy et al (2014) Nature Protocols 9:
Requires molecular barcodes for increased sensitivity and accuracy
For Research Use Only.
Not for use in diagnostic procedures.

8. Basic molecular barcode analysisT A C G
Group reads with the same molecular barcode
Barcode family
Consensus read
True variant
Random error
Molecular barcode
Sample Index
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)
For Research Use Only.
Not for use in diagnostic procedures.

9. Benefits of molecular barcode analysis
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
For Research Use Only.
Not for use in diagnostic procedures.

10. Agenda Introduction How HaloPlexHS works Performance data1
Introduction 2
How HaloPlexHS works 3
Performance data 5
A flexible and accelerated solution
For Research Use Only.
Not for use in diagnostic procedures.

11. Introducing HaloPlexHS – High Sensitivity Next Gen PCR
Key Features :
More than a million unique 10nt molecular barcodes are incorporated into DNA library fragments
Requires only 50ng starting DNA input
Rapid 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.

12. Unparalleled Sensitivity
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.

13. SureDesign – Create a custom design in minutes
Select the HaloPlexHS design workflow
Input gene ID/name/coordinate
Define regions of interest (eg. Exons, UTRs, etc)
Click “Start Design”
Design report in 10 minutes
For Research Use Only.
Not for use in diagnostic procedures.

14. How HaloPlexHS works For Research Use Only.
Not for use in diagnostic procedures.

15. The HaloPlexHS Workflow
Each 50ng DNA sample is fragmented in eight double-digest reactions 1
Amplicon tiling
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
For Research Use Only.
Not for use in diagnostic procedures.

16. Basics of HaloPlex technology – amplicon redundancy
DNA variant
DNA variant 2 2 3 3
TARGET
TARGET 1 1
HaloPlex
Others
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
For Research Use Only.
Not for use in diagnostic procedures.

17. Amplicon redundancy provides excellent coverage.
Increased Confidence in Mutation Calling
Amplicon redundancy provides excellent coverage.
Read coverage
Genomic region
Target
For Research Use Only.
Not for use in diagnostic procedures.

18. DNA fragments are mixed with custom HaloPlex probes and primer cassettes containing the molecular barcodes. 2
Hybridization
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
For Research Use Only.
Not for use in diagnostic procedures.

19. 3 Ligation, capture and wash
Probe/fragment hybrids are ligated and retrieved with streptavidin magnetic beads, followed by high stringency wash.
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.

20. 4 PCR amplification Ready for sequencing in <6hr!
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.

21. Agenda Introduction How HaloPlexHS works Performance data1
Introduction 2
How HaloPlexHS works 3
Performance data 5
A flexible and accelerated solution
For Research Use Only.
Not for use in diagnostic procedures.

22. 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
For Research Use Only.
Not for use in diagnostic procedures.

23. HaloPlexHS 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.

24. HaloPlexHS Performance – Detection down to 0
HaloPlexHS Performance – Detection down to 0.5% variant allele frequency
Detection of down to 0.5% allele frequency in HapMap dilutions
Expected 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.

25. HaloPlexHS Performance – Detection down to 0
HaloPlexHS Performance – Detection down to 0.5% variant allele frequency
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.
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)

26. Simplify Data Analysis with SureCall
For Research Use Only.
Not for use in diagnostic procedures.

27. Agenda Introduction How HaloPlexHS works Performance data1
Introduction 2
How HaloPlexHS works 3
Performance data 5
A flexible and accelerated solution
For Research Use Only.
Not for use in diagnostic procedures.

28. 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.

29. Accelerate Time to Results
Prepare
Sequence
Analyze 1 2
Day 1
Day 2
Prepare DNA libraries in <6hr
Begin sequencing on a desktop sequencer
Analyze your data
For Research Use Only.
Not for use in diagnostic procedures.

30. 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.