Halo Plex HS Get to Know Your DNA

Halo. Plex. HS Get to Know Your DNA. Every Single Fragment .

Agenda 1 Introduction 2 How Halo. Plex. HS works 3 Performance data 5 A flexible and accelerated solution For Research Use Only. Not for use in diagnostic procedu

From Discovery to Clinical Research DISCOVERY § § § FOLLOW-UP § Follow-up Exome Whole Exome § Follow-Up Whole Genome WGAS GWAS § Follow-up GWAS CLINICAL RESEARCH § Clinical Research Panels For Research Use Only. Not for use in diagnostic procedu

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 procedu

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

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: 1432 -1438 For Research Use Only. Not for use in diagnostic procedu

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 100 -1000 sequenced bases) Kennedy et al (2014) Nature Protocols 9: 2586 - 2606 Requires molecular barcodes for increased sensitivity and accuracy For Research Use Only. Not for use in diagnostic procedu

Basic molecular barcode analysis Random True error variant Molecular barcode 1. 2. Align reads 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) Barcode family C A T Sample Index G T Group reads with the same molecular barcode Consensus read T For Research Use Only. Not for use in diagnostic procedu

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

Agenda 1 Introduction 2 How Halo. Plex. HS works 3 Performance data 5 A flexible and accelerated solution For Research Use Only. Not for use in diagnostic procedu

Introducing Halo. Plex. HS – High Sensitivity Next Gen PCR Key Features : o o o More than a million unique 10 nt molecular barcodes are incorporated into DNA library fragments Requires only 50 ng starting DNA input Rapid workflow : From sample to sequencing-ready libraries in <6 hr Compatible with FFPE samples More sensitive and accurate than other conventional NGS TE methods For Research Use Only. Not for use in diagnostic procedu

Key Benefits Unparalleled Sensitivity Superior Accuracy Accelerated Solution • 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 • Differentiation of true variants from PCR or formalin fixation artifacts by targeting both DNA strands • Complete target enrichment in less than 6 hr from only 50 ng of g. DNA • From raw data to categorized mutations in 3 steps using Sure. Call data analysis software For Research Use Only. Not for use in diagnostic procedu

Sure. Design – Create a custom design in minutes 1. Select the Halo. Plex. HS 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 For Research Use Only. Not for use in diagnostic procedu

How Halo. Plex. HS works For Research Use Only. Not for use in diagnostic procedu

The Halo. Plex. HS Workflow 1 Each 50 ng DNA sample is fragmented in eight double-digest reactions 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 procedu

Basics of Halo. Plex technology – amplicon redundancy DNA variant 2 2 3 TARGET 3 1 1 Halo. Plex 1. With Halo. Plex 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 Halo. Plex Others 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 For Research Use Only. Not for use in diagnostic procedu

Increased Confidence in Mutation Calling Amplicon redundancy provides excellent coverage. 000111112232222233345542222111110000 Read coverage Genomic region Target For Research Use Only. Not for use in diagnostic procedu

2 DNA fragments are mixed with custom Halo. Plex probes and primer cassettes containing the molecular barcodes. 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 procedu

3 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 procedu

4 Only fully circularized DNA targets are amplified on -bead. PCR amplification § Thousands of different amplicons, one primer pair § On-bead PCR of ligated fragments simplifies workflow Ready for sequencing in <6 hr! For Research Use Only. Not for use in diagnostic procedu

Agenda 1 Introduction 2 How Halo. Plex. HS works 3 Performance data 5 A flexible and accelerated solution For Research Use Only. Not for use in diagnostic procedu

Halo. Plex. HS Performance - High Uniformity and Specificity Halo. Plex. HS sequencing performance 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% 9. 9 kb 48. 1 kb On-target specificity 142. 7 kb 10% of ave. depth 260 kb 1. 8 Mb Coverage at 10 X 4. 8 Mb Coverage at 20 x 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 procedu

Halo. Plex. HS Performance – Excellent coverage even with FFPE samples Halo. Plex. HS performance with FFPE samples 100% 600 90% Coverage 70% 400 60% 50% 300 40% 200 30% 20% Sequencing Depth 500 80% 100 10% 0% DIN 0 5. 8 3. 6 1. 9 2. 1 8. 2 FFPE Sample. FFPE 1 Sample. FFPE 2 Sample. FFPE 3 Sample. FFPE 4 Sample Cell 5 line NA 18507 Specificity bp (%) Coverage at 20 x (%) Coverage at 100 x (%) Average depth Seq Region Excellent coverage of target bases (>90% covered at 100 x) 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 g. DNA and completely degraded g. DNA respectively. For Research Use Only. Not for use in diagnostic procedu

Halo. Plex. HS Performance – Detection down to 0. 5% variant allele frequency • Detection of down to 0. 5% allele frequency in Hap. Map dilutions Expected allele frequency Detection down to 0. 5% allele frequency 8. 0% 7. 0% 6. 0% 5. 0% 4. 0% 3. 0% 2. 0% 1. 0% 0. 025 0. 01 1 04 4 16 5 36 31 6 86 3 68 71 5 21 1 99 42 4 chr 7 23 3 chr 7 69 chr 7 49 4 chr 6 56 chr 4 53 chr 3 0. 05 2 2 42 9 92 24 4 55 24 2 60 1 46 8 55 24 0 26 55 23 8 25 55 22 9 90 21 2 5 77 0 04 15 15 2 55 17 89 1 70 05 0. 0% chr 11 chr 12 chr 16 chr 21 0. 005 Hap. Map cell lines, NA 18507 and NA 10831, 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 Halo. Plex. HS for low frequency variant detection. Data shown is representative of replicates (sequencing depth = 2000 x – 4000 x) For Research Use Only. Not for use in diagnostic procedu

Halo. Plex. HS Performance – Detection down to 0. 5% variant allele frequency Number of total unique reads covering the target region The unique reads covering each allele fraction at the va chromosomal positions are highlighted (green) For Research Use Only. Not for use in diagnostic procedu

Simplify Data Analysis with Sure. Call For Research Use Only. Not for use in diagnostic procedu

Agenda 1 Introduction 2 How Halo. Plex. HS works 3 Performance data 5 A flexible and accelerated solution For Research Use Only. Not for use in diagnostic procedu

Halo. Plex. HS - a flexible solution ü Compatible with both ILM and ION PGM platforms ü Create custom designs up to 5 Mb (2. 5 Mb for ION) ü NGS Disease Research Panels are available in catalog or made-to-order format ü Multiplex up to 96 samples for ILM and 16 samples for ION For Research Use Only. Not for use in diagnostic procedu

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

Summary • Halo. Plex. HS Target Enrichment System is a high sensitivity method for the accurate identification of low allele frequency variants • Halo. Plex. HS incorporates unique molecular barcodes into each DNA library fragment • Halo. Plex. HS 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 procedu