HaloPlexHS Get to Know Your DNA. Every Single Fragment.

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Presentation transcript:

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.

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

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.

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.

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

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 procedures.

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 procedures.

Basic molecular barcode analysis T 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.

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.

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

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.

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.

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 www.agilent.com/genomics/suredesign 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 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.

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.

Amplicon redundancy provides excellent coverage. Increased Confidence in Mutation Calling Amplicon redundancy provides excellent coverage. 0001111122322222333455422221111222111110000 Read coverage Genomic region Target 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. 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.

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.

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.

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

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.

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.

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)

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

Agenda Introduction How HaloPlexHS works Performance data 1 Introduction 2 How HaloPlexHS works 3 Performance data 5 A flexible and accelerated solution 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 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.

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.