HaloPlex HS Get to Know Your DNA. Every Single Fragment. Kevin Poon, Ph.D. Sr. Global Product Manager Diagnostics & Genomics Group Agilent Technologies For Research Use Only. Not for Use in Diagnostic Purposes.
Agenda 1 2 Introduction How HaloPlex HS works 3 Performance data 4 A flexible and accelerated solution
From Discovery to Clinical Research DISCOVERY FOLLOW-UP CLINICAL RESEARCH Whole Exome Whole Genome GWAS Follow-up Exome Follow-Up WGAS Follow-up GWAS Clinical Research Panels
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
The need for sensitivity and accuracy
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
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
Molecular Barcodes Molecular barcodes are degenerate oligonucleotide sequences (10-16bp) attached to individual DNA molecules Allow for accounting of sequencer and PCR errors in high coverage NGS data Courtesy of Dr. Eric Duncavage (AGBT 2015)
Molecular Barcode Example Genomic DNA PCR product PCR product PCR product PCR product Sequencer Sequence Read Sequence Read Sequence Read Sequence Read Sequence Read Sequence Read Sequence Read Sequence Read Sequence Read Sequence Read Sequence Read Sequence Read Sequence Read Sequence Read Sequence Read Sequence Read Courtesy of Dr. Eric Duncavage (AGBT 2015)
Molecular Barcode Example Without Barcodes: VAFs 1.5% 3% 0.3% 0.9% 10% 1.1% With Barcodes we know all sequences descended from the same piece of DNA. Therefore a true base change should be Present in all reads with that barcode VAFs 10% Sequencer Sequence Read Sequence Read Sequence Read Sequence Read Sequence Read Sequence Read Sequence Read Sequence Read Sequence Read Sequence Read Sequence Read Sequence Read Sequence Read Sequence Read Sequence Read Sequence Read Courtesy of Dr. Eric Duncavage (AGBT 2015)
Basic molecular barcode analysis 1. Align reads Molecular barcode Random True error variant A T G Sample Index 2. Group read pairs to designed probes based on read start-stop position Barcode family C A T T 3. For each probe: group reads with identical molecular barcode sequence Group reads with the same molecular barcode 4. Consolidate read information to one read per molecule (remove PCR duplicates) Consensus read T
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
Agenda 1 2 Introduction How HaloPlex HS works 3 Performance data 4 A flexible and accelerated solution
HaloPlex HS High Sensitivity Target Enrichment Key Features : o o o o o 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
Key Benefits Unparalleled Sensitivity Confidently detect mutations present at below 1% frequency in genetically heterogeneous samples Preserve your tissue: only 50ng of gdna, compatible with FFPE samples Superior Accuracy Differentiation of true variants from PCR or sequencing errors Differentiation of formalin fixation artifacts by targeting both DNA strands Accelerated Solution Have sequencable libraries in <1 day automation available No need to shear! Less equipment & less hassle. Easy analysis from raw data to categorized mutations in 3 steps using SureCall software
SureDesign Create a custom design in minutes 1. Select the HaloPlex 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
How HaloPlex HS works
The HaloPlex HS Workflow 1 Each 50ng 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
Basics of HaloPlex technology amplicon redundancy 3 TARGET DNA variant 2 3 TARGET DNA variant 2 1 1 HaloPlex 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 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
Increased Confidence in Mutation Calling Amplicon redundancy provides excellent coverage. 0001111122322222333455422221111222111110000 Read coverage Genomic region Target
2 DNA fragments are mixed with custom HaloPlex HS 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
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
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 <6hr!
Agenda 1 2 Introduction How HaloPlex HS works 3 Performance data 4 A flexible and accelerated solution
HaloPlex HS Performance: High Uniformity & Specificity Across Wide Design Range HaloPlex HS sequencing performance 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% 9.9kb 48.1kb 142.7kb 260kb 1.8Mb 4.8Mb On-target specificity Coverage at 10X Coverage at 20x Uniform coverage of targeted bases: >95% covered at 10X (Normalized to 200X) High Specificity: >80% on-target specificity Important since deep sequencing is required for low frequency variant detection
Coverage Sequencing Depth HaloPlex HS Performance Excellent coverage even with FFPE samples HaloPlex HS performance with FFPE samples 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 600 500 400 300 200 100 DIN 0% 5.8 3.8 3.6 1.9 2.1 8.2 0 FFPE Sample 1 FFPE Sample 2 FFPE Sample 3 FFPE Sample 4 FFPE Sample 5 Cell line NA18507 Specificity bp (%) Coverage at 20x (%) Coverage at 100x (%) Average depth Seq Region 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.
- HaloPlex & HaloPlexHS have the option to optimize a design for FFPE samples - Selection of smaller fragment lengths - Targeting both strands for each target fragment - Designs exhibit lower false-positive errors caused by FFPEinduced artifacts and lower detection rate of somatic mutations The Journal of Molecular Diagnostics, Vol. 17, No. 6, November 2015
178917005 55152040 152129077 55229255 55238268 55240461 55242609 92244422 534242 56494991 69233215 69233716 68863314 36165041 Expected allele frequency HaloPlex HS Performance Detection down to 0.5% variant allele frequency Detection of down to 0.5% allele frequency in HapMap dilutions Detection down to 0.5% allele frequency 8.0% 7.0% 6.0% 5.0% 4.0% 3.0% 2.0% 1.0% 0.0% chr3 chr4 chr6 chr7 chr7 chr7 chr7 chr7 chr11 chr12 chr12 chr12 chr16 chr21 5% 2.5% 1% 0.5% 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 HaloPlex HS for low frequency variant detection. Data shown is representative of replicates (sequencing depth = 2000x 4000x)
HaloPlex HS : Published study of somatic mosaicism Tumors associated with DICER1 Syndrome Deep sequencing of gdna from, blood, tumor and normal tissue Detected RNaseIIIb mutations at 0.2 13% frequency in constitutional DNA Agilent Webinar Available: Dr. Ioannis Ragoussis Mosaic DICER1 RNaseIIIb missense mutations important genetic cause of DICER1 syndrome de Kock L, et al. J Med Genet 2015;0:1 10. doi:10.1136/jmedgenet-2015-103428 JMG Online First, published on October 16, 2015
Simplify Data Analysis with SureCall
Agenda 1 2 Introduction How HaloPlex HS works 3 Performance data 4 A flexible and accelerated solution
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
HaloPlex HS Automation Available Bravo - NGS Option A Validated protocol ready-for-use Up to 96-samples multiplexed
Accelerate Time to Results Prepare Sequence Analyze Prepare DNA libraries in <6hr 1 2 Day 1 Begin sequencing on a desktop sequencer Day 2 Analyze your data
Made-to-Order Catalog Custom Ordering Information HaloPlex HS 16-rxn 48-rxn 96-rxn HaloPlexHS 1-250kb, ION G9932C G9932B HaloPlexHS 1-500kb, ILM G9931C G9931B HaloPlexHS 2.6Mb - 5Mb, ILM G9951C G9951B HaloPlexHS 251kb - 2.5Mb, ION G9942C G9942B HaloPlexHS 501kb - 2.5Mb, ILM G9941C G9941B ClearSeq AML, HS, ILM G9963A G9963B ClearSeq AML, HS, ION G9964A G9964B ClearSeq Cancer, HS, ILM G9933A G9933B ClearSeq Cancer, HS, ION G9934A G9934B ClearSeq Cardiomyopathy, HS, ILM G9943A G9943B ClearSeq Cardiomyopathy, HS, ION G9944A G9944B ClearSeq Arrhythmia HS, ILM G9954C G9954B ClearSeq Arrhythmia HS, ION G9954C G9954B ClearSeq Chromosome X HS, ILM G9954C G9954B ClearSeq Connective Disorder HS, ILM G9954C G9954B ClearSeq Connective Disorder HS, ION G9954C G9954B ClearSeq ICCG HS, ILM G9954C G9954B ClearSeq ICCG HS, ION G9954C G9954B ClearSeq Noonan Syndrome HS, ILM G9954C G9954B ClearSeq Noonan Syndrome HS, ION G9954C G9954B
Summary HaloPlex HS Target Enrichment System is a high sensitivity method for the accurate identification of low allele frequency variants using molecular barcode technology HaloPlex HS performs with high coverage, on-target specificity and with a sensitivity that allow detection of alleles down to below 1% allele frequency HaloPlex HS allows users to perform powerful analysis with SureCall, enabling quick starts via wizards, easy exploration through intuitive interfaces, and high productivity through customizable report generation HaloPlex HS is ideally suited for cancer research and studies that involve the detection of somatic variants in heterogeneous samples
Thank you! Questions? HaloPlex HS NGS Target Enrichment System Get to Know Your DNA. Every Single Fragment. For Research Use Only. Not for use in