Agilent Technologies, Inc. · NYSE:A
Agilent Technologies — Technical Presentation (March 2014)
28 slides from Agilent Technologies's technical presentation, Mar 5, 2014
Slide 1: eSeminar: Diseño de un nuevo test NGS para cáncer hereditario
eSeminar: Diseño de un nuevo test NGS para el análisis rutinario de cáncer hereditario 5 marzo 2014 Ponente Invitado Dr. Alberto Acedo Amparo Dávila Co-fundador y Dr. Técnico Especialista Producto Genómica para Iberia AC-Gen Reading Life S.L. Agilent Technologies HaloPlex 1 For research use only
Slide 2: The NGS Applications Workflow
The NGS Applications Workflow DISCOVERY FOLLOW-UP CLINICAL Whole Exome Follow-up Exome Clinical Research Whole Genome Follow-Up WGS Panels GWAS Follow-up GWAS HaloPlex For research use only
Slide 3: The advantages with Traditional PCR
The advantages with Traditional PCR High Coverage High Specificity Fast Cost Effective Simple Well established HaloPlex For research use only
Slide 4: The problems with Multiplex PCR
The problems with Multiplex PCR Primer Cross reactivity Limited in # target regions Long optimization Hard to change Dropouts Artifacts HaloPlex For research use only
Slide 5: HaloPlex Workflow: A Simple & Fast 4-step protocol
HaloPlex Workflow A Simple & Fast 4-step protocol Sample is fragmented using restriction 1. Digest DNA enzymes 1 Probe library is added and hybridized 2. Hybridize probes to the targeted fragments making them 2 form a Halo shape. Probe/Fragment hybrids are retrieved 3. Purify and ligate with magneti
Slide 6: Molecular Concept: Digest DNA
Molecular Concept Each DNA sample is fragmented in eight 1. Digest DNA double-digest reactions Design Wizard - In Silico digestion and HaloPlex probe design Define region of interest (ROI) Genome digested in silico Fragments chosen for enrichment HaloPlex For research use only
Slide 7: Molecular Concept: Amplicon tiling
Molecular Concept Each DNA sample is fragmented in eight 1. Digest DNA double-digest reactions Amplicon tiling Amplicon tiling improves coverage by design Amplicon redundancy reduces risk of loosing completeness if a probe fails; protects against primer site mutations Specificity of the restri
Slide 8: Molecular Concept: Hybridize probes
Molecular Concept Fragments are mixed with custom 2. Hybridize probes HaloPlex probes and a Primer Cassette. Hybridization overnight. Red parts – Hybridization guides circularization complementary to the targeted fragment Same hybridization requirement as in regular PCR – high specificity! Grey pa
Slide 9: Molecular Concept: Purify and ligate
Molecular Concept Probe/Fragment hybrids are retrieved 3. Purify and ligate with magnetic streptavidin beads. The targets circular molecules are then closed by a DNA ligase. Purification and DNA Ligation Level of enrichment is increased by removal of non-targeted fragments Only perfectly hybridi
Slide 10: Molecular Concept: Amplify targeted fragments
Molecular Concept Only circular DNA targets are 4. Amplify targeted amplified. Sample barcodes are introduced. Final product is ready for fragments sequencing 1) Bridge PCR primer Thousands of amplicons – one 2) Sequencing primer primer pair 2 PCR is primed from the Primer 1 Cassette Tailed prim
Slide 11: HaloPlex Technology Overview
HaloPlex Technology Overview Eight different fragments cover each target position. Amplicons are tiled over the region. Genomic region Target exon HaloPlex For research use only
Slide 12: HaloPlex Technology Overview
HaloPlex Technology Overview A subset covering the target region is selected using HaloPlex probes More than one different fragment cover the target region Genomic region Target exon HaloPlex For research use only
Slide 13: HaloPlex Technology Overview
HaloPlex Technology Overview HaloPlex baits guide the circularization Genomic region Target exon HaloPlex For research use only
Slide 14: HaloPlex Technology Overview
HaloPlex Technology Overview The selected subset is equipped with sequencing primer sequences by the selector probes. 1 2 1 1) library bridge PCR primer 2)Sequencing primer+Sample barcode Genomic region Target exon HaloPlex For research use only
Slide 15: HaloPlex Technology Overview
HaloPlex Technology Overview The sequencing primers are used to sequence in from the fragment ends (paired-end sequencing). Genomic region Target exon HaloPlex For research use only
Slide 16: HaloPlex Technology Overview
HaloPlex Technology Overview 0001111122322222333455422221111222111110000 Read coverage Genomic region Target exon HaloPlex For research use only
Slide 17: HaloPlex Technology Overview
HaloPlex Technology Overview By tiling amplicons high target coverage is achieved 0001111122322222333455422221111222111110000 Genomic region Target exon HaloPlex For research use only
Slide 18: What makes HaloPlex better than other PCR methods
What makes HaloPlex better than other PCR methods! HaloPlex Others (TruSeq, AmpliSeq, Fluidigm, RainDance) TARGET TARGET With HaloPlex each target base is With other multiplex PCR based covered by multiple amplicons technologies, each target base is (different start and stop sites)! covered by only
Slide 19: What makes HaloPlex better than other PCR methods
What makes HaloPlex better than other PCR methods! HaloPlex Others (TruSeq, AmpliSeq, Fluidigm, RainDance) DNA variant DNA variant TARGET TARGET If a variant occurs – it can be If a variant occurs, it is hard to checked by multiple amplicons know if it is a real mutations and with HaloPlex not a PCR
Slide 20: What makes HaloPlex better than other PCR methods
What makes HaloPlex better than other PCR methods! HaloPlex Others (TruSeq, AmpliSeq, Fluidigm, RainDance) TARGET TARGET If an unknown mutation appears If an unknown mutation appears in a restriction site, it may affect in a primer site it causes a one or two fragments but all complete dropout in th
Slide 21: Consistent Results among Different Design Sizes
Consistent Results among Different Design Sizes Extremely high Performance 100% 90% 80% 70% 60% 4.4K 50% 21Kb 60Kb 40% 120K 30% 280K 20% 396K 10% 0% Covered by % Bases on % Bases % Bases % Bases % Bases % Bases design Target covered 1x covered 20x covered 100x covered at 10% covered at 20% of mean o
Slide 22: Don't limit yourself: thousands of amplicons
Don’t limit yourself: thousands of amplicons How many 100% amplicons are 90% amplified in a 80% single tube? 70% 60% ¡73,328 amplicons! 50% 1.5 Mb 3.2 Mb ¡100,550 amplicons! 40% 30% 5.0 Mb ¡193,775 20% amplicons! 10% 0% Coverage by Specificity Coverage 1x Coverage at design 10% of mean 25
Slide 23: Publications
Publications HaloPlex Page 26 For research use only
Slide 24: SureDesign: Create your design in only 10 minutes
SureDesign Create your design in only 10 minutes https://earray.chem.agilent.com/suredesign/ 1. Input gene ID/name/coordinate 2. Define regions of interest (eg. Exons, UTRs, etc) 3. Click “Start Design” 4. Receive design report in 10 minutes HaloPlex For research use only
Slide 25: FFPE Samples Using HaloPlex
FFPE Samples Using HaloPlex FFPE Challenges • DNA quality issues (age, storage, fixation process) • Sample degradation can impact sequencing quality (sensitivity, quality, allelic balance, etc) HaloPlex with FFPE samples • Validated protocol • Adapted Design for FFPE • Shorter probes • Both DNA stra
Slide 26: Designed for FFPE compatibility
Designed for FFPE compatibility Standard design FFPE design Target Region Target Region Exon Exon Normally fragments in the For FFPE samples shorter fragments range of 100-400bp are used (50-400bp) are included HaloPlex For research use only
Slide 27: Designed for FFPE compatibility
Designed for FFPE compatibility Probes are designed for both target fragment polarities • Improves capture efficiency • Eliminate strand specific FFPE artifacts DNA TARGET FRAGMENT AntiSense Sense Strand Strand One probe is targeting the sense and one the anti-sense strand of the target DNA HaloPlex
Slide 28: Thank You!
Muchas gracias por su atención Dirección de contacto: amparo_davila@agilent.com Thank You! HaloPlex For research use only
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