Setting Up Copy Number Variation Assays

Slides:

Advertisements

Similar presentations

Agilent’s MX QPCR Software Tutorial Field Application Scientist

Advertisements

REAL TIME PCR ………A step forward in medicine

Droplet digital PCR Overview and applications

RNA Lab (Isolation, quantification and qPCR analysis) MCB7300.

Real-Time PCR mRNA quantification. What do mRNA levels tell us? DNA  mRNA  protein Reflect level of gene expression Information about cell response.

Data analytical issues with high-density oligonucleotide arrays A model for gene expression analysis and data quality assessment.

PCR quantitative en temps réel Lydie Pradel. PCR.

Carolyn Dunn, Annabel Whibley, Lionel Willatt and Ingrid Simonic

Results Interpretation and Database Management

Analysis of Gene Expression - Overview -

Dr. Chaim Wachtel Introduction to PCR and qPCR Part II: PCR!!

What Can You Do With qPCR?

Q-PCR Bige Vardar

COBAS TaqMan 48 Sales Brochure

The polymerase chain reaction (PCR) rapidly

Principles and Important Considerations

Real Time PCR = Quantitative PCR.

Real-Time PCR (Quantitative PCR)

Quantitative PCR Session 2: Overview of qPCR

Real-Time Quantitative RT-PCR

DNA-based Methods for Quantifying Microbes in Atmospheric Samples Tom Hill, Helen Ahern and Bruce Moffett University of East London.

Dr. Sumbul Fatma Department of Medical Biochemistry.

Real time RT-PCR Quantitating Gene Expression.

Quantification of RNA by real-time PCR

Chapter 13: DNA Quantitation.  Quantitation determines the amount of human DNA present in an extract  A narrow concentration range is required to “seed”

qPCR SNAPSHOTS LIVE MAY 13, 2003.

Real-Time Quantitative PCR Basis

Results 1 comparison for 5 systems containing the SNP at postion 1 to 5 of the up-stream probe  systems 2-5 work well, system 3 offers most stable & reliable.

Molecular Testing and Clinical Diagnosis

From the Seed Sample to DNA II: DNA Isolation, Quantification, & Normalization Beni Kaufman.

Droplet Digital PCR Competition Overview

Chapter 13: DNA Quantitation.  Determining the amount of DNA is a sample is essential  A narrow concentration range is required for PCR  Must use human-specific.

Promega Corporation ©2014 Promega Corporation. Overview of Promega's Science and Business June 2015 Tom Livelli, VP Life Sciences.

Good qPCR The Necessary and the Reasonable

QUANTITATIVE ANALYSIS OF AFRICAN SWINE FEVER VIRUS BY DIGITAL PCR

Introduction to GENDX HLA typing products

Synthetic Circulating Cell-free DNA as Quality Control Materials for Somatic Mutation Detection in Liquid Biopsy for Cancer R. Zhang, R. Peng, Z. Li, P.

WP2 – ddPCR and microRNA C.Divieto, INRIM, Torino, Italy.

Principles of Real-Time Quantitative PCR Techniques

Israel maritime college

Multiplexed Elimination of Wild-Type DNA and High-Resolution Melting Prior to Targeted Resequencing of Liquid Biopsies I. Ladas, M. Fitarelli-Kiehl, C.

Assistant Prof. Dr. Nibras Saleam Al-Ammar PhD in Clinical Immunology

John P. Jakupciak, Wendy Wang, Peter E

PCR uses polymerases to copy DNA segments.

Reverse Complement PCR: fast, low cost amplicon based NGS

Using Galaxy for Molecular Assay Design

Title Detection of HLA-B*58:01 with TaqMan assay and its association with allopurinol-induced sCADR.

Single-Color Digital PCR Provides High-Performance Detection of Cancer Mutations from Circulating DNA Christina Wood-Bouwens, Billy T. Lau, Christine.

Gene quantification using real-time quantitative PCR

Elena Castellanos-Rizaldos, Cloud Paweletz, Chen Song, Geoffrey R

Development and Clinical Utility of a Blood-Based Test Service for the Rapid Identification of Actionable Mutations in Non–Small Cell Lung Carcinoma

Accurate Quantification of T Cells by Measuring Loss of Germline T-Cell Receptor Loci with Generic Single Duplex Droplet Digital PCR Assays Willem H.

Noninvasive prenatal diagnosis of hemophilia by microfluidics digital PCR analysis of maternal plasma DNA by Nancy B. Y. Tsui, Rezan A. Kadir, K. C. Allen.

PCR uses polymerases to copy DNA segments.

PCR uses polymerases to copy DNA segments.

Droplet Digital PCR Is a Reliable Tool for Monitoring Minimal Residual Disease in Acute Promyelocytic Leukemia Claudia Brunetti, Luisa Anelli, Antonella.

Highly Sensitive Droplet Digital PCR Method for Detection of EGFR-Activating Mutations in Plasma Cell–Free DNA from Patients with Advanced Non–Small Cell.

Figure 1. Plasma next-generation sequencing (NGS) assay workflow, comparison of variant allelic fraction with ... Figure 1. Plasma next-generation sequencing.

PCR uses polymerases to copy DNA segments.

Low Incidence of Minor BRAF V600 Mutation-Positive Subclones in Primary and Metastatic Melanoma Determined by Sensitive and Quantitative Real-Time PCR

Dale Muzzey, Shera Kash, Jillian I. Johnson, Laura M

Luigi Warren, David Bryder, Irving L. Weissman, and Stephen R. Quake

Development and Clinical Utility of a Blood-Based Test Service for the Rapid Identification of Actionable Mutations in Non–Small Cell Lung Carcinoma

PCR uses polymerases to copy DNA segments.

PCR uses polymerases to copy DNA segments.

Bio-Rad Overview and Statement of Interests

Rapid pathogen-specific phenotypic antibiotic susceptibility testing using digital LAMP quantification in clinical samples by Nathan G. Schoepp, Travis.

PCR uses polymerases to copy DNA segments.

Presentation transcript:

Setting Up Copy Number Variation Assays Droplet Digital PCR Setting Up Copy Number Variation Assays Ben Bunce

Outline Droplet Digital PCR (ddPCR) overview Applications Implementation of a new workflow Requirements Challenges Solutions Case studies X-LAG tNGS confirmations Summary

ddPCR Principle Standard PCR qPCR ddPCR Quantitation of an end product in a bulk reaction qPCR Quantitation of product in real time in a bulk reaction ddPCR Bulk reaction separated into smaller partitions. Each partition intended to contain zero or one copy of DNA. The fraction of positive partitions is fitted to a Poisson distribution to determine the absolute starting copy number of DNA.

ddPCR Principle: Process Bio-Rad QX200 System

Bio-Rad Quantasoft 1D Amplification Channel One Channel Two

Bio-Rad Quantasoft Concentration Channel 1 concentration

Bio-Rad Quantasoft Events Total droplets Channel 1 droplets

ddPCR Applications Detection of Single Nucleotide Variants (SNV) Utilise TaqMan-type hydrolysis probes Greater statistical power for low level detection (ctDNA, cfDNA, transcript abundance) Absolute quantitation possible without standard curves Detection of gene dosage/CNV Utilise either dye-binding (external reference) or hydrolysis probes (internal reference) comparison of positive partitions for target and reference allow gene dosage calculations absolute quantitation possible without standard curves Phasing of variants

Integration: Requirements Exporting Quantasoft Setup File Bio-Rad QX200 System Quantasoft Analysis Package Import patient results

Challenges Quantasoft Setup File Patient demographics Primers/Probes Reagents Experiment type Results Import File Patient demographics Quality control data Assay results Software derived Externally calculated Interpretation of numerical results Manual entry Modify an existing LIMS export function Commission LIMs provider to create new feature Quantasoft output – “example” Contains redundant information Hydrolysis probe assay results are calculate within the software (internal reference) i.e. Duplex probes. EvaGreen assays use an external and can’t be calculated within the software. Requires outside calculating. All data is numerical – requires interpretation Quality data, pass and fail samples - OK for a few samples, but for the possibility of larger batches in the future it is more prudent to automate this process too. Consistency between users. Manual entry Modify existing LIMS export function Commission LIMS to create new feature

Challenge: Importing results 63 columns of data

Solution: Importing results 15 columns of data

Minor Challenges Technical challenges DNA quantification Increased automation Transitioning from each step of implementation Informatics challenges Networking

High throughput ddPCR copy number assay services Solution: Importing results Modified Quantasoft output Quantasoft Analysis Package Quantasoft Setup File High throughput ddPCR copy number assay services Bio-Rad QX200 System

Case Study 1: X-Linked Acrogiganitsm

Case Study 1: X-Linked Acrogiganitsm Positive female Positive male Normal female Normal males

Case Study 2: tNGS confimations CDC73 exon 8 duplication CDC73 exon 8 primer Reference primer

Summary QX200 system - a replacement for current qPCR and dosage techniques? Increased sensitivity and statistical robustness Workflow implementation Underestimating IT project time frames Utilising staff with additional skills (e.g. advanced knowledge of Excel) Communicating concepts clearly between clinical, technical and bioinformatic staff Any questions?