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qPCR
dPCR
& NGS Application Workshops
The workshops
are aimed at giving participants a deep and objective understanding of
real-time quantitative PCR, digital PCR, Next-Generation Sequencing
(NGS), biostatistics, expression profiling, and their applications in
various fields of molecular diagnostics. The courses are intended for
academic or industrial researchers considering working with qPCR, dPCR
or NGS or scientists currently working with these technologies who seek
a deeper understanding. All workshops offer extensive hands-on training
by experts in the field on Thu
21st to Fri 22nd March 2019.
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The
workshop laboratories and seminar rooms are very close to the
central lecture hall.
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The workshops are
hosted by:
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In the qPCR data
analysis workshop -- Analysis of Gene Expression Data -- the data
conversion, normalisation procedure,
biostatistical calculations and the expression profiling will be done
with the newest GenEx software by MultiD.
You can download a free trial version
from our webpage => Genex.gene-quantification.info
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The
courses cover all aspects in qPCR and take two days to complete. Each
course is app. 50% hands-on and is limited to app. 20 participants,
resulting in very interactive teaching where everybody is given the
opportunity to try the instrumentation.
After the course, participants will be able to plan and perform qPCR
experiments themselves, as well as interpret and analyze data. Detailed
course material and full catering (lunch, coffee, soft drinks and
snacks) are included in the course fee.
Course
dates
21st and 22nd March 2019
Course starts
at 9
am
Course ends
around 5 pm
Please
register for the Symposium & Workshops using the ConfTool
registration platform => Registration.qPCR-dPCR-NGS-2019.net
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Workshop short agendas:
Basic
real-time
qPCR Application Workshop
(2-days) hosted
by TATAA
Biocenter
Seminar room
-- S2
Description.
This is a basic real-time qPCR course for
people starting with qPCR or
is working with the technique and want to understand it deeper. The
course starts with describing how the technology works, different types
of detection technologies and instruments. Then you will learn how to
design primers and probes and optimize new assays. You will get an
understanding of the qPCR reaction so you are able to troubleshoot and
improve your own experiments. During the first day a standard curve
hands-on experiment will be performed where we will practically show
how to evaluate amplification curves and handle qPCR instrument
software.
The second day will cover how to do relative quantification with qPCR,
how to find stable reference genes and do proper normalization. Several
quantification examples will be demonstrated with calculations of ΔCq,
ΔΔCq and efficiency corrected ratios. During hands-on lab you will
receive relative quantification data that you will perform calculations
with yourself with a few different methods. You will get an
understanding of how Cq-values, thresholds and efficiency affect your
quantification results. During the day you will also learn how to do
absolute quantification, work with standard curves and how to properly
validate your qPCR assay performance.
Day 1
Introduction to PCR and qPCR
- How does qPCR work?
- Different detection chemistries, dyes or probes?
- Different applications
qPCR data evaluation
- How does qPCR software process the data
- How to evaluate curves and set threshold
Primer and probe design
- How to do proper primer design
- How to avoid primer dimer formation
- Other important considerations for primer design
- How to design hydrolysis probes
- Practical exercises in primer design
Optimization of qPCR protocols
- Which factors affect the PCR?
- Which factors can be optimized? |
Day 2
Normalization
- Different ways to normalize
- How to find stable reference genes
Basic quantification theory
- Quantification methods and equations
- How to do interplate calibration
Absolute quantification strategies
- How to do absolute quantification
- What is a suitable standard?
Validation of assays
- How to determine LOD and LOQ
- Precision estimation
- Which controls to use
Quantification calculation examples
- Practical examples with relative quantification
calculations
- Calculations with own generated qPCR data |
Analysis of Gene
Expression Data -- qPCR, RNA-Seq, Microarray, Nanostring;
From pre-processing and quality control to normalization, statistical
analysis and modelling
(2-days) hosted
by TATAA Biocenter
Computer
seminar room -- PU26
Description:
This training course is aimed at those
working with real-time qPCR and
would like to learn how appropriate statistics shall be selected and
applied correctly to get the most out of the qPCR data. It is a
comprehensive course teaching the basics of statistics including the
most common methods to analyze qPCR data generated both in large and
small studies. Participants are expected to have knowledge and
experience of the qPCR method.
The course includes extensive computer based exercises using the
software GenEx (MultiD Analyses). You can download a free trial version
from our webpage => Genex.gene-quantification.info
The first day of the course focuses on the principles of statistics
going through the terminology used in statistics and how to approach
statistics when analysing qPCR data. Day one will describe the most
common terms used in descriptive statistics and the most frequently
used statistical tests to extrapolate from sample to population. It
will also discuss how to setup qPCR experiments to be able to make
statistical analysis of the data.
In addition day one focuses on the ability to detect difference and
factors that influence the power analysis that can be made to optimize
an experiment. The last part will discuss the pre-treatment that
is required before analysing qPCR data, how to use relative
quantification to compare samples and the use of reference genes to
normalise between samples. Day one will also discuss the importance of
controlling for genomic DNA contamination in gene expression analysis
and how we can correct for gDNA presence using NoRT controls or
ValidPrime.
Day two of this course focuses mainly on two things. The first is
absolute quantification using standard curves that is used to quantify
unknown samples. To do this in a proper way one need to know the limits
between which we can make a good detection and quantification. How to
identify limit of detection (LOD), limit of quantification (LOQ) and
the dynamic range of a qPCR test will be handled.
Second main topic of the day is gene expression profiling where more
than one gene is analysed in many samples. Analysing many genes in many
samples often mean that the analysis cannot be performed with a single
plate, to account for this a proper design is vital for your
multi-plate experiment and analysis. We will finally discuss the most
common multivariate methods used in expression profiling where samples
(or genes) are classified based on the similar response of different
genes (or samples)
Day 1
Principles of statistics
- Introduction to the basic principles of statistics
- What does the basic terms mean?
Design of experiments
- How should the experiment be designed?
- How to do statistical hypothesis testing
Statistical tests
- Overview of the most common statistical tests
- How do we apply the different tests and when are
they valid?
Ability to detect a difference (Power Testing)
- How to define the power of a test
- How to test the null hypothesis
- Type I and typ II errors
Relative quantification
- How are the calculations for relative
quantification performed?
- How should qPCR data be pre-treated before
comparison? |
Day 2
Multiplate measurements
- How do we get Cq-values, what is delta-Cq and
delta-delta-Cq?
- How should we plan the experiment to analyze
multi-plate measurements in the best way?
- How do we use interplate calibrators
Absolute quantification
- How is absolute quantification performed?
- How to interpret standard curves
Limit of detection
- How to calculate LOD and LOQ
- How to identify the source of variance in
experiments
Reference genes
- How to find and validate optimal reference genes
Expression profiling
- How can gene expression profiling be performed?
- How to classify genes or samples with scatter plots
and dendograms
- How to handle missing data in multivariate analysis
- Practical computer based training in principal
component analysis, self-organizing maps and clustering analysis |
Digital
PCR (2-days) --
hosted by Bio-Rad
Seminar room
-- S1
Description:
Digital PCR is a technology that is gaining momentum in multiple areas
of nucleic acid analysis. This course introduces the user to digital
PCR technology with an solid review to the theory, overview of old and
new applications, hands on wet chemistry experiments (absolute
quantitation, Copy Number Variation and Rare Event Detection, Genome
Editing Screening for NHEJ/HDR), data analysis and math and stats
related to dPCR.
Day 1
- Welcome and Introductions
- Introduction to Digital PCR
- Experimental reaction setup
- Abs and CNV Quant Experiment (wet)
- ABS, CNV and RED applications overview
- Data collection for ABS and CNV experiment
- RED and GE experiment (wet lab)
- ABS and CNV results analysis
- Data collection for RED experiment
- Review of the day
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Day 2
- dPCR assay design and optimization
- ddPCR: basic statistics
- RED results analysis
- Other ddPCR Applications
- When qPCR and when ddPCR? Moving from qPCR to
ddPCR
- Open Q&A session
- Review of the workshop
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NGS – Library
Construction and Quality Control
(2-days) hosted by TATAA Biocenter
Seminar
room -- S3
Description:
This course gives an introduction to massively
parallel
sequencing (also called Next Generation Sequencing, NGS), and its many
applications. The course consists of a theoretical part, which will
focus on considerations for the NGS experiment design, the different
sequencing platforms, quality control of samples, library preparation
techniques, and quantification of libraries for sequencing. The course
also includes practical parts where the participants will prepare
libraries and perform quality control and compare libraries.
Day 1
Introduction to NGS:
- The history of DNA sequencing
- What are the advantages with NGS? ...
Disadvantages?
- What is the difference between microarray and
NGS?
Applications:
- What are the possibilities for analysis of the
genome?
Transcriptome? Epigenome? Metagenome?
- RNA sequencing; investigating differential
expression, splice
variants, novel transcripts and much more.
- DNA sequencing; finding Single-Nucleotide
Polymorphisms (SNPs),
Copy Number Variants (CNVs), structural variations and more.
- NGS for epigenetic studies; analyzing the
methylation state of
DNA or discovering protein-binding sites in DNA.
- Sample preparation and quality control
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Day 2
How to prepare samples for NGS:
- Which starting material can be used for NGS?
- Which quality controls are included in the
experimental workflow?
- How to choose the right library prep protocol.
- Practical experiment to learn the basics of
library preparation
and quality control.
Instrumentation:
- Which platforms are available?
- How do they work?
- Which platform is suitable for my experiment?
- Experimental design
What to think
about when
designing your NGS experiment:
- How much to sequence?
- How to calculate coverage and depth?
- How many samples can I multiplex?
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Next-Generation Sequencing Data
Analysis -- How to Call Genomic Variations and Uncover Their
Effects (2 days) -- hosted by ecSeq
Computer
seminar room – PU26A -- GIS room
Description: On the
first day of this hands-on course, the participants will get an
introduction to Next-Generation Sequencing (NGS) together with an
overview of its assets and drawbacks. To understand the necessary
bioinformatics analysis, the participants will perform the first steps
of a typical NGS analysis pipeline on their own. They will not only
understand the NGS data formats, but also dip into sophisticated
bioinformatics algorithms, preprocess real-life sequencing data, run a
mapping tool and visualize the mapped reads in a genome browser. On the
second day, the participants will work with the genetic testing
software 'Seamless NGS' (www.seamless-ngs.com) for
performing a complete DNA variation analysis. After a first
introduction to the software, whole exome sequencing (WES) data will be
analysed, including detailed variant exploration, filtering and
interpretation.
Day 1
NGS Technology:
- Short
History of Next Generation Sequencing
- Introduction to Illumina Sequencing from a
Data Analysts View
- View Into the Near Future
- Common NGS Data Analysis Issues
- Notations and NGS Terminology
Hands-On
Introduction to NGS Data Analysis:
- Raw
Sequence Files (FASTQ Format)
- Preprocessing of raw reads: quality control,
adapter clipping, quality trimming
- Introduction to Read Alignment (Methods,
Heuristics)
- Read Mapping
- Mapping Output (SAM/BAM Format)
- Visualization
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Day 2
NGS Analysis with Seamless NGS Software:
- User
Management
- Data Import and Projects
- Starting and Queuing Analyses
- Quality Control
DNA Variant
Analysis with Seamless NGS Software:
- DNA
Variant Calling Theory
- Calling Parameters and Configuration
- Defining Virtual Gene Panels/ROI
- Quality Control Metrics
- Tracking QC Over Time
- Handling Non-Callable Regions
- Exploring DNA Variants
- Filtering DNA Variants
- Computational Predictions
- Classification of DNA Variants
- Visualization
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Please
direct your enquiry to our
scientific organisation team, headed by Michael W.
Pfaffl qPCR-dPCR-NGS-2017@wzw.tum.de
©
2018-2019
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