Reproducibility, Quality Control and Importance of Automation

Sample to Insight
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Pierre-Henri Ferdinand
Global Product Management
Pierre-henri.ferdinand@qiagen.com
Reproducibility, quality control and importance of automation
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Legal Disclaimer
2
▪ QIAGEN products shown here are intended for molecular
biology applications. These products are not intended for
the diagnosis, prevention, or treatment of a disease.
▪ For up-to-date licensing information and product-specific
disclaimers, see the respective QIAGEN kit handbook or
user manual. QIAGEN kit handbooks and user manuals are
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Reproducibility, quality control and importance of automation

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Outline
Title, Location, Date 4
Why is sample QC so important?1
Main sample QC parameters2
Advantages of laboratory automation for quality
samples and quality results
3
Wrap up4

Sample to Insight
Outline
3
Wrap up4

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http://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.1002165
“Low reproducibility rates within life science research undermine cumulative
knowledge production and contribute to both delays and costs of therapeutic
drug development. An analysis of past studies indicates that the cumulative
(total) prevalence of irreproducible preclinical research exceeds 50%, resulting in
approximately US$28 Billion/year spent on preclinical research that is not
reproducible—in the United States alone.”
Freedman LP, et al. (2015) The Economics of Reproducibility in Preclinical Research. PLoS Biol.

Sample to Insight
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http://www.sciencemag.org/news/2015/06/study-claims-28-billion-year-spent-irreproducible-biomedical-research
http://www.nature.com/news/reproducibility-1.17552
http://www.nature.com/news/1-500-scientists-lift-the-lid-on-reproducibility-1.19970

Sample to Insight
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Non reproducibleReproducible
biomedical
research
50%
• Culture of “publish or perish”
• Inefficiencies in designing, conducting, and reporting of
studies
• Lack of standardization of sample quality for inter-lab
reproducibility
Some solutions for tackling the issue:
• Implementing quality control procedures at key steps of laboratory workflows to help
standardizing the parameters of the samples, and thus the quality of the data
generated.
• Combining lab automation with quality chemistries to enhance reproducibility of
experiments, bringing confidence in data interpretation.

Sample to Insight
9
?
Purity?
Quantity?
Integrity?
Sequence?
Are these parameters in the range of my
experiment’s requirements for
generating high-quality results?
Sample
Quality?
• Human errors
• Operator-to-operator variations
• Tools imprecisions/misuse
• Environmental changes
• Sample to sample variations
Risks of degrading sample quality
and generating results of poor quality
What makes sample QC relevant?

Sample to Insight
Why is sample QC it so important ?
 Risk of failure
 Workflows are complex, error- and failure-prone multi-step procedures
 A lot of things can potentially go wrong – at each step
 DNA and especially RNA can be unstable: sensitive to heat, radiations and
nucleases
 Confidence in results
 At the end, only the final result matters
 Reliability and reproducibility of results are affected by poor quality samples
or sample sets for which quality is not standardized.
 Robust results have more impact and only best results are worth sharing
 Cost aspects
 Detection technologies get more and more sensitive but not necessarily
more robust
 Downstream analysis can incur high price per sample (NGS, Arrays, etc.)
 Failure means waste of time and resources

Sample to Insight
At least one QC step required in each workflow
PowerPoint Style Guide 11
Sample
collection/
stabilization
DNA
purification
Amplification
Amplicons
detection and
analysis
Data analysis
&
interpretation
Genotyping
QC
Sample
collection/
stabilization
RNA
purification,
cDNA & cRNA
synthesis
Quantification,
Normalization,
Fragmentation
Array
Hybridization
and scanning
Data analysis
&
interpretation
Gene expression Microarrays
QC QC V&V
Sample
collection/
stabilization
RNA
purification
Amplification Analysis
Data analysis
&
interpretation
Gene expression RT-PCR
QC V&V
Sample
collection/
stabilization
DNA/RNA
purification
NGS Library
Prep
Sequencing
Data analysis
&
interpretation
Next Generation Sequencing
QC QC V&V
V&V

Sample to Insight
Cost of insufficient
or inappropriate QC
Cost of QC
Instruments, consumables,
training, time…
?
The overall cost of not performing QC, i.e. risking processing poor quality samples
along downstream applications, is often overlooked if calculated at all.
• Can you exactly tell the cost/sample of your qPCR experiment?
• Time spent interpreting challenging results?
• Price of your confidence and peace of mind?

Sample to Insight
Cost of insufficient
or inappropriate QC
Cost of QC
Instruments, consumables,
training, time…
?
The overall cost of not performing QC, i.e. risking processing poor quality samples
along downstream applications, is often overlooked if calculated at all.
Price per sample Time
Downstream
application
qPCR 1.5 $US 4-5 hrs
Pyrosequencing 3.0 $US 7-8 hrs
Sanger Sequencing 5-6 $US 4-6 hrs
Next-Gen Sequencing >200 $US(5plex) 2-3 working days
A trade-off must be found between the cost of QC procedure
and the cost of not doing QC

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Outline
3
Wrap up4

Sample to Insight
Sample QC requirements vary from one workflow to another and small
deviations in key sample parameters can have important impact on final results.
Appropriate QC procedures and accurate monitoring of key quality
parameters is essential in order to save time, money, and peace of mind
when turning samples into insights.

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Main quality parameters to look at for NGS libraries
16
Purity
Quantity
Size
Distribution
/ Integrity
What is my purification yield and sample concentration? Is it enough
material?
NA overestimation: weak amplification and weak signal strength.
NA underestimation: use too much of precious libraries, increase concentration
of inhibitors, unfavourable ratios with adapters, etc.
What’s really in my tube? Could contamination or impurities interfere with
my sequencing reaction?
Phenol, ethanol, salts, etc. can jeopardize sensitivity and efficiency of
downstream enzymatic reactions (restriction, ligase, polymerase).
Contamination by unwanted RNA, ssDNA or dsDNA templates lead to
overestimation of molecule of interest and may interfere with downstream
applications.
Does my sample have the right size distribution? Has my sample been
degraded? Did my enzymatic reaction work properly?
Samples of poor integrity can affect quality of the restriction, amplification of
sequencing reaction, introduce mutations during replication (i.e., sequencing
errors). Degraded samples can produce false-negative results.
QIAGEN - Automated NGS libraries QC

Sample to Insight
Assessing quality parameters
17
Purity
Quantity
Integrity
Sequence
There is no one-for-all solution
QC Criteria Gels Dye-based
spectro-
photometry
UV/Vis
spectro-
photometry
UV/Vis
spectro-
photometry
+SCP
CE / µ-
fluidic
Pyro-
sequencing
Protein contaminants
(A260/A280) ✓ ✓
Salts and other
contaminants
(A260/A230)
✓ ✓
Quantity of dsDNA
vs. other nucleic
acids
✓ (✓)
Yield
(✓) ✓ ✓ ✓ ✓
Degradation/
integrity (✓) ✓
Sizing
✓ ✓
Sample sequence
(V&V) ✓

Sample to Insight
Assessing quality parameters
18
QC Criteria Gels Dye-based
spectro-
photometry
UV/Vis
spectro-
photometry
UV/Vis
spectro-
photometry
+SCP
CE / µ-
fluidic
Pyro-
sequencing
(A260/A280) ✓ ✓
Salts and other
contaminants
(A260/A230)
✓ ✓
Quantity of dsDNA
vs. other nucleic
acids
✓ (✓)
Yield
(✓) ✓ ✓ ✓ ✓
Degradation/
integrity (✓) ✓
Sizing
✓ ✓
Sample sequence
(V&V) ✓
Purity
Quantity
Integrity
Sequence
There is no one-for-all solution, but QIAGEN covers all quality control needs!

Sample to Insight
Sample quality control in workflows
Gene Expression
Genotyping
Next-Gen Sequencing
gDNA integrity
DNA/RNA integrity
Library QC
RNA QC
V&V
Detection
V&V
V&V
Quantity &
Purity
Integrity & size
distribution
Sequence
Extraction yield
contamination
Extraction yield
contamination
Extraction yield
contamination
QIAxpert QIAxcel Advanced PyroMark Q48 AP

Sample to Insight
Sample
collection/
stabilization
DNA/RNA
purification
Amplification/
NGS Library
Prep
Analysis
Data analysis
&
interpretation
QC V&VQC
Quantity &
Purity
Integrity & size
distribution
Sequence

Sample to Insight
Innovative microfluidic sample carrier grants valuable benefits
21
1
2
3
4
5
1
2
3
4
5
Sample loading well
Capillaries
Microcuvette
Overflow
Vent hole for vacuum
QIAxpert - High Performance Spectrophotometer - INTERNAL USE ONLY
Features offered by the innovative design:
 Flexible loading of 1 to 16 samples, only 2 µl of sample required
 Fast analysis in as little as 90 sec. for up to 16 samples
 Dynamic range: 1.5 ng/μl up to 2000 ng/μl (A260)
 Samples stable in carrier up to 2 hours with no evaporation
 No drop & clean actions ; no cross contamination
 Un-used positions can be re-used
But the QIAxpert actually does more than just spectrophotometry...

Sample to Insight
The QIAxpert can detect contaminants and discriminate RNA from DNA
QIAxpert - High Performance Spectrophotometer - INTERNAL USE ONLY 22
Nucleic-acid sample
Raw spectrum
RNA SaltsDNA
IgG Phenol
…
Reference spectra (from selected QIAxpert app on instr.)
Spectral Content Profiling (SCP)
Raw spectrum
Target molecule (DNA or RNA)
Impurities (detected contaminants)
Residues (uncharacterized contaminants)
Deconvolution of absorption spectra
i.e. umixing the spectral/absorbance contribution of the different
molecule absorbing UV/Vis light present in the sample
SCP is performed on the instrument and doesn‘t require extra SW

Sample to Insight
Accurate quantification
and qualitation!
QIAxpert – Unique spectral content profiling protocols
23
Classic spectrophotometry
Biased quantification
Spectral Content Profiling (SCP)
For internal use only
• DNA or RNA spectrum
• Impurities spectrum
• Residue spectrum
With SCP, the QIAxpert offers more than classic spectrophotometers

Sample to Insight
Simplicity
Speed and throughput
More sample information
• Intuitive
• Easy integration in any workflow
• Simplifies sample QC
• Flexible throughput, up to 16 samples at the time
• No drop & clean action required ; no cross contamination
• Results in less than 2 minutes
• Detect potential contaminants and Discriminate DNA from RNA
• Adjusted and more accurate concentration measurements
• Comprehensive sample info for well-informed decision making
24
Why QIAxpert helps achieving better sample QC?

Sample to Insight
Sample
collection/
stabilization
DNA/RNA
purification
Amplification/
NGS Library
Prep
Analysis
Data analysis
&
interpretation
QC V&VQC
Quantity &
Purity
Integrity & size
distribution
Sequence

Sample to Insight
The QIAxcel Advanced capillary electrophoresis principle
Genotyping: Challenges and streamlined solutions with the QIAxcel Advanced system 26

Sample to Insight
QIAxcel - Automated capillary electrophoresis system - INTERNAL USE ONLY
gDNA
RNA
Restriction fragments
PCR/RT-PCR amplicons
Assays
Genotyping, detection,
CRISPR, gene expression
and regulation, Next
Generation Sequencing
cDNA
NGS library
Automated analysis of up to 96
samples unattended
As little as 3 minutes to analyse
12 samples at the time
Reliable detection with a
sensitivity down to 0.1ng/µL
Resolution down to 3-5bp for
fragments <500bp
QIAxcel Advanced analyses any nucleic acid samples

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Three DNA and one RNA cartridges address ALL needs
High Resolution kit
1200 samples/cartridge
Screening kit
Fast analysis kit
Resolution
Speed of analysis
QIAxcel Advanced uses ready-to-use
cartridges accelerates assay setup.
No handling of hazardous chemicals. It
improves safety and simplifies waste disposal.
Cartridges are reusable. It improves cost
efficiency and environmental footprint

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QIAxcel Advanced: How does it work?
29
Operating the QIAxcel Advanced system
Place the gel
cartridge into the
instrument
Load samples,
buffers and
markers
Select the
Process Profile
of your choice ...
and GO!
Look at the
report just a few
minutes later!
Genotyping: Challenges and streamlined solutions with the QIAxcel Advanced system

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QIAxcel Advanced overcomes the limitations of slab gels
30
1
2
3
4
5
1 Variations in pipetting
2 Gel-to-gel variation, changes in buffer composition,
variations in migration parameters (time, current,
intensity, etc.)
3 Error-prone manual steps
4 Quality of the data, objectivity in interpreting results
5 Traceability of results
QIAxcel Advanced addresses the lack of reproducibility of slab gels electrophoresis

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Material Amount Unit
Package
price
Price/
unit
Net price
0.5–10 µl Disposable tips 13 pc 39.66 960 0.54
1−200 µl Disposable tips 1 pc 34.02 960 0.04
Adhesive acryl tape 18 cm 6.96 6600 0.02
Weighing boats 1 pc 7.44 100 0.07
Gloves 2 pc 18.96 100 0.38
Tris 1.21 g 78.82 1000 0.10
Na2EDTA.2H2O 0.19 g 135 1000 0.03
Agarose 1.25 g 188.52 100 2.36
Acetic acid 0.28 ml 29.64 1000 0.01
100 bp ladder 0.25 µg 52.8 50 0.26
EtBr 0.005 ml 37.32 10 0.02
Sample no. 12
Sub total 3.81
Labor cost 25 min 20 60 8.33
Total (for all samples) 12.15
Total/sample 1.01
• Materials
• Agarose or high-resolution agarose
• Reagents
• Thermal-printer paper
• Tips, gloves, weighing boats, tape, etc.
• Repeating experiments due to manual errors
• Labor
• Disposal of waste (liquid and solid)
QIAxcel Advanced addresses cost aspects of slab-gel electrophoresis

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1
2
3
1 UV-light
2 Hazardous nucleic acid intercalating
agents such as EtBr
3 Handling requires protective
clothing, eye protection and gloves
4 Contamination of lab equipment;
requires a designated area and
specific waste disposal4
Ethidium bromide (EtBr) − Safety concerns
• Irritant to eyes, skin and respiratory tract
• Harmful if swallowed or inhaled
• Potential mutagen, carcinogen and teratogen
QIAxcel Advanced addresses safety concerns of slab gels electrophoresis

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Capillary Electrophoresis overcome bottleneck of gel electrophoresis
33
Time to result
Sample info
Cost per sample
Capillary
Electrophoresis
Slab gel
electrophoresis
Resolution / Multiplexing
Safety
Reproducibility
Capillary electrophoresis overcomes all bottlenecks of slab-gel electrophoresis.

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The solution of choice for genotyping
34
PCR products generated using the QIAGEN Multiplex PCR Kit and analyzed on:
2% agarose gel QIAxcel system with the QIAxcel DNA High Resolution Kit
Advantages of the QIAxcel Advanced
• Higher sensitivity and resolution with minimal false negative and false positive errors
• Higher reproducibility − both intra- and inter-lab
• Higher throughput and shorter time to results
• Traceability of results − thanks to an efficient data management system

Sample to Insight
Quality Control of genomic DNA
35
Assessment of DNA degraded by sonication
A. Agarose gel photo showing gDNA in
various states of degradation
Lane 1 non-degraded gDNA
Lane 3 gDNA partially degraded
Lane 5 gDNA fully degraded
B. QIAxcel gel photo showing gDNA
samples in various states of degradation
Data kindly provided by Andre Schaller, Christopher Jackson, Division of Human Genetics, Inselspital Bern, Bern, Switzerland
C. Electropherograms of gDNA samples
Lane 1: Very good quality gDNA
Lane 3: Partially degraded gDNA
Lane 5: Highly degraded gDNA
QIAxcel for NGS workflow QC
Lane 1 Lane 3 Lane 5

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RNA Quality Control - RNA Integrity Score (RIS)
36
A1 RIS: 9.5 A7 RIS: 5.8
A11 RIS: 3.6 Superimposed view
Lane Name RIS
A1 rat_liver _1 9.5
A7 rat_liver _4 5.8
A11 rat_liver _6 3.6
QIAxcel for NGS workflow QC
RIS
10 1
RNA Integrity

Sample to Insight
QIAxcel Advanced - Quality Control of RNA
• RIS (RNA Integrity Score) value directly reflects RNA integrity
• Provides objective quality measurement for RNA samples
• Eliminates need for human interpretation and enables implementation of
rigorous QC
• Allows comparison between samples and standardization of experiments
• High correlation with RIN number
RIS
RIN
R² = 92.92%

Sample to Insight
RIS is more robust than RIN for assessing RNA integrity
38
• Depending on the degradation mechanism (heat, nuclease or UV), total RNA samples have different
electrophoretic behavior
• Different RNA degradation methods result in different ranges of ΔΔCt values for identical RIS/RIN values
• “Overall, RIS was more robust than RIN for assessing RNA integrity” Unger, C et al., 2015
Learn more about RNA quality control by listening to our RNA QC webinar!
http://www.ncbi.nlm.nih.gov/pubmed/25998866
www.researchgate.net/publication/277080839_Ultraviolet_C_radiation_influences_the_robustness_of_RNA_integrity_measurement

Sample to Insight
QIAxcel Advanced enables objective QC of NGS libraries
Single peak of the
expected molecular weight
Primer dimers
Adapter dimers Broader bands of
higher MW than the
expected peak
Quality Control can be done visually, or automated for fast, reliable and objective QC
µg
NGS library QC requirements may vary from one library to another and from
one sequencing platform to another while small deviations in key sample
parameters can have large impacts on the final results.

Sample to Insight
GeneReader – QC of a high-quality library
AoI1
“Small”
AoI2
“Library”
AoI3
“Large”
170bp 300bp
Height
≥ 40 S/N
AoI = Area of Interest
40QIAGEN - Automated NGS libraries QC
Molarity
< 20%
Molarity
< 10%

Sample to Insight
• Run analysis of NGS libraries on the QIAxcel
Advanced
• The software analyzes each sample
individually and tests them against defined
QC criteria to pass or fail samples
• QC report is generated. Both run and QC
results can be exported
Performing QC of NGS libraries with QIAxcel ScreenGel software
• Quality control analysis of NGS libraries can start automatically after the sample run.
• The software shows QC results and the passed/failed status for each library.
• Results can be reported as Excel, PDF and other document formats.
41QIAGEN - Automated NGS libraries QC
QIAxcel Advanced automates objective QC of NGS libraries

Sample to Insight
The QIAxcel Advanced overcomes limitations of classic electrophoresis
QIAxcel
Advanced
High resolution and sensitivity
Accurate sizing and discrimination fragments with a resolution down
to 3 bp. Get reproducible results with as little as 0.1 ng/µL per sample
Low cost-per-sample
Smart technology pays off
Fast sample analysis
Get 12 samples analyzed in a few minutes. Accelerate your
science, increase throughput and productivity of your lab
Standardization of results
Normalize your results, so you can compare samples with each other,
between experiments and between labs
Full automation
No manual preparation of sample required, they can be processed
straight out of the thermocycler or fridge, in 96-well plates or strip tubes
ScreenGel
software
Instrument and data integrity
User management and protection of data to
secure your lab, data and ensure traceability
Digital data output and advanced analyses
State-of-the-art algorithms provide both qualitative and quantitative
results. Samples can be automatically analyzed which simplifies analysis,
QC and data interpretation.
Report/export of data
Export your analysis results to customizable reports
and in various formats
Cartridges
ONE instrument
to address all
electrophoresis
needs

Sample to Insight
Outline
3
Wrap up4

Sample to Insight
44
Let lab automation work for you and do the job they are excellent at:
Delivering reliable and reproducible results
Perform accurate measurements, keep track of your sample quality, standardize
your experiments and increase reproducibility of your results.
Increase your lab productivity: automation frees your hands and your mind!

Sample to Insight
Outline
3
Wrap up4

Sample to Insight
Sample QC can save you time, money, and peace of mind
46
Application Price per sample Time
Downstream
application
qPCR 1.5 $US 4-5 hrs
Sanger Sequencing 5-6 $US 4-6 hrs
Next-Gen Sequencing >200 $US(5plex) 2-3 working days
Affymetrix GeneChip
Analysis
500-800 $US/sample > 2 working days
Sample Quality
Control
QIAxpert <1$US
90 seconds
(16 samples in parallel)
QIAxcel Advanced <1$US
3-10 minutes
(12 samples in parallel)
Purity &
quantity
Integrity
QC gives insights into
sample suitability for
downstream application
• Brings confidence in data analysis and interpretation, troubleshooting
• Saves time, money, and peace of mind by excluding samples of poor quality
“Save time, money, and peace of mind only dealing with the
samples you deserve: the ones of highest quality”

Sample to Insight
Key quality indicators
QC Criteria UV spectro. Gels Dye-based
spectro
CE / µ-
fluidic
UV/VIS
spectro.
Pyro-
sequencing
(A260/280) ✓ ✓
Salts & other
contaminants
(A260/230)
✓ ✓
Quantity of dsDNA
vs. other NA ✓
Yield
✓ (✓) ✓ ✓ ✓
Degradation/
Size distribution (✓) ✓
Sizing
✓ ✓
Sample sequence
✓
There is no one-for-all solution
purity
quantity
integrity
sequence

Sample to Insight
Key quality indicators
QC Criteria UV spectro. Gels Dye-based
spectro
QIAxcel
Advanced
QIAxpert PyroMark
Q48 AP
(A260/280) ✓ ✓
Salts & other
contaminants
(A260/230)
✓ ✓
Quantity of dsDNA
vs. other NA ✓
Yield
✓ (✓) ✓ ✓ ✓
Degradation/
Size distribution (✓) ✓
Sizing
✓ ✓
Sample sequence
✓
There is no one-for-all solution – QIAGEN covers all your needs for sample Quality Control!
purity
quantity
integrity
sequence

Sample to Insight
Sample Insight
Sample
collection/
stabilization
DNA/RNA
purification
Amplification/
NGS Library
Prep
Analysis
Data analysis
&
interpretation
From Sample to Insight, QIAGEN offers you flexible automated solutions for
increasing the reproducibility of your research and standardizing your workflows.
V&VQC QC

Sample to Insight
Quantity &
Purity
Integrity & size
distribution
Sequence
QIAxpert QIAxcel Advanced
More info on:
QIAGEN.com
Discover how these instruments can help you improve the
reproducibility of your experiments and achieve higher
quality results with our interactive online demo tools and
request a free demo in your lab!
PyroMark Q48 AP

Sample to Insight
51
Questions?
Thank you for your attention!
All our QC solutions on:
QIAGEN.com/QCsolutions

Reproducibility, Quality Control and Importance of Automation

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Reproducibility, Quality Control and Importance of Automation