The Importance of Quality Control Steps in Experiments

Sample to Insight

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Dr. Pierre-Henri FerdinandGlobal Product Management

[email protected]

You Can’t Always Trust your DNA to be There For You - The Importance of Quality Control Steps in Experiments

Trust your samples, trust your results - nucleic acids quality control

mailto:[email protected]

Sample to Insight

Legal Disclaimer

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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 available at www.QIAGEN.com or can be requested from QIAGEN Technical Services or your local distributor.


http://www.qiagen.com/

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Trust your samples, trust your results - nucleic acids quality control 3

Outline

Why is sample QC so important?1

Main sample QC parameters2

Advantages of laboratory automation for quality samples and quality results

3

Wrap up4

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Outline




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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.





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


http://www.sciencemag.org/news/2015/06/study-claims-28-billion-year-spent-irreproducible-biomedical-research

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/reproducibility-1.17552

http://www.nature.com/news/1-500-scientists-lift-the-lid-on-reproducibility-1.19970

http://www.nature.com/news/1-500-scientists-lift-the-lid-on-reproducibility-1.19970

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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.


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QC emphasizes on gaining insight into a sample quality and assess its suitability for downstream applications and its potential for generating reliable results.

QC plays an important role when it comes to developing routine procedures or troubleshooting an experiment.

µg

?

Purity?

Quantity?

Integrity?

Sequence?

Are these parameters in the range of my experiment’s requirements for generating high-quality results?

Sample Quality?

• Sample to sample variations• Human errors• Operator-to-operator variations• Tools imprecisions/misuse• Environmental changes

Risks of degrading sample quality and generating results of poor quality

What is sample QC?


Sample to Insight

Why is sample QC so important?


? % success

µg

Assess sample quality according to relevant parameters and thresholds

Process downstream only samples of good quality, maximizing success of assays

Highest quality results are only achieved from quality samples Only quality results are worth sharing

Analyse, interpret, validate and share results with confidence

Track sample quality along the workflow

Sample to Insight

At least one QC step required in each workflow


Sample collection/

stabilization

DNA purification Amplification

Ampliconsdetection 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

Why is 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


Cost of insufficient or inappropriate QC

Cost of QCInstruments, 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 QCInstruments, 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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Wrap up4

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Parameters are of prime relevance for sample Quality Control


QC Criteria UV spectro. Gels Dye-based spectro.

CE / µ-fluidic UV/VISspectro.

Pyro-sequencing

Protein contaminants(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 – 3 technologies cover all sample QC needs

purity

quantity

integrity

sequence

Sample to Insight

Their impact on downstream applications


AGCC

GA

GATC

CA

CCTT

CA

Purity

Quantity

Integrity

Sequence

Phenol, ethanol, salts, etc. can jeopardize sensitivity and efficiency of downstream enzymatic reactions.Large amounts of unwanted RNA, ssDNA or dsDNA template lead to overestimation of the molecule of interest and interferes with downstream applications.

When the concentration of NA is overestimated, the input amount will be too low. This leads to weak amplification and weak signal strength.When the concentration of DNA is underestimated the input amount will be too high and can lead to overuse of precious samples, increased concentration of inhibitors, false priming, unfavorable ratios, etc.

NA samples of poor integrity can affect PCR reactions and lead to errors in replication and/or lower yields e.g. irrelevant CT values. Degraded samples can produce false negative results.NGS libraries of incorrect size distribution or containing contaminating NA (primers and/or adaptor dimers) can lead to loss in sequencing coverage.

Failing at detecting mutations can be misleading when linking an observed phenotype to a genotype or highlighting mutations/variant which could explain an observed phenotype.

http://www.google.ch/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0ahUKEwjyt-uHv5DLAhWBWxoKHSKzD1IQjRwIBw&url=http://digitalsplashmedia.com/2014/04/free-health-icon-dna-molecule/&bvm=bv.114733917,d.ZWU&psig=AFQjCNGMDD4JoM3RgxS1JPHQ7mzi-k35Pw&ust=1456406462993670




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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.

Sample to Insight

Sample quality control in workflows


Gene Expression

Genotyping

Next-Gen Sequencing

gDNA integrity

DNA/RNA integrityLibrary QC

RNA QC

V&VDetection

V&V

V&V

Quantity & Purity

Integrity & size distribution Sequence

Extraction yieldcontamination



QIAxpert QIAxcel Advanced PyroMark Q48 AP

Sample to Insight

These instrument match requirements for efficient QC


Short time to result:

High performance:

Value for money:

Quantity & Purity

Integrity & size distribution Sequence


• Ease of use, don’t require extensive trainings

• Convenient data management for traceability

Sample to Insight

How does QIAGEN helps you achieving reliable QC?


Sample collection/

stabilization


Amplification/ NGS Library

PrepAnalysis

Data analysis &

interpretationQC V&VQC

Quantity & Purity

Integrity & size distribution

Sequence


Sample to Insight

With QIAxpert – tell DNA from RNA and other contaminants without a dye

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High-speed microfluidics• No drop & clean action required, no cross contamination• Evaporation-safe for 2h• Flexible input & up to 16 samples in <2 min

Comprehensive Export• Reports can be viewed on any browser• USB and Network data output• No extra software or computer required

Easy interaction• Inbuilt touchscreen• Simple GUI • Variety of pre-installed protocols

Spectral content profiling• Differentiation between molecules• Quantify and subtract identified impurities• Give best insight into samples’ quality


Sample to Insight

QIAxpert uses innovative microfluidic sample carrier

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Key features:

1

2

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5

1

2

3

4

5

Sample loading wellCapillariesMicrocuvetteOverflowVent hole for vacuum

Only 2 µl of sample required

DNA range: 1.5 ng/μl up to 2000 ng/μl dsDNA (A260)

Fast analysis, up to 16 samples in less than 2 minutes

Algorithm can unmixe sample’s spectra to differentiate contribution of different molecules.

QIAxpert is a high-speed spectrophotometer for DNA, RNA and

protein analysis. It is fast, accurate and sensitive.


Sample to Insight


QIAxpert – Unique spectral content profiling protocols

A230: impurities

A280: proteins

A260: nucleic acids

Sample to Insight


1. Absorbance measurement (and background correction)

2. Content profiling of the measured spectrum by fitting of reference spectra into

• Specific DNA or RNA spectrum• Impurities spectrum • Residue spectrum

3. Quality control• Impurities spectrum• Residue spectrum • Backround spectrum• A260/A280 ratio for protein

contamination


Quantity of DNA or RNA you think you have

Quantity of DNA or RNA you actually have

Sample to Insight

Quantity & Purity !


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Classic spectrophotometry

Quantity

Spectral Content Profiling

For internal use only

• DNA or RNA spectrum• Impurities spectrum • Residue spectrum


Sample to Insight



Sample collection/

stabilization



PrepAnalysis

Data analysis &


Quantity & Purity


Sequence


Sample to Insight

QIAxcel Advanced allows separation, detection and analysis of NA

27Trust your samples, trust your results - nucleic acids quality control

QIAxcel Advanced is an automated capillary electrophoresis system for

DNA and RNA analysis.

Sample to Insight

QIAxcel Advanced allows separation, detection and analysis of NA

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QIAxcel Advanced

Fully automated DNA and RNA analysis

Fast processing: 12 samples in 8 – 13 min

Up to 96 samples per run, more with lab automation

High resolution down to 3 – 5 bp up to 500 bp

Safety and convenience with ready-to-use gel cartridges

Digital data output

QIAxcel Advanced allows analysis of gDNA, RNA and NGS libraries


Sample to Insight

How does it work?

Place the gel cartridge into the instrument

Load samples (tube strips or 96-well plates), buffers, and markers

Select the Process Profile of your choice... and GO!

Look at the results in real-time on the screen and report data just a few minutes later

Operating the QIAxcel Advanced system:


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QIAxcel Advanced - Quality Control of genomic DNA

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A&B. Agarose gel (left) and QIAxcel gel (right) photo showing gDNA in various states of degradation (sonication)

Lane 1 non-degraded gDNA Lane 3 gDNA partially degraded Lane 5 gDNA fully degraded

C. Electropherograms of gDNA samples Lane 1: Very good quality gDNA Lane 3: Partially degraded gDNA Lane 5: Highly degraded gDNA


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RNA Quality Control - RNA Integrity Score (RIS)

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A1 RIS: 9.5 A7 RIS: 5.8

A11 RIS: 3.6 Superimposed view


RIS10 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

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• 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/25998866www.researchgate.net/publication/277080839_Ultraviolet_C_radiation_influences_the_robustness_of_RNA_integrity_measurement


http://www.ncbi.nlm.nih.gov/pubmed/25998866

http://www.ncbi.nlm.nih.gov/pubmed/25998866

http://www.researchgate.net/publication/277080839_Ultraviolet_C_radiation_influences_the_robustness_of_RNA_integrity_measurement



Sample to Insight

QIAxcel Advanced - Quality Control of NGS libraries

34Trust your samples, trust your results - nucleic acids quality control

Recommended quality control steps

Primer and/or adaptors dimers?

Concentration/Molarity of the library?

Large fragments?

Small fragments?

Illumina HiSeq—Whole Genome Sequencing

Sample to Insight

GeneReader – QC of a high quality library


AoI1 „Small“

AoI2 „Library“

AoI3„Large“

170bp 300bp

Molarity<20%

Molarity<10%

Height≥ 40 S/N

AoI = Area of Interest

QIAxcel Advanced automates objective and reliable QC of NGS libraries

Sample to Insight



Sample collection/

stabilization



PrepAnalysis

Data analysis &


Quantity & Purity


Sequence


Sample to Insight

Pyrosequencing provides the most detailed validation data

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Sanger sequencing Pyrosequencing

PCR Real-time PCR

• Quantification of sequence variations o LOD down to 1–2%o short to medium sequences

• Detection of sequence variationso LOD approximately 20%o medium to long sequences

• Detection of single sequence variationso LOD <1%

• Quantification of single sequence variations o LOD typically <1%

Complex analysis

Non-quantitative Quantitative

Simple analysis

Seq

uenc

e va

riatio

n

Result requirement

xsingle mutation or SNP

xx x xx xmultiple mutations or SNPs

ABC D E F


Sample to Insight


Outline




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Wrap up4

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Quantity & Purity


Sequence


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

Frame of reference of QIAGEN automated solutions:• Flexible throughput along your workflow

• Shorter time to results : get faster insights into your samples

• Increases productivity : free up your hands so you can focus on your other tasks

• High reproducibility: automation decreases manual errors and standardizes your results

• Compatibility with QIAGEN quality chemistries


Sample to Insight


Outline




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Wrap up4

Sample to Insight

Key quality indicators


QC Criteria UV spectro. Gels Dye-based spectro

CE / µ-fluidic UV/VISspectro.

Pyro-sequencing



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



There is no one-for-all solution – QIAGEN covers all your needs for sample Quality Control!

purity

quantity

integrity

sequence

Sample to Insight

Sample QC can save you time, money, and peace of mind

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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

QIAGEN helps you achieving reliable QC


Quantity & Purity


Sequence

QIAxpert QIAxcel Advanced

More info on:QIAGEN.com/QCSolutions

• Learn about sample Quality Control• Understand how QIAGEN helps you improving the

reproducibility of your experiments and achieving quality results• Discover your instruments with our interactive online demo tools

and request a free demo in your lab!

PyroMark Q48 AP

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Questions?

Thank you for your attention!

Pierre-Henri FerdinandGlobal Product Management

[email protected]


More about nucleic acids QC:QIAGEN.com/QCSolutions

mailto:[email protected]

The Importance of Quality Control Steps in Experiments

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