Reproducibility, Quality Control and Importance of Automation

Sample to Insight

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Pierre-Henri Ferdinand

Global Product Management

[email protected]

Reproducibility, quality control and importance of automation

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mailto:[email protected]

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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

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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[email protected]

Reproducibility, quality control and importance of automation


Sample to Insight

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 cumulativeknowledge production and contribute to both delays and costs of therapeuticdrug development. An analysis of past studies indicates that the cumulative(total) prevalence of irreproducible preclinical research exceeds 50%, resulting inapproximately US$28 Billion/year spent on preclinical research that is notreproducible—in the United States alone.”

Freedman LP, et al. (2015) The Economics of Reproducibility in Preclinical Research. PLoS Biol.

http://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.1002165

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

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

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?

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

purificationAmplification

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

purificationAmplification Analysis

Data analysis

&

interpretation

Gene expression RT-PCR

QC V&V

Sample

collection/

stabilization

DNA/RNA

purification

NGS Library

PrepSequencing

Data analysis

&

interpretation

Next Generation Sequencing

QC QC V&V

V&V

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

Sample to Insight

Outline





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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.

Sample to Insight

Main quality parameters to look at for NGS libraries

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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

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



Sample to Insight

Assessing quality parameters

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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

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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

distributionSequence

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

&

interpretationQC V&VQC

Quantity &

Purity

Integrity & size

distribution

Sequence


Sample to Insight

Innovative microfluidic sample carrier grants valuable benefits

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1

2

34

51

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 spectrai.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

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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

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Why QIAxpert helps achieving better sample QC?

Sample to Insight


Sample

collection/

stabilization

DNA/RNA

purification

Amplification/

NGS Library

Prep

Analysis

Data analysis

&

interpretationQC 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

AssaysGenotyping, 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

Sample to Insight

Three DNA and one RNA cartridges address ALL needs


High Resolution kit1200 samples/cartridge

Screening kit2400 samples/cartridge

Fast analysis kit3000 samples/cartridge

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

Sample to Insight

QIAxcel Advanced: How does it work?

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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

Sample to Insight

QIAxcel Advanced overcomes the limitations of slab gels

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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

Sample to Insight


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Material Amount UnitPackage

price

Price/

unitNet 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

Sample to Insight


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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

Sample to Insight

Capillary Electrophoresis overcome bottleneck of gel electrophoresis

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Time to result

Sample info

Cost per sample

Capillary

ElectrophoresisSlab gel

electrophoresis

Resolution / Multiplexing

Safety

Reproducibility

Capillary electrophoresis overcomes all bottlenecks of slab-gel electrophoresis.

Sample to Insight

The solution of choice for genotyping

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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

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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

Sample to Insight

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

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

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/25998866

www.researchgate.net/publication/277080839_Ultraviolet_C_radiation_influences_the_robustness_of_RNA_integrity_measurement

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 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

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Wrap up4

Sample to Insight

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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

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Wrap up4

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$US90 seconds

(16 samples in parallel)

QIAxcel Advanced <1$US3-10 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!



[email protected]

All our QC solutions on:

QIAGEN.com/QCsolutions


Reproducibility, Quality Control and Importance of Automation

Health & Medicine

Transcript of Reproducibility, Quality Control and Importance of Automation