Maximize the Success of Your NGS Experiments with State-of-the-Art NGS Library Quality Control...
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Transcript of Maximize the Success of Your NGS Experiments with State-of-the-Art NGS Library Quality Control...
Sample to Insight
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Dr. Pierre-Henri FerdinandGlobal Product Management
Maximize the success of your NGS experiments by ensuring quality of your libraries
Sample to Insight
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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.
Disclaimer
Sample to Insight
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Agenda
Reproducibility in biomedical research1
Why is sample Quality Control (QC) so important?2
Main QC parameters for NGS workflows3
Fast and reliable QC in NGS4
Concluding remarks5
Sample to Insight
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Agenda
Reproducibility in biomedical research1
Why is sample Quality Control (QC) so important?2
Main QC parameters for NGS workflows3
Fast and reliable QC in NGS4
Concluding remarks5
Sample to Insight
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“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 L.P. et al. (2015) The Economics of Reproducibility in Preclinical Research. PLoS Biol 13(6): e1002165.
Sample to Insight
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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
Sample to Insight
Non-reproducibleReproducible biomedical
research
50% • Culture of “publish or perish”• Inefficiencies in designing, conducting,
validating 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 in lab workflows to
help standardize the parameters of the samples and the quality of the data they generate.
• Using lab automation with quality chemistries to increase reproducibility of experiments, bringing confidence in results interpretation.
• Verify and validate molecular insights using a different technology
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Sample to Insight
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Agenda
Reproducibility in biomedical research1
Why is sample Quality Control (QC) so important?2
Main QC parameters for NGS workflows3
Fast and reliable QC in NGS4
Concluding remarks5
Sample to Insight
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QC consists of gaining insight into a sample’s quality and assessing its suitability for downstream applications and potential for generating reliable results.
QC plays an important role in developing routine procedures or troubleshooting experiments.
µg
?
Purity?
Quantity?
Integrity?
Size?
Are these parameters in the range of the experiment’s requirements for generating quality results?
Sample Quality
What is sample QC ?
Sample to Insight
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Why is sample QC so important?
Risk of failure• NGS workflows are complex, multistep procedures prone to errors and failures• Many things can potentially go wrong during library preparation steps• DNA and (especially) RNA can be unstable and sensitive to heat, radiation and nucleases
Confidence in results• In the end, only the quality of the sequence reads matters• The read quality and coverage depth are strongly affected by the sample quality
Cost aspects• Sequencers get more and more sensitive but not necessarily more robust• Failures waste time and resources
Sample collection/
stabilization
DNA/RNA purification
NGS library preparation Sequencing Data analysis
& interpretationQC QC V&V
Sample to Insight
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Fragment(shear) gDNA
End-repairAdenylate
ends(add A)
Adapter ligation
Library amplification
gDNA
Size selection
FragmentmRNA
2nd strand cDNA
synthesis
Adenylate ends
(add A)
Adapter ligation
Library amplification
mRNA
Size selection
1st strand cDNA
synthesisEnd-repair
Sample collection/
stabilization
DNA/RNA purification
NGS library preparation Sequencing Data analysis &
interpretationQC QC
Recommended QC steps during library preparation
Quantity and purity check
Concentration and size distribution check
V&V
Sample to Insight
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Agenda
Reproducibility in biomedical research1
Why is sample Quality Control (QC) so important?2
Main QC parameters for NGS workflows3
Fast and reliable QC in NGS4
Concluding remarks5
Sample to Insight
Seven parameters are of prime relevance for sample QC
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QC Criteria UV spectro-photometry
Gels Dye-based spectro-
photometry
CE / microfluidic
UV/Visspectro-
photometry
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
Purity
Quantity
Size distribution
Sequence
There is no one-for-all solution – 3 technologies cover all sample QC needs
Sample to Insight
Main quality parameters to look at for NGS samples
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Purity
Quantity
Size Distribution
What is my purification yield and sample concentration? Is it enough material?
DNA overestimation: weak amplification and weak signal strength.DNA 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 NGS library have the right size distribution? Has my sample been degraded? Did my enzymatic reaction work properly? Libraries of poor integrity can affect quality of the reads and sequencing depth, introduce mutations during replication (i.e., sequencing errors). Degraded samples can produce false-negative results.NGS libraries of incorrect size distribution or containing contaminating nucleic acids (primers and/or adapter dimers) can lead to loss in sequencing coverage.
Sample to Insight
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NGS library QC requirements may vary from one library to another and from one sequencing platform to another. Small deviations in key sample parameters can have large impacts on the final results.
Appropriate QC procedures and accurate monitoring of key quality parameters are essential for saving time and money and keeping peace of mind when turning samples into insights with NGS technology.
Sample to Insight
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Agenda
Reproducibility in biomedical research1
Why is sample Quality Control (QC) so important?2
Main QC parameters for NGS workflows3
Fast and reliable QC in NGS4
Concluding remarks5
Sample to Insight
Fast and reliable sample QC for results of highest quality
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Next-generation sequencing DNA/RNA integrity,Library QC V&V
Quantity and purity
Integrity, size distribution and concentration
Sequence
Extraction yieldcontamination
QIAxpert QIAxcel Advanced PyroMark Q48 Autoprep
Sample to Insight
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Sample collection/
stabilization
DNA/RNA purification
NGS library prep Sequencing
Data analysis &
interpretationQC V&VQC
Quantity and purity
Integrity, size distribution and concentration
Sequence
QIAxpert QIAxcel Advanced PyroMark Q48 Autoprep
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.
Quality Control of RNA samples
Sample to Insight
Quality Control of RNA samples 20
Classic spectrophotometry
A230: impurities
A280: proteins
A260: nucleic acids
Sample to Insight
Quality Control of RNA samples 21
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
QIAxpert – Unique spectral content profiling protocols
Quantity of DNA or RNA you think you have
Quantity of DNA or RNA you actually have
Sample to Insight
With QIAxpert – tell RNA from DNA 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
Quality Control of RNA samples
Sample to Insight
Quantity & Purity !
QIAxpert – Unique spectral content profiling protocols
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Classic spectrophotometry
Quantity
Spectral Content Profiling
For internal use only
• DNA or RNA spectrum• Impurities spectrum • Residue spectrum
Quality Control of RNA samples
Sample to Insight
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Sample collection/
stabilization
DNA/RNA purification
NGS library prep Sequencing
Data analysis &
interpretationQC V&VQC
Quantity and purity
Sequence
QIAxpert QIAxcel Advanced PyroMark Q48 Autoprep
Integrity, size distribution and concentration
Sample to Insight
QIAxcel Advanced allows separation, detection and analysis of NA
25Trust 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
Trust your samples, trust your results - nucleic acids quality control
• Reusable 12-capillary cartridges
• Fast processing: 12 samples in 8-13 min
• Detection limit of 0.1 ng/µl
• Sample consumption < 0.1µl
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!
See the results in real-time on the screen and the report data just a few minutes later
Operating the QIAxcel Advanced system:
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Sample to Insight
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Fragment(shear) gDNA)
End-repairAdenylate
ends(add A)
Adapter ligation
Library amplification
gDNA
Size selection
FragmentmRNA
2nd strand cDNA
synthesis
Adenylate ends
(add A)
Adapter ligation
Library amplification
mRNA
Size selection
1st strand cDNA
synthesisEnd-repair
Sample collection/
stabilization
DNA/RNA purification
NGS library preparation Sequencing Data analysis &
interpretationQC QC
Recommended QC steps during library preparation
Quantity and purity check
Concentration and size distribution check
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
Lane 1 Lane 3 Lane 5
Sample to Insight
RNA quality control – RNA Integrity Score (RIS)
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
RIS10 1
RNA Integrity
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Sample to Insight
QIAxcel Advanced – quality control of RNA
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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• Allow 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 et al, 2015
http://www.ncbi.nlm.nih.gov/pubmed/25998866www.researchgate.net/publication/277080839_Ultraviolet_C_radiation_influences_the_robustness_of_RNA_integrity_measurement
Learn more about RNA quality control from our RNA QC webinar. Watch it at www.qiagen.com.
Sample to Insight
QIAxcel Advanced – quality control of NGS libraries
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Incomplete shearing?
Efficient size selection?
Primer–dimers?
gDNA
Sheared DNA
Final NGS library
Recommended quality control steps
Sample to Insight
Illumina HiSeq – NGS library QC with QIAxcel Advanced
Different dilutions of the same library after shearing, adapter ligation, amplification and size selection
Adapter ligated libraries (A9) Amplified adapter ligated libraries Size-selected libraries (A12)Sheared DNA by Covaris (A2)
Black arrows indicate presence of adapters and primer–dimers in the samples after ligation (7–9) and amplification (10)
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Lane Lybrary Conc. (ng/μ)lA1 DNA Size Marker 10A2 Covaris - 8μl 18.93A3 Covaris - 4μl 8.68A4 Covaris - 2μl 4.6A5 Covaris - 1μl 1.51A6 Covaris - 0.5μl 1.47A7 Adaptor ligation - 8μl 18.2A8 Adaptor ligation - 4μl 6.89A9 Adaptor ligation - 2μl 3.19A10 PCR - 8μl 5.15A11 Size selection - 8μl 0.28A12 Size selection - 4μl 0.26
Lane Library Conc. (ng/μl)
Sample to Insight
Primer and/or adapter–dimers?
Concentration/molarity of the library?
Large fragments?
Small fragments?
Illumina HiSeq – whole genome sequencing
?
Purity?
Quantity?
Integrity?
Size?
Are these parameters in the range of the experiment’s requirements for generating quality results?
Sample Quality
Quality control of final libraries
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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
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Sample to Insight
Automated QC of libraries with the distribution analysis feature
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Distribution analysis: define the QC criteria
Height AoI2 = 879 S/NRatio Small/Total = 0.01
Ratio Large/Total = 0.03
1) Standard library
Library passed
Height AoI2 = 13 S/NRatio Small/Total = 0.26
Ratio Large/Total = 0.04
2) No / low library analyzedLibrary failed
Note: Height check of library peak (AoI2) failed. Repeat QIAxcel run using increased injection time of up to 40 sec and/or reduce sample dilution.
Ratio Small/Total = AoI1/total molarityRatio Large/Total = AoI3/total molarity
Sample to Insight
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Distribution analysis
Height AoI2 = 1779 S/NRatio Small/Total = 0.26
Ratio Large/Total = 0.04
3) Small fragments within final library
Library failed
Height AoI2 = 2393 S/NRatio Small/Total = 0.12
Ratio Large/Total = 0.21
4) Large fragments within final libraryLibrary failed
Note: These peaks represent library artifacts from nonspecific amplification that occurred during target enrichment. Size selection of larger fragments after multiplex PCR was insufficient.
Note: These peaks represent artifacts (e.g., library adapters and adapter–dimers) that occur when there is no, or insufficient size selection after library preparation.
Ratio Small/Total = AoI1/total molarityRatio Large/Total = AoI3/total molarity
Automated QC of libraries with the distribution analysis feature
Sample to Insight
Performing QC of NGS libraries with QIAxcel ScreenGel software
1. Define the size range of your library in bp and its signal threshold (signal-to-noise ratio)
2. Define other Areas of Interest flanking your library to complete your QC
3. Defined your QC criteria using parameters such as the AoI and total sample ratio
AoI1 AoI2 AoI3
QIAxcel ScreenGel software includes TE and LP QC criteria for GeneReader libraries
QC criteria can be created or modified to fit QC needs for libraries of all NGS workflows, including Illumina, Ion torrent etc.
QIAxcel Advanced automates NGS library QC
39
Sample to Insight
4. Run analysis of NGS libraries on the QIAxcel Advanced
5. The software analyzes each sample individually and tests them against defined QC criteria to pass or fail samples
6. Both run and QC results can be exported and reported
Performing QC of NGS libraries with QIAxcel ScreenGel software
Quality control analysis of NGS libraries can be performed according to the users criteria automatically after the sample run. The software shows QC results and the passed/failed status of each sample.Results can be reported as Excel, PDF and other document formats.
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Sample to Insight
Possible root causes• Excessive shearing of starting gDNA or cDNA• Adapter concentration too high or contamination by
adapters/primers• Inefficient size selection
Recommendation:• Repeat size selection or• Repeat library preparation
Possible root causes:• Insufficient shearing of starting gDNA or cDNA• Too much input material• Adapter concentration too low• Wrong PCR protocol used for target enrichment
Recommendation:Large DNA products introduced into library prep cannot be removed during the process! The library preparation or target enrichment must be repeated.
Molarity of SHORT fragments is too high Molarity of LONG fragments is too high
Troubleshooting of libraries that fail the QC check
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Sample to Insight
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Sample collection/
stabilization
DNA/RNA purification
NGS library prep Sequencing
Data analysis &
interpretationQC V&VQC
Quantity and purity
Sequence
QIAxpert QIAxcel Advanced PyroMark Q48 Autoprep
Integrity, size distribution and concentration
Sample to Insight
Verification and validation is an important step
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How can I ensure that the NGS results I want to publish are correct?
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
Trust your samples, trust your results - nucleic acids quality control
Sample to Insight
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Agenda
Reproducibility in biomedical research1
Why is sample Quality Control (QC) so important?2
Main QC parameters for NGS workflows3
Fast and reliable QC in NGS4
Concluding remarks5
Sample to Insight
46
Why is sample QC so important?
Sample collection/
stabilization
DNA/RNA purification
NGS Library Preparation Sequencing Data analysis
& interpretationQC QC
Risk of failure
• NGS workflows are complex, multistep procedures prone to errors and failures
• Many things can potentially go wrong during library preparation steps
• DNA and (especially) RNA can be unstable and sensitive to heat, radiation and nucleases
Confidence in results
• In the end, only the quality of the sequence reads matters
• The read quality and coverage depth are affected by the sample quality
Cost aspects
• Sequencers get more and more sensitive but not necessarily more robust
• Failures waste time and resources
Sample to Insight
Main key quality indicators
47
QC Criteria UV spectro-photometry
Gels Dye-based spectro-
photometry
CE / microfluidic
UV/Visspectro-
photometry
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
Purity
Quantity
Integrity
Sequence
There is no one-for-all solution
Sample to Insight
Main key quality indicators
48
QC Criteria UV spectro-photometry
Gels Dye-based spectro-
photometry
QIAxcel Advanced
QIAxpert PyroMark Q48
Autoprep
Protein contaminants(A260/A280) Salts and other contaminants(A260/A230)
Quantity of dsDNA vs. other nucleic acids
()
Yield () Degradation/integrity () Sizing Sample sequence
Purity
Quantity
Integrity
Sequence
There is no one-for-all solution, but QIAGEN covers all quality control needs!
Sample to Insight
How does QIAGEN helps you achieving reliable QC?
Quality Control of RNA samples 49
Quantity & Purity
Integrity & size distribution
QIAxpert QIAxcel Advanced
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!
Sequence
PyroMark Q48 Autoprep
Sample to Insight
Sample QC saves time and money and gives 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-generation sequencing >200 $US (5-plex) 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 8–13 minutes(12 samples in parallel)
Purity & quantity
Integrity
QC gives insights into sample suitability for
downstream applications
Brings confidence in results analysis and interpretation, troubleshooting
Saves time and money and gives peace of mind by excluding poor-quality samples
Save time and money and gain peace of mind by only dealing with the highest quality samples
Sample to Insight
51
Quantity and purity
Integrity and size distribution
Sequence
QIAxpert QIAxcel Advanced
Discover these instruments and how they can help you improve the quality and reproducibility of your NGS results. Check out the interactive online demo tools and request a free demo in your lab!
PyroMark Q48 Autoprep
Learn more about nucleic acids QC on: qiagen.com/QCSolutions
Sample to Insight
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Questions?
Thank you for your attention!
Pierre-Henri FerdinandGlobal Product Management
Learn more about nucleic acids QC on: qiagen.com/QCSolutions
Quality Control of RNA samples