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

[email protected]

Maximize the success of your NGS experiments by ensuring quality of your libraries

mailto:[email protected]

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

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Agenda





Concluding remarks5

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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.

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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

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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Agenda





Concluding remarks5

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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 ?

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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


mRNA

Size selection

1st strand cDNA

synthesisEnd-repair

Sample collection/

stabilization


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

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Agenda





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.

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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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.

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Agenda





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

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Sample collection/

stabilization


NGS library prep Sequencing

Data analysis &

interpretationQC V&VQC

Quantity and purity


Sequence


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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

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Quality Control of RNA samples 20

Classic spectrophotometry

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

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


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


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Sample collection/

stabilization



Data analysis &


Quantity and purity

Sequence



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


gDNA

Size selection

FragmentmRNA

2nd strand cDNA

synthesis

Adenylate ends

(add A)

Adapter ligation


mRNA

Size selection

1st strand cDNA

synthesisEnd-repair

Sample collection/

stabilization


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.

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

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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



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

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Distribution analysis



3) Small fragments within final library

Library failed



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

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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 collection/

stabilization



Data analysis &


Quantity and purity

Sequence



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





Concluding remarks5

Sample to Insight

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Why is sample QC so important?

Sample collection/

stabilization


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

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photometry

CE / microfluidic

UV/Visspectro-

photometry

Pyro-sequencing



()


Purity

Quantity

Integrity

Sequence

There is no one-for-all solution

Sample to Insight

Main key quality indicators

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photometry

QIAxcel Advanced

QIAxpert PyroMark Q48

Autoprep



()


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?


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

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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

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Questions?

Thank you for your attention!

Pierre-Henri FerdinandGlobal Product Management

[email protected]

Learn more about nucleic acids QC on: qiagen.com/QCSolutions


mailto:[email protected]

Maximize the Success of Your NGS Experiments with State-of-the-Art NGS Library Quality Control...

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