The Importance of Quality Control Steps in Experiments
1 like1,400 views
The document discusses the importance of quality control (QC) in nucleic acid experiments, emphasizing that inadequate QC can lead to unreliable results and high costs in research. It highlights the need for standardization in sample handling and introduces QIAGEN's solutions, like qiaxpert and qiaxcel, for effective sample analysis and automation. Overall, implementing stringent QC methodologies is essential to enhance reproducibility and data integrity in molecular biology applications.
The Importance of Quality Control Steps in Experiments
- 1. Sample to Insight 1 Dr. Pierre-Henri Ferdinand Global Product Management Pierre-henri.ferdinand@qiagen.com 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 acidsquality control
- 2. Sample to Insight 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. Trust your samples, trust your results - nucleic acidsquality control
- 3. Sample to Insight Outline Trust your samples, trust your results - nucleic acidsquality control 3 Why is sample QC so important?1 Main sample QC parameters2 Advantages of laboratory automation for quality samples and quality results 3 Wrap up4
- 4. Sample to Insight Outline Trust your samples, trust your results - nucleic acidsquality control 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
- 5. Sample to Insight 5 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—inthe UnitedStatesalone.” Freedman LP, et al. (2015) The Economics of Reproducibility in Preclinical Research. PLoS Biol. Trust your samples, trust your results - nucleic acidsquality control
- 6. Sample to Insight 6 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 Trust your samples, trust your results - nucleic acidsquality control
- 7. Sample to Insight 7 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. Trust your samples, trust your results - nucleic acidsquality control
- 8. Sample to Insight 8 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? Trust your samples, trust your results - nucleic acidsquality control
- 9. Sample to Insight Why is sample QC so important? Trust your samples, trust your results - nucleic acidsquality control 9 ? % 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
- 10. Sample to Insight At least one QC step required in each workflow Trust your samples, trust your results - nucleic acidsquality control 10 Sample collection/ stabilization DNA purification Amplification Amplicons detection and analysis Data analysis & interpretation Genotyping QC Sample collection/ stabilization RNA purification, cDNA & cRNA synthesis Quantification, Normalization, Fragmentation Array Hybridization and scanning Data analysis & interpretation Gene expression Microarrays QC QC V&V Sample collection/ stabilization RNA purification Amplification Analysis Data analysis & interpretation Gene expression RT-PCR QC V&V Sample collection/ stabilization DNA/RNA purification NGS Library Prep Sequencing Data analysis & interpretation Next Generation Sequencing QC QC V&V V&V
- 11. Sample to Insight Why is QC it so important ? Trust your samples, trust your results - nucleic acidsquality control 11 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 RNAcan 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 worthsharing Cost aspects Detection technologies get more and more sensitive but not necessarily more robust Downstreamanalysis can incur high price per sample (NGS, Arrays, etc.) Failure means waste of time and resources
- 12. Sample to Insight Trust your samples, trust your results - nucleic acidsquality control 12 Cost of insufficient or inappropriate QC Cost of QC Instruments, consumables, training, time… ? The overall cost of not performing QC, i.e. risking processing poor quality samples along downstream applications, is often overlooked if calculated at all. • Can you exactly tell the cost/sample of your qPCR experiment? • Time spent interpreting challenging results? • Price of your confidence and peace of mind?
- 13. Sample to Insight Trust your samples, trust your results - nucleic acidsquality control 13 Cost of insufficient or inappropriate QC Cost of QC Instruments, consumables, training, time… ? The overall cost of not performing QC, i.e. risking processing poor quality samples along downstream applications, is often overlooked if calculated at all. Price per sample Time Downstream application qPCR 1.5 $US 4-5 hrs Pyrosequencing 3.0 $US 7-8 hrs Sanger Sequencing 5-6 $US 4-6 hrs Next-Gen Sequencing >200 $US(5plex) 2-3 working days A trade-off must be found between the cost of QC procedure and the cost of not doing QC
- 14. Sample to Insight Outline Trust your samples, trust your results - nucleic acidsquality control 14 Why is sample QC so important?1 Main sample QC parameters2 Advantages of laboratory automation for quality samples and quality results 3 Wrap up4
- 15. Sample to Insight Parameters are of prime relevance for sample Quality Control Trust your samples, trust your results - nucleic acidsquality control 15 QC Criteria UV spectro. Gels Dye-based spectro. CE / µ- fluidic UV/VIS spectro. 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
- 16. Sample to Insight Their impact on downstream applications Trust your samples, trust your results - nucleic acidsquality control 16 AGCCGA GATCCA CCTTCA Purity Quantity Integrity Sequence Phenol, ethanol, salts, etc. can jeopardize sensitivity and efficiency of downstream enzymatic reactions. Large amounts of unwanted RNA, ssDNAor 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.
- 17. Sample to Insight Trust your samples, trust your results - nucleic acidsquality control 17 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 parametersis essential in order to save time, money, and peace of mind when turningsamples into insights.
- 18. Sample to Insight Sample quality control in workflows Trust your samples, trust your results - nucleic acidsquality control 18 Gene Expression Genotyping Next-GenSequencing gDNA integrity DNA/RNA integrity Library QC RNA QC V&V Detection V&V V&V Quantity & Purity Integrity & size distribution Sequence Extraction yield contamination Extraction yield contamination Extraction yield contamination QIAxpert QIAxcel Advanced PyroMark Q48 AP
- 19. Sample to Insight These instrument match requirements for efficient QC Trust your samples, trust your results - nucleic acidsquality control 19 Short time to result: High performance: Value for money: Quantity & Purity Integrity & size distribution Sequence QIAxpert QIAxcel Advanced PyroMark Q48 AP • Ease of use, don’t require extensive trainings • Convenient data management for traceability
- 20. Sample to Insight How does QIAGEN helps you achieving reliable QC? Trust your samples, trust your results - nucleic acidsquality control 20 Sample collection/ stabilization DNA/RNA purification Amplification/ NGS Library Prep Analysis Data analysis & interpretation QC V&VQC Quantity & Purity Integrity & size distribution Sequence QIAxpert QIAxcel Advanced PyroMark Q48 AP
- 21. Sample to Insight With QIAxpert – tell DNA from RNA and other contaminantswithout a dye 21 High-speed microfluidics • No drop & clean action required, no cross contamination • Evaporation-safefor 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 contentprofiling • Differentiationbetween molecules • Quantify and subtract identified impurities • Give best insight into samples’ quality Trust your samples, trust your results - nucleic acidsquality control
- 22. Sample to Insight QIAxpert uses innovative microfluidic sample carrier 22 Key features: 1 2 3 4 5 1 2 3 4 5 Sample loading well Capillaries Microcuvette Overflow Vent 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. Trust your samples, trust your results - nucleic acidsquality control
- 23. Sample to Insight Trust your samples, trust your results - nucleic acidsquality control 23 QIAxpert – Unique spectral content profiling protocols A230: impurities A280: proteins A260: nucleic acids
- 24. Sample to Insight Trust your samples, trust your results - nucleic acidsquality control 24 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
- 25. Sample to Insight Quantity & Purity ! QIAxpert – Unique spectral content profiling protocols 25 Classic spectrophotometry Quantity Spectral Content Profiling For internal use only • DNA or RNA spectrum • Impurities spectrum • Residue spectrum Trust your samples, trust your results - nucleic acidsquality control
- 26. Sample to Insight How does QIAGEN helps you achieving reliable QC? Trust your samples, trust your results - nucleic acidsquality control 26 Sample collection/ stabilization DNA/RNA purification Amplification/ NGS Library Prep Analysis Data analysis & interpretation QC V&VQC Quantity & Purity Integrity & size distribution Sequence QIAxpert QIAxcel Advanced PyroMark Q48 AP
- 27. Sample to Insight QIAxcel Advanced allows separation, detection and analysis of NA 27Trust your samples, trust your results - nucleic acidsquality control QIAxcel Advanced is an automated capillary electrophoresis systemfor DNA and RNA analysis.
- 28. Sample to Insight QIAxcel Advanced allows separation, detection and analysis of NA 28 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, RNAand NGS libraries Trust your samples, trust your results - nucleic acidsquality control
- 29. 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: Trust your samples, trust your results - nucleic acidsquality control 29
- 30. Sample to Insight QIAxcel Advanced - Quality Control of genomic DNA 30 A&B. Agarose gel (left) and QIAxcel gel (right) photo showing gDNAin various states of degradation (sonication) Lane 1 non-degraded gDNA Lane 3 gDNA partially degraded Lane 5 gDNA fully degraded C. Electropherograms of gDNAsamples Lane 1: Very good quality gDNA Lane 3: Partially degraded gDNA Lane 5: Highly degraded gDNA Trust your samples, trust your results - nucleic acidsquality control
- 31. Sample to Insight RNA Quality Control - RNA Integrity Score (RIS) 31 A1 RIS: 9.5 A7 RIS: 5.8 A11 RIS: 3.6 Superimposed view Trust your samples, trust your results - nucleic acidsquality control RIS 10 1 RNA Integrity
- 32. Sample to Insight QIAxcel Advanced - Quality Control of RNA Trust your samples, trust your results - nucleic acidsquality control 32 • RIS (RNA Integrity Score) value directly reflects RNAintegrity • 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%
- 33. Sample to Insight RIS is more robust than RIN for assessing RNA integrity 33 • Depending on the degradation mechanism(heat, nuclease or UV), total RNA samples have different electrophoreticbehavior • Different RNAdegradation methods result in different ranges ofΔΔCt values for identical RIS/RIN values • “Overall, RIS was more robust than RIN for assessingRNAintegrity” 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 Trust your samples, trust your results - nucleic acidsquality control
- 34. Sample to Insight QIAxcel Advanced - Quality Control of NGS libraries 34Trust your samples, trust your results - nucleic acidsquality control Recommended quality control steps Primer and/or adaptors dimers? Concentration/Molarity of the library? Large fragments? Small fragments? Illumina HiSeq—Whole Genome Sequencing
- 35. Sample to Insight GeneReader – QC of a high quality library Trust your samples, trust your results - nucleic acidsquality control 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
- 36. Sample to Insight How does QIAGEN helps you achieving reliable QC? Trust your samples, trust your results - nucleic acidsquality control 36 Sample collection/ stabilization DNA/RNA purification Amplification/ NGS Library Prep Analysis Data analysis & interpretation QC V&VQC Quantity & Purity Integrity & size distribution Sequence QIAxpert QIAxcel Advanced PyroMark Q48 AP
- 37. Sample to Insight Pyrosequencing provides the most detailed validation data 37 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 variations o LOD approximately 20% o medium to long sequences • Detection of single sequence variations o LOD <1% • Quantification of single sequence variations o LOD typically <1% Complex analysis Non-quantitative Quantitative Simple analysis Sequencevariation Result requirement x single mutation or SNP xx x xx x multiple mutations or SNPs ABC D E F Trust your samples, trust your results - nucleic acidsquality control
- 38. Sample to Insight Outline Trust your samples, trust your results - nucleic acidsquality control 38 Why is sample QC so important?1 Main sample QC parameters2 Advantages of laboratory automation for quality samples and quality results 3 Wrap up4
- 39. Sample to Insight How does QIAGEN helps you achieving reliable QC? Trust your samples, trust your results - nucleic acidsquality control 39 Quantity & Purity Integrity & size distribution Sequence QIAxpert QIAxcel Advanced PyroMark Q48 AP Let lab automation work for you and do the job they are excellent at: Delivering reliable and reproducible results Performaccurate 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!
- 40. 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 Trust your samples, trust your results - nucleic acidsquality control
- 41. Sample to Insight Outline Trust your samples, trust your results - nucleic acidsquality control 41 Why is sample QC so important?1 Main sample QC parameters2 Advantages of laboratory automation for quality samples and quality results 3 Wrap up4
- 42. Sample to Insight Key quality indicators Trust your samples, trust your results - nucleic acidsquality control 42 QC Criteria UV spectro. Gels Dye-based spectro CE / µ- fluidic UV/VIS spectro. 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 purity quantity integrity sequence
- 43. Sample to Insight Key quality indicators Trust your samples, trust your results - nucleic acidsquality control 43 QC Criteria UV spectro. Gels Dye-based spectro QIAxcel Advanced QIAxpert PyroMark Q48 AP 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 – QIAGEN covers all your needs for sample Quality Control! purity quantity integrity sequence
- 44. Sample to Insight Sample QC can save you time, money, and peace of mind 44 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” Trust your samples, trust your results - nucleic acidsquality control
- 45. Sample to Insight QIAGEN helps you achieving reliable QC Trust your samples, trust your results - nucleic acidsquality control 45 Quantity & Purity Integrity & size distribution 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
- 46. Sample to Insight 46 Questions? Thank you for your attention! Pierre-Henri Ferdinand Global Product Management Pierre-henri.ferdinand@qiagen.com Trust your samples, trust your results - nucleic acidsquality control More about nucleic acids QC: QIAGEN.com/QCSolutions