DNA Workflow. Marine Biological Laboratory. Mark Bratz Applications Scientist, Promega Corporation. August 2016

Transcription

1 DNA Workflow Marine Biological Laboratory Mark Bratz Applications Scientist, August 2016

2 Scientific Applications Support Mission Realize customer driven applications of Promega technologies to enhance the value of our technologies and strengthen our customer relationships Customer-specific testing & protocol development Scientific Training Custom field support (demos, seminars & experiments) 2

3 Presentation Outline DNA Workflow Purification Quantitation Key considerations at each step Ways to overcome major challenges Examples of challenging samples 3

4 DNA Workflow Each Step Affects the Quality of the Final Data PCR Amplify Cloning Purify Quantify qpcr Sequencing Microarray 4

5 Downstream Applications Importance of Input DNA Characteristics PCR qpcr Sequencing Arrays Quantity of DNA Integrity of DNA +/- Depending on amplicon - Typically small amplicons More important with longer read technologies, but many providers assume large fragments Typically small fragments, but providers expect minimum fragment sizes Lack of Inhibitors Accurate Quantitation Ultimately, sequencing data is dependent on the integrity and quality of the starting material 5

6 Purification: Setting the Stage for Downstream Success 6

7 Purification Yield, Integrity & Purity are Critical to Success Key Challenges Purifying sufficient DNA from: Low biomass samples Difficult samples Degraded samples DNA integrity Isolating pure DNA No enzyme inhibitors to affect downstream applications No contaminating RNA 7

8 DNA Purification Technologies All Provide Advantages Depending on Specific Needs Manual Small automated 96 well manual Automated 8

9 Purification, Manual Low Investment and Scalability are Attractive Manual columns and scalable solution-based purification are attractive low-throughput options for standard or difficult samples Advantages Low initial investment vs. automation Flexibility in sample processing Lower price per prep Minimal set up time Reasons to Consider Other Options Greater throughput desired Time constraints Error reduction Many sample types supported: Blood, Tissue, FFPE, Plant 9

10 DNA isolation from coffee beans ReliaPrep gdna Tissue Miniprep system 10

11 DNA isolation from coffee beans DNA was isolated from coffee beans and was amplifiable using plant universal primers. 11

12 DNA Purification, Small Scale Automation Small Automated Systems Offer Major Benefits Small, dedicated purification instruments allow individuals to automate purification and increase productivity Advantages Minimal initial investment Frees time for other activities Fewer purification errors Increases sample throughput Reasons to Consider Other Options Not enough throughput to justify Even greater throughput desired Input sample volume incompatibility Maxwell RSC: 5 minute setup minutes to extract 1-16 samples 12

13 Versatility of the Maxwell RSC instrument Clinical Research Applied Academic 13

14 Maxwell RSC Instrument 14

15 Microbes in water from Trunk River Purpose: Isolate DNA from microbes in Trunk River water using Maxwell RSC instrument. Experimental variables = Amount of water and homogenizing matrix. Homogenization Lysate 15

16 Microbes in water from Trunk River DNA was extracted using all homogenization matrices, but at different amounts. Sulfurreducing bacteria was detected in the samples using qpcr. 16

17 DNA Purification, Manual 96 Well Vacuum Purification Increases Sample Throughput Advantages Low initial investment High sample throughput Offers performance equal to spin columns Reasons to Consider Other Options 96 well processing can be tedious Desire to reduce errors Staff time has become rate limiting Greater throughput desired Input sample volume incompatibility The Wizard SV 96 Genomic system can isolate gdna from many sample types in less than 60 minutes 96 well manual 17

18 DNA Purification, Automated 96 Well Increases Laboratory Throughput and Lowers Costs Advantages Increases laboratory productivity Aids in sample tracking Increases consistency of results Can automate many activities Reasons to Consider Other Options High initial cost Not enough throughput to justify Automated 18

19 Purification of DNA from food using automation qpcr 19

20 Purification of DNA from food using automation DNA was isolated from these food samples using an automated platform. The DNA was amplifiable using Universal Plant and Animal primers. 20

21 What sample types have challenged you? 21

22 Quantitation: A Simple But Critical Step in Analysis 22

23 Quantitation A Simple But Critical Step in Analysis PCR Amplify Cloning Purify Quantify qpcr Sequencing Microarray 23

24 Key Challenges Include Sensitivity, Accuracy, and Nucleic Acid Specificity Quantify Challenges Sensitivity effectively measuring small nucleic acid amounts Accuracy affected by purity detection range Specificity dsdna vs ssdna vs RNA human vs non-human 24

25 Three Common Methods of Quantitation Utilize UV Absorbance, Fluorescent Dyes and qpcr UV Absorbance Spectrophotometer NanoDrop /NanoVue Fluorescent Dye-based Quantitation Plate Reader Hand-held Instruments Real-Time PCR 25

26 UV Absorbance Measures Different Components with Distinct Wavelengths Wavelength 260nm 280nm 230nm 320nm Measurement Amount of nucleic acid present in a sample A 260nm of 1.0 = 50µg/ml for dsdna 40µg/ml for RNA 33µg/ml for ssdna Amount of protein present in a sample Amount of other contaminants present in a sample Amount of light scattering components present in a sample; used for background subtraction 26

27 Fluorescent Dye-based Quantitation is a More Sensitive Method Dye binds nucleic acid the resulting conformation shift produces in fluorescence when excited Fluorescence is directly proportional to the amount of nucleic acid in the sample Easy Protocol: Add, Mix, Measure Incubate at room temp for 5 minutes Higher signal = more nucleic acid present Unbound dye does not fluoresce Low background increases sensitivity 504nm Excitation 531nm Unbound dyex 504nm 28

28 Real-Time PCR (quantitative PCR, qpcr) Quantitation Involves Detection of Product at Each Cycle What is end-point PCR? The amount of amplified product is typically determined only after a set number of amplification cycles is completed What is Real-Time PCR? The amount of amplified product is measured after each PCR amplification cycle 29

29 Quantitation Activity DNA Analysis Methods Equipment/Sup plies Required Relative Cost per Assay Assay Time Hands-On Time Ability to Automate Absorbance Dye-Based Quantitation qpcr Spectrophotome ter Fluorometer, Dye kits Instrument, reaction comp. Low <1min <1min Y Medium 10min 5min Y High 1-2hr min Y DNA Analysis Methods Concentration Purity/ Contamination Integrity Specificity DNA/RNA Sensitivity Absorbance Dye-Based Quantitation qpcr 30

30 Quantitation Activity DNA Analysis Methods Equipment/Sup plies Required Relative Cost per Assay Assay Time Hands-On Time Ability to Automate Absorbance Dye-Based Quantitation qpcr Spectrophotome ter Fluorometer, Dye kits Instrument, reaction comp. Low <1min <1min Y Medium 10min 5min Y High 1-2hr min Y DNA Analysis Methods Concentration Purity/ Contamination Integrity Specificity DNA/RNA Sensitivity Absorbance Y Dye-Based Quantitation Y qpcr Y 31

31 Quantitation Activity DNA Analysis Methods Equipment/Sup plies Required Relative Cost per Assay Assay Time Hands-On Time Ability to Automate Absorbance Dye-Based Quantitation qpcr Spectrophotome ter Fluorometer, Dye kits Instrument, reaction comp. Low <1min <1min Y Medium 10min 5min Y High 1-2hr min Y DNA Analysis Methods Concentration Purity/ Contamination Integrity Specificity DNA/RNA Sensitivity Absorbance Y Y Dye-Based Quantitation Y N qpcr Y Y/N 32

32 Quantitation of DNA by Absorbance Can Be Overestimated Due to Contaminants gdna Extraction from Matched Lung Tissue FFPE Slides Chemistry A Chemistry B No peak at 230nm Large peak at 230nm Absorbance is unreliable for Chemistry B is qpcr a better choice? 33

33 ng/ul ng/ul Absorbance at 260nm May Not Be an Accurate Measure of Amplifiable Yield Quantitation by Absorbance breast breast colon colon lung lung Chem A Chem B Chem A Chem B Chem A Chem B Tissue/method Quantitation by Amplification Chem A Chem B Large difference in quantitation Absorbance at 260nm may not be an accurate measure of amplifiable yield Absorbance and amplifiability may correlate, but several other factors play a role breast colon lung 34

34 The NanoDrop Instrument Often Overestimates the Amount of DNA in Solution Accurate quantitation is critical for many downstream applications Many FFPE tissue sections are small, and isolated DNA samples have concentrations well below the limit of detection of traditional spectrophotometric assays Even with highly purified DNA, the NanoDrop consistently overestimates the amount of DNA in solution 35

35 Quantitation Activity DNA Analysis Methods Equipment/Sup plies Required Relative Cost per Assay Assay Time Hands-On Time Ability to Automate Absorbance Dye-Based Quantitation qpcr Spectrophotome ter Fluorometer, Dye kits Instrument, reaction comp. Low <1min <1min Y Medium 10min 5min Y High 1-2hr min Y DNA Analysis Methods Concentration Purity/ Contamination Integrity Specificity DNA/RNA Sensitivity Absorbance Y Y Dye-Based Quantitation Y N qpcr Y Y/N 36

36 Quantitation Activity DNA Analysis Methods Equipment/Sup plies Required Relative Cost per Assay Assay Time Hands-On Time Ability to Automate Absorbance Dye-Based Quantitation qpcr Spectrophotome ter Fluorometer, Dye kits Instrument, reaction comp. Low <1min <1min Y Medium 10min 5min Y High 1-2hr min Y DNA Analysis Methods Concentration Purity/ Contamination Integrity Specificity DNA/RNA Sensitivity Absorbance Y Y N Dye-Based Quantitation Y N N qpcr Y Y/N Y/N 37

37 Quantitation Activity DNA Analysis Methods Equipment/Sup plies Required Relative Cost per Assay Assay Time Hands-On Time Ability to Automate Absorbance Dye-Based Quantitation qpcr Spectrophotome ter Fluorometer, Dye kits Instrument, reaction comp. Low <1min <1min Y Medium 10min 5min Y High 1-2hr min Y DNA Analysis Methods Concentration Purity/ Contamination Integrity Specificity DNA/RNA Sensitivity Absorbance Y Y N N Dye-Based Quantitation Y N N Y/N qpcr Y Y/N Y/N Y 38

38 A 260 Absorbance is Not Specific and Cannot Distinguish Between dsdna, RNA, or ssdna DNA Sample A Sample B RNase NanoDrop (ng/µl) QuantiFluor dsdna (ng/µl) Sample A Sample B RNA 260nm reading represents total amount of all nucleic acid present in sample Cannot distinguish between dsdna, ssdna, or RNA 39

39 Quantitation Activity DNA Analysis Methods Equipment/Sup plies Required Relative Cost per Assay Assay Time Hands-On Time Ability to Automate Absorbance Dye-Based Quantitation qpcr Spectrophotome ter Fluorometer, Dye kits Instrument, reaction comp. Low <1min <1min Y Medium 10min 5min Y High 1-2hr min Y DNA Analysis Methods Concentration Purity/ Contamination Integrity Specificity DNA/RNA Sensitivity Absorbance Y Y N N Dye-Based Quantitation Y N N Y/N qpcr Y Y/N Y/N Y 40

40 Quantitation Activity DNA Analysis Methods Equipment/Sup plies Required Relative Cost per Assay Assay Time Hands-On Time Ability to Automate Absorbance Dye-Based Quantitation qpcr Spectrophotome ter Fluorometer, Dye kits Instrument, reaction comp. Low <1min <1min Y Medium 10min 5min Y High 1-2hr min Y DNA Analysis Methods Concentration Purity/ Contamination Integrity Specificity DNA/RNA Sensitivity Absorbance Y Y N N Good 10-12,000ng/ul Dye-Based Quantitation Y N N Y/N Better ng qpcr Y Y/N Y/N Y Best Depends 41

41 Disadvantages of Absorbance Include Lack of Specificity, Overestimation, and Lack of Integrity Information Lack of Specificity Cannot distinguish between dsdna, RNA or ssdna Nucleic acid contamination cannot be determined Overestimation of nucleic acid concentration due to contaminants Many contaminants absorb at or around 260nm No information on integrity Nucleotides and small nucleic acid fragments still contribute to the 260nm reading 42

42 Disadvantages of Fluorescent Dye-based Quantitation Include No Information on Purity or Integrity Must create standards No information on purity Separate dye-based quantification systems are available for ssdna, RNA and protein No information on integrity Lack of specificity with dyes Fluorescent dyes are potentially hazardous 43

43 Disadvantages of Real-Time PCR Quantitation Include Expensive Equipment and Sensitivity to Inhibitors Requires specialized instrumentation Higher cost compared to UV absorbance and fluorescent dye-based methods ABI 7500 Real Time System Sensitivity to inhibitors Bio-Rad MyiQ2 Bio-Rad QX100 Droplet Digital PCR System 44

44 Several Different Metrics Can Be Used to Predict the Likelihood of Success in Downstream Assays Quantitation by: Absorbance Fluorescent dye Amplification (qpcr) Purity by: Absorbance ratio Factors that impact quality: Purification chemistry contaminants Sample specific inhibitors Fragmentation All are used to predict likelihood of success in downstream assays 45

45 NGS Workflow The QuantiFluor dsdna dye system is designed to measure total double-stranded DNA concentration without regard for species, size, or amplifiability of the DNA, whereas a qpcr assay (84 bp target) is a human-specific qpcr test designed to measure amplifiable DNA. Samples with significantly lower qpcr quantitation results vs. fluorescent dye-based quantitation are indicative of degraded DNA. Ratios of small to large amplicons as measured in the DNA QC assay are predictive of coverage uniformity and sequencing quality. For FFPE samples, a lower ratio of small (75 bp) to large (300 bp) amplicon target is indicative of less degradation of the DNA. Green = Amplicon ratios 47, coverage uniformity 93% Yellow = Amplicon ratios , coverage uniformity 85-92% Red = Amplicon ratios 1000, coverage uniformity 84% 46

46 Promega Application Lab at MBL Contact: Mark Bratz July 31 August 12 Office/Lab: Loeb

47 Questions 48