Post on 16-Apr-2018
Promega CorporationPromega Corporation
DNA Workflow
Marine Biological Laboratory
Mark BratzApplications Scientist, Promega Corporation
August 2016
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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)
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Presentation Outline
DNA Workflow• Purification• Quantitation
Key considerations at each step Ways to overcome major challenges Examples of challenging samples
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DNA WorkflowEach Step Affects the Quality of the Final Data
Purify Quantify
PCR Amplify
qPCR
Sequencing
Microarray
Cloning
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Downstream ApplicationsImportance 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
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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
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DNA Purification TechnologiesAll Provide Advantages Depending on Specific Needs
ManualSmall
automated96 well manual
Automated
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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 Reasons to Consider Other Options
Low initial investment vs. automation Greater throughput desired
Flexibility in sample processing Time constraints
Lower price per prep Error reduction
Minimal set up time
Many sample types supported: Blood, Tissue, FFPE, Plant…
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DNA isolation from coffee beans
ReliaPrep gDNA Tissue Miniprep system
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DNA isolation from coffee beans
DNA was isolated from coffee beans and was amplifiable using plant universal primers.
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DNA Purification, Small Scale AutomationSmall Automated Systems Offer Major Benefits
Small, dedicated purification instruments allow individuals to automate purification and increase productivity
Advantages Reasons to Consider Other Options
Minimal initial investment Not enough throughput to justify
Frees time for other activities Even greater throughput desired
Fewer purification errors Input sample volume incompatibility
Increases sample throughput
Maxwell® RSC: 5 minute setup – 30-45 minutes to extract 1-16 samples
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Clinical
Research
AcademicApplied
Versatility of the Maxwell® RSC instrument
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Microbes in water from Trunk River
Lysate
Purpose: Isolate DNA from microbes in Trunk River water using Maxwell RSC instrument. Experimental variables = Amount of water and homogenizing matrix.
Homogenization
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Microbes in water from Trunk River
DNA was extracted using all homogenization matrices, but at different amounts. Sulfur-reducing bacteria was detected in the samples using qPCR.
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DNA Purification, Manual 96 Well Vacuum Purification Increases Sample Throughput
Advantages Reasons to Consider Other Options
Low initial investment 96 well processing can be tedious
High sample throughput Desire to reduce errors
Offers performance equal to spin columns
Staff time has become rate limiting
Greater throughput desired
Input sample volume incompatibility
96 well manual
The Wizard® SV 96 Genomic system can isolate gDNAfrom many sample types in less than 60 minutes
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DNA Purification, Automated 96 Well Increases Laboratory Throughput and Lowers Costs
Advantages Reasons to Consider Other Options
Increases laboratory productivity High initial cost
Aids in sample tracking Not enough throughput to justify
Increases consistency of results
Can automate many activities
Automated
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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.
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QuantitationA Simple But Critical Step in Analysis
Purify Quantify
PCR Amplify
qPCR
Sequencing
Microarray
Cloning
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Challenges
Sensitivity
• effectively measuring small nucleic acid amounts
Accuracy
• affected by purity
• detection range
Specificity
• dsDNA vs ssDNA vs RNA
• human vs non-human
Quantify
Key Challenges Include Sensitivity, Accuracy, and Nucleic Acid Specificity
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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
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UV Absorbance Measures Different Components with Distinct Wavelengths
Wavelength Measurement
260nm
Amount of nucleic acid present in a sample
A260nm of 1.0 = 50µg/ml for dsDNA
40µg/ml for RNA
33µg/ml for ssDNA
280nm Amount of protein present in a sample
230nm Amount of other contaminants present in a sample
320nmAmount of light scattering components present in a sample; used for
background subtraction
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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
• Higher signal = more nucleic acid present
• Unbound dye does not fluoresce
• Low background increases sensitivity
Incubate at room temp for 5 minutes
504nmExcitation
Emits @ 531nm
504nm
XUnbound dye
Easy Protocol: Add, Mix, Measure
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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
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Quantitation Activity
DNA Analysis Methods
Concentration Purity/ Contamination
Integrity Specificity –DNA/RNA
Sensitivity
Absorbance
Dye-Based Quantitation
qPCR
DNA Analysis Methods
Equipment/Supplies Required
Relative Cost per Assay
Assay Time Hands-On Time Ability to Automate
Absorbance Spectrophotometer
Low <1min <1min Y
Dye-Based Quantitation
Fluorometer, Dye kits
Medium 10min 5min Y
qPCR Instrument, reaction comp.
High 1-2hr 15-30 min Y
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Quantitation Activity
DNA Analysis Methods
Concentration Purity/ Contamination
Integrity Specificity –DNA/RNA
Sensitivity
Absorbance Y
Dye-Based Quantitation
Y
qPCR Y
DNA Analysis Methods
Equipment/Supplies Required
Relative Cost per Assay
Assay Time Hands-On Time Ability to Automate
Absorbance Spectrophotometer
Low <1min <1min Y
Dye-Based Quantitation
Fluorometer, Dye kits
Medium 10min 5min Y
qPCR Instrument, reaction comp.
High 1-2hr 15-30 min Y
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Quantitation Activity
DNA Analysis Methods
Concentration Purity/ Contamination
Integrity Specificity –DNA/RNA
Sensitivity
Absorbance Y Y
Dye-Based Quantitation
Y N
qPCR Y Y/N
DNA Analysis Methods
Equipment/Supplies Required
Relative Cost per Assay
Assay Time Hands-On Time Ability to Automate
Absorbance Spectrophotometer
Low <1min <1min Y
Dye-Based Quantitation
Fluorometer, Dye kits
Medium 10min 5min Y
qPCR Instrument, reaction comp.
High 1-2hr 15-30 min Y
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Quantitation of DNA by Absorbance Can Be Overestimated Due to Contaminants
Chemistry A Chemistry B
Large peak at 230nmNo peak at 230nm
gDNA Extraction from Matched Lung Tissue FFPE Slides
Absorbance is unreliable for Chemistry B – is qPCR a better choice?
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Absorbance at 260nm May Not Be an Accurate Measure of Amplifiable Yield
0.0
20.0
40.0
60.0
80.0
100.0
120.0
140.0
breast breast colon colon lung lung
Chem A Chem B Chem A Chem B Chem A Chem B
ng/
ul
Tissue/method
Quantitation by Absorbance
0.0
1.0
2.0
3.0
4.0
5.0
6.0
breast colon lung
ng/
ul
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 amplifiabilitymay correlate, but several other factors play a role
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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
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Quantitation Activity
DNA Analysis Methods
Concentration Purity/ Contamination
Integrity Specificity –DNA/RNA
Sensitivity
Absorbance Y Y
Dye-Based Quantitation
Y N
qPCR Y Y/N
DNA Analysis Methods
Equipment/Supplies Required
Relative Cost per Assay
Assay Time Hands-On Time Ability to Automate
Absorbance Spectrophotometer
Low <1min <1min Y
Dye-Based Quantitation
Fluorometer, Dye kits
Medium 10min 5min Y
qPCR Instrument, reaction comp.
High 1-2hr 15-30 min Y
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Quantitation Activity
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
DNA Analysis Methods
Equipment/Supplies Required
Relative Cost per Assay
Assay Time Hands-On Time Ability to Automate
Absorbance Spectrophotometer
Low <1min <1min Y
Dye-Based Quantitation
Fluorometer, Dye kits
Medium 10min 5min Y
qPCR Instrument, reaction comp.
High 1-2hr 15-30 min Y
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Quantitation Activity
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
DNA Analysis Methods
Equipment/Supplies Required
Relative Cost per Assay
Assay Time Hands-On Time Ability to Automate
Absorbance Spectrophotometer
Low <1min <1min Y
Dye-Based Quantitation
Fluorometer, Dye kits
Medium 10min 5min Y
qPCR Instrument, reaction comp.
High 1-2hr 15-30 min Y
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A260 Absorbance is Not Specific and Cannot Distinguish Between dsDNA, RNA, or ssDNA
RNA
Sample A Sample B - + - + RNase
NanoDrop®
(ng/µl)
QuantiFluor™
dsDNA (ng/µl)
Sample A 213.5 158.0
Sample B 87.5 66.0
260nm reading represents total amount of all nucleic acid present in sample
Cannot distinguish between dsDNA, ssDNA, or RNA
DNA
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Quantitation Activity
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
DNA Analysis Methods
Equipment/Supplies Required
Relative Cost per Assay
Assay Time Hands-On Time Ability to Automate
Absorbance Spectrophotometer
Low <1min <1min Y
Dye-Based Quantitation
Fluorometer, Dye kits
Medium 10min 5min Y
qPCR Instrument, reaction comp.
High 1-2hr 15-30 min Y
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Quantitation Activity
DNA Analysis Methods
Concentration Purity/ Contamination
Integrity Specificity –DNA/RNA
Sensitivity
Absorbance Y Y N N Good10-12,000ng/ul
Dye-Based Quantitation
Y N N Y/N Better0.1-500ng
qPCR Y Y/N Y/N Y BestDepends
DNA Analysis Methods
Equipment/Supplies Required
Relative Cost per Assay
Assay Time Hands-On Time Ability to Automate
Absorbance Spectrophotometer
Low <1min <1min Y
Dye-Based Quantitation
Fluorometer, Dye kits
Medium 10min 5min Y
qPCR Instrument, reaction comp.
High 1-2hr 15-30 min Y
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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
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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
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Disadvantages of Real-Time PCR Quantitation Include Expensive Equipment and Sensitivity to Inhibitors
ABI 7500 Real Time System
Bio-Rad MyiQ2
Requires specialized instrumentation
Higher cost compared to UV absorbance and fluorescent dye-based methods
Sensitivity to inhibitors
Bio-Rad QX100™ Droplet
Digital™ PCR System
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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
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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 125-766, coverage uniformity 85-92% • Red = Amplicon ratios ≥ 1000, coverage uniformity ≤84%
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Promega Application Lab at MBL
Contact: Mark Bratz
mark.bratz@promega.com
608-320-0697
July 31 – August 12
Office/Lab: Loeb 252